Lipase variants, polynucleotides encoding same and uses thereof

Abstract

The present invention relates to lipase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

[0001] References to sequence lists

[0002] This application includes a sequence list in a computer-readable form, which is incorporated herein by reference. Background of the Invention Technical Field

[0004] This invention relates to lipase variants, polynucleotides encoding these variants, methods for generating these variants, and methods for using these variants. Background Technology

[0006] Lipases are important biocatalysts, and they have been shown to be useful in a wide range of applications. A large number of different lipases have been identified, and many have been commercialized. However, novel lipases suitable for use in different compositions adapted to current operating conditions are desirable.

[0007] Lipases are included in detergent compositions to improve washing performance and specifically to enhance the removal of lipid stains. Current detergent, cleaning, and / or fabric care compositions contain numerous active ingredients that interfere with the ability of lipases to remove lipid stains. Among other things, builders are included in detergent compositions to reduce calcium concentration, as calcium deposition can cause a "graying" appearance on treated surfaces. Low levels of calcium have been shown to lead to reduced lipase activity.

[0008] In many lipases, the catalytic site is masked by a cap domain (cap region or cap), and studies have shown that this cap is important for lipase activity and plays a role in activating lipases. The enhanced catalytic activity in the presence of the water / lipid interface is called "interfacial activation," and describes the opening of the amphiphilic surface ring, i.e., the cap, upon contact with the interface. Shu et al., 2011, Enzyme and Microbial Technology, 48:129-133, generated four Aspergillus niger lipase (ANL) mutants, one without a cap and the other three with a cap in an open conformation, aiming to identify lipase mutants independent of interfacial activation.

[0009] Therefore, there is a need for lipases with improved lipase activity, particularly for use in detergent compositions. Summary of the Invention

[0010] In one aspect, the present invention relates to lipase variants comprising modifications at one or more positions corresponding to positions 1, 2, 3, 5, 8, 43, 45, 105, 167, 178, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 239 and 244 of SEQ ID NO:1, wherein these variants have lipase activity.

[0011] In a second aspect, the present invention relates to lipase variants comprising the contents corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 30, 31, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 106, 167, 178, 180, 192, 193, 194, 199, 200, 202, 210, 211 of SEQ ID NO:1. Modifications at two or more sites of 217, 218, 220, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254, and 256, wherein these variants have lipase activity.

[0012] In a third aspect, the present invention relates to lipase variants comprising the contents corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 30, 31, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 106, 167, 178, 180, 192, 193, 194, 199, 200, 202, 210, 211, 2 Arg is substituted at two or more positions at positions 17, 218, 220, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254, and 256, where these variants have lipase activity.

[0013] The present invention also relates to isolated polynucleotides encoding these variants; nucleic acid constructs, vectors, and host cells containing these polynucleotides; and methods for producing and using these variants.

[0014] definition

[0015] Lipase: The terms "lipase," "lipolytic enzyme," "lipid esterase," "lipolytic polypeptide," and "lipolytic protein" refer to an enzyme in class EC 3.1.1 as defined by enzyme nomenclature. It can possess lipase activity (triacylglycerol lipase, EC 3.1.1.3), keratinase activity (EC 3.1.1.74), sterol esterase activity (EC 3.1.1.13), and / or wax ester hydrolase activity (EC 3.1.1.50). For the purposes of this invention, lipase activity is determined according to the procedure described in the Examples section: substrates with different chain lengths can be used, and hydrolytic activity is determined using the PnP assay. In one aspect, the variants of the invention have at least 20% of the lipase activity of the parent lipase, for example at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In another aspect, the parent lipase is a polypeptide of SEQ ID NO:1.

[0016] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (encoding a polypeptide with no change) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0017] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature spliced ​​mRNA molecules derived from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. The initial RNA transcript is a precursor to mRNA, which undergoes a series of processing steps, including splicing, before becoming mature spliced ​​mRNA.

[0018] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame (OPG), which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0019] Control Sequence: The term "control sequence" refers to the nucleic acid sequence required for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to: leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. Control sequences may be provided with adapters for the purpose of introducing specific restriction sites that facilitate the connection of the control sequence to the coding region of the polynucleotide encoding the variant.

[0020] Expression: The term “expression” includes any steps involved in the generation of variants, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0021] Expression vector: The term “expression vector” refers to a straight or circular DNA molecule that includes polynucleotides encoding a variant and is operatively linked to a control sequence for its expression.

[0022] Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids absent at the amino and / or carboxyl termini of the polypeptide; wherein the fragment has lipase activity. In one aspect, the fragment comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids present in the parental lipase, but less than 100%. In one aspect, the parental lipase is the polypeptide of SEQ ID NO:1.

[0023] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0024] Improved properties: The term "improved properties" refers to characteristics associated with variants that are improved relative to the parental lipase. Such improved properties include, but are not limited to, lipase activity and non-Ca dependence. Lipase activity can be increased lipase activity; increased lipase activity under reduced / low levels of Ca; or increased lipase activity in the presence of EDTA. Non-Ca dependence can be increased non-Ca dependence. The term "reduced or low levels of Ca" means that the concentration of Ca in the solution has been reduced or lowered compared to a control solution. Such reduced or low levels of Ca can be obtained by adding a reagent that consumes some or all of the Ca from the solution. Such a reagent can be a washing aid as described in the "Compositions" section of this application.

[0025] Separate: The term “separate” means a substance in a form or environment that does not exist in nature. Non-limiting examples of separated substances include (1) any substance that is not naturally occurring, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, said substance being at least partially removed from one or more or all naturally occurring components associated with its properties; (3) any substance that has been artificially modified relative to a substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant generation in a host cell; multiple copies of the gene encoding said substance; and the use of a promoter stronger than that naturally associated with the gene encoding said substance).

[0026] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one respect, the mature polypeptide is SEQ ID NO:1. It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0027] Mutant: The term “mutant” refers to a polynucleotide that encodes a variant.

[0028] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain segments of nucleic acid, or is synthesized and contains one or more control sequences.

[0029] Operable ligation: The term “operable ligation” refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence guides the expression of the coding sequence.

[0030] Parent or parental lipase: The term "parent" or "parental lipase" refers to a lipase that has been modified to produce the enzyme variant of the present invention. The parent can be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof. An example of such a parental lipase is the amino acid sequence of SEQ ID NO:1.

[0031] Sequence identity: The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0032] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) is used to determine sequence identity between two amino acid sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the percentage identity and calculated as follows:

[0033] (Same residue × 100) / (Alignment length - Total number of vacancies in the alignment)

[0034] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later) is used to determine sequence identity between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -non-simplification option) is used as the percentage identity and calculated as follows:

[0035] (Same deoxyribonucleotide × 100) / (Alignment length - Total number of vacancies in the alignment)

[0036] Variants: The term "variant" refers to a polypeptide having lipase activity that contains modifications (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. These variants of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the lipase activity of the parent lipase polypeptide. In one aspect, the parent lipase comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0037] Wild-type lipase: The term "wild-type" lipase refers to lipases expressed by naturally occurring microorganisms (such as bacteria, yeast, or filamentous fungi) found in nature.

[0038] Variant Naming Rules

[0039] For the purposes of this invention, SEQ ID NO:1 was used to determine the corresponding amino acid residues in another lipase. The amino acid sequence of the other lipase was aligned with SEQ ID NO:1, and based on this alignment, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) was used to determine the amino acid position number corresponding to any amino acid residue in SEQ ID NO:1. The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0040] The identification of corresponding amino acid residues in another lipase can be determined by using several computer programs that compare multiple peptide sequences with their respective default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparison by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-2797), MAFTT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology). Biology [Molecular Biology Methods] 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680).

[0041] When other enzymes deviate from SEQ ID NO:1, rendering traditional sequence-based comparison methods unable to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. [Journal of Molecular Biology] 295:613-615), alternative pairwise sequence comparison algorithms can be used. Higher sensitivity in sequence-based searches can be achieved using search programs that employ probabilistic representations (spectrums) of peptide families to search databases. For example, the PSI-BLAST program generates multiple spectra through an iterative database search process and can detect distant homologs (Atschul et al., 1997, Nucleic Acids Res. [Nucleic Acids Research] 25:3389-3402). Even higher sensitivity can be achieved if the peptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. [Journal of Molecular Biology] 287:797-815; McGuffin and Jones, 2003, Bioinformatics [Bioinformatics] 19:874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J. Mol. Biol. [Journal of Molecular Biology] 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using a variety of tools developed for this purpose.

[0042] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms can be used to align two or more protein structures, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), and implementations of these algorithms can also be used to query structure databases containing structures of interest to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0043] In describing variations of the invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are used.

[0044] replace: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations / modifications are separated by plus signs ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" (or G205S411F) indicate that glycine (G) and serine (S) at positions 205 and 411 are substituted with arginine (R) and phenylalanine (F), respectively.

[0045] Missing: For amino acid deletions, use the following nomenclature: original amino acid, position, *. Therefore, a deletion of glycine at position 195 would be represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*".

[0046] insert: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, inserting lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, inserting lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0047] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding the inserted amino acid residue. In the example above, the sequence would therefore be:

[0048] 195 195 195a 195b G GKA

[0049] Multiple modifications: Variants containing multiple modifications are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0050] Different modifiers:In cases where different modifications may be introduced at a certain position, these different modifications are separated by commas. For example, "Arg170Tyr,Glu" indicates that arginine at position 170 is replaced by tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant:

[0051] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”. Detailed Implementation

[0052] This invention relates to lipase variants that, compared to the parent enzyme, have increased lipase activity and / or increased non-Ca dependence.

[0053] variants

[0054] In a first aspect, the present invention relates to isolated lipase variants comprising modifications at one or more (e.g., several) positions corresponding to positions 1, 2, 3, 5, 8, 43, 45, 105, 167, 178, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 239, and 244 of SEQ ID NO:1, wherein these variants have lipase activity and wherein:

[0055] a. The modification at the position corresponding to position 1 is missing, such as E1*, or Arg is inserted, such as E1ER;

[0056] b. The modification at position 2 is missing, for example, V2*;

[0057] c. The modification at position 3 is missing, for example, S3*;

[0058] d. The modification at position 5 is missing, for example, D5*;

[0059] e. The modification at position 8 is replaced by Arg or Lys, for example N8R,K, especially by Arg;

[0060] f. The modification at position 43 is missing, for example, E43*;

[0061] g. The modification at position 45 is missing, for example, E45*;

[0062] h. The modification at position 167 is missing, for example, D167*;

[0063] i. The modification at the position corresponding to position 224 is missing, such as S224*, or Arg is inserted, such as S224SR;

[0064] j. The modification at the position corresponding to position 225 is missing, such as G225*, or Arg is inserted, such as G225GR;

[0065] k. The modification at the position corresponding to position 226 is missing, such as T226*, or Arg insertion, such as T226TR;

[0066] l. The modification at the position corresponding to position 227 is missing, such as L227*, or Arg insertion, such as L227LR;

[0067] m. The modification at the position corresponding to position 228 is missing, such as V228*, or Arg insertion, such as V228VR;

[0068] n. The modification at the position corresponding to position 229 is missing, e.g., P229*, or Arg insertion, e.g., P229PR;

[0069] o. The modification at the position corresponding to position 230 is missing, such as V230*, or replaced by Arg or Lys, such as V230K,R, especially replaced by Arg;

[0070] p. The modification at the position corresponding to position 231 is one or more Arg insertions, such as T231TR or T231TRR;

[0071] q. The modification at the position corresponding to position 232 is an Arg insertion, for example, R232RR;

[0072] r. The modification at position 234 is missing, for example, D234*;

[0073] s. The modification at position 236 is replaced by Arg or Lys, for example, V236K or V236R, especially by Arg; and

[0074] t. The modification at position 239 is missing, for example, E239*;

[0075] u. The modification at the position corresponding to position 105 is replaced by Arg, such as S105R, and / or Arg is inserted, such as S105SR or S105RR; and

[0076] v. The modifier at position 178 is replaced by Arg, for example, N178R;

[0077] x. The modification at position 244 is an Arg insertion, for example, T244TR.

[0078] In a second aspect, the present invention relates to isolated lipase variants comprising positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 31, 30, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 167, 178, 180, 192, 193, 194, 199, 200, 202, 210, 211, 217 corresponding to SEQ ID NO: 1. Modifications at two or more (e.g., several) positions of 218, 220, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254, and 256, wherein these variants possess lipase activity and wherein

[0079] a. The modification at the position corresponding to position 1 is missing, such as E1*, Arg is inserted, such as E1ER, or is replaced by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, especially replaced by Arg, Gln, Lys or Pro, most especially replaced by Gln or Pro, such as E1P or E1Q;

[0080] b. The modification at the position corresponding to position 2 is missing, such as V2*, or replaced by Arg or Lys, such as V2R or V2K;

[0081] c. The modification at the position corresponding to position 3 is missing, such as S3*, or replaced by Arg or Lys, such as S3R or S3K, especially replaced by Arg;

[0082] d. The modification at position 4 is replaced by Arg or Lys, for example Q4R or Q4K, especially by Arg;

[0083] e. The modification at position 5 is missing, such as D5*, or is replaced by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, especially replaced by Arg or Lys, such as D5K or D5R, most especially replaced by Arg;

[0084] f. The modification at position 6 is replaced by Arg or Lys, for example L6R or L6K, especially by Arg;

[0085] g. The modification at the position corresponding to position 7 is replaced by Arg or Lys, for example F7R or F7K, especially by Arg;

[0086] h. The modification at the position corresponding to position 8 is replaced by Arg or Lys, such as N8R or N8K, especially by Arg;

[0087] i. The modification at the position corresponding to position 9 is replaced by Arg or Lys, such as Q9R or Q9K, especially by Arg;

[0088] j. The modification at the position corresponding to position 11 is replaced by Arg or Lys, such as N11R or N11K, especially by Arg;

[0089] k. The modification at the position corresponding to position 12 is replaced by Arg or Lys, such as L12R or L12K, especially by Arg;

[0090] l. The modification at the position corresponding to position 15 is replaced by Arg or Lys, such as Q15R or Q15K, especially by Arg;

[0091] m. The modification at the position corresponding to position 37 is replaced by Arg or Lys, such as T37R or T37K, especially by Arg;

[0092] n. The modification at the position corresponding to position 38 is replaced by Arg or Lys, such as Q38R or Q38K, especially by Arg;

[0093] o. The modification at the position corresponding to position 39 is replaced by Arg or Lys, such as N39R or N39K, especially by Arg;

[0094] p. The modification at the position corresponding to position 40 is replaced by Arg or Lys, such as A40R or A40K, especially by Arg;

[0095] q. The modification at position 42 is replaced by Arg or Lys, for example P42R or P42K, especially by Arg;

[0096] r. The modification at position 43 is missing, for example, E43*, or is replaced at position 43 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, especially by Arg or Lys, for example, E43K or E43R, most especially by Arg;

[0097] s. The modification at position 45 is missing, for example, E45*, or is replaced at position 45 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, especially by Arg or Lys, for example, E45K or E45R, most especially by Arg;

[0098] t. The modification at the position corresponding to position 73 is replaced by Arg or Lys, such as N73R or N73K, especially by Arg;

[0099] u. The modification at position 167 is missing, such as D167*, or is replaced at position 167 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, especially by Arg or Lys, such as D167K or D167R, most especially by Arg;

[0100] v. The modification at the position corresponding to position 192 is replaced by Arg or Lys, for example T192R or T192K, especially by Arg;

[0101] w. The modification at the position corresponding to position 193 is replaced by Arg or Lys, such as L193R or L193K, especially by Arg;

[0102] x. The modification at the position corresponding to position 194 is replaced by Arg or Lys, such as Y194R or Y194K, especially by Arg;

[0103] y. The modification at the position corresponding to position 199 is replaced by Arg or Lys, such as T199R or T199K, especially by Arg;

[0104] z. The modification at the position corresponding to position 200 is replaced by Arg or Lys, such as N200R or N200K, especially by Arg;

[0105] aa. The modification at the position corresponding to position 202 is replaced by Arg or Lys, such as I202R or I202K, especially by Arg;

[0106] ab. The modification at the position corresponding to position 217 is replaced by Arg or Lys, such as S217R or S217K, especially by Arg;

[0107] ac. The modification at the position corresponding to position 218 is replaced by Arg or Lys, such as P218R or P218K, especially by Arg;

[0108] ad. The modification at the position corresponding to position 220 is replaced by Arg or Lys, such as Y220R or Y220K, especially by Arg;

[0109] ae. The modification at the position corresponding to position 221 is replaced by Arg or Lys, for example W221R or W221K, especially by Arg;

[0110] af. The modification at the position corresponding to position 224 is missing, e.g. S224*, Arg is inserted, e.g. S224SR, or replaced by Arg or Lys, e.g. S224K or S224R, especially replaced by Arg;

[0111] ag. The modification at the position corresponding to position 225 is missing, such as G225*, Arg is inserted, such as G225GR, or replaced by Arg or Lys, such as G225K or G225R, especially replaced by Arg;

[0112] ah. The modification at the position corresponding to position 226 is missing, such as T226*, Arg is inserted, such as T226TR, or replaced by Arg or Lys, such as T226K or T226R, especially replaced by Arg;

[0113] ai. The modification at the position corresponding to position 227 is missing, such as L227*, Arg is inserted, such as L227LR, or replaced by Arg or Lys, such as L227K or L227R, especially replaced by Arg;

[0114] aj. The modification at the position corresponding to position 228 is missing, e.g. V228*, Arg is inserted, e.g. V228VR, or replaced by Arg or Lys, e.g. V228K or V228R, especially replaced by Arg;

[0115] ak. The modification at the position corresponding to position 229 is missing, such as P229*, Arg is inserted, such as P229PR, or replaced by Arg or Lys, such as P229K or P229R, especially replaced by Arg;

[0116] al. The modification at the position corresponding to position 230 is missing, such as V230*, Arg is inserted, such as V230VR, or replaced by Arg or Lys, such as V230K or V230R, especially replaced by Arg;

[0117] The modification at the position corresponding to position 231 is one or two Arg insertions, such as T231TR or T231TRR, or is replaced by Arg or Lys, such as T231K or T231R, especially by Arg;

[0118] an. The modification at the position corresponding to position 232 is an Arg insertion, for example, R232RR;

[0119] The modification at position 233 is replaced by Arg or Lys, for example, N233K or D233R, especially by Arg;

[0120] The modification at position 234 is missing, for example, D234*, or is replaced at position 234 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, especially by Arg or Lys, for example, D234K or D234R, most especially by Arg;

[0121] The modification at position 236 in aq. is replaced by Arg or Lys, for example V236K or V236R, especially by Arg;

[0122] The modification at the position corresponding to position 238 is replaced by Arg or Lys, such as I238K or I238R, especially by Arg;

[0123] The modification at position 239 is missing, for example, E239*, or is replaced at position 239 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, especially by Arg or Lys, for example, E239K or E239R, most especially by Arg;

[0124] The modification at the position corresponding to position 243 is an Arg insertion, such as A243AR, or replaced by Arg or Lys, such as A243K or A243R, especially replaced by Arg;

[0125] The modification at position 244 is an Arg insertion, such as T244TR, or replaced by Arg or Lys, such as T244K or T244R, especially replaced by Arg;

[0126] The modification at position 245 is replaced by Arg or Lys, such as G245K or G245R, especially Arg;

[0127] The modification at position 248 is replaced by Arg or Lys, such as N248K or N248R, especially by Arg;

[0128] The modification at position 249 is replaced by Arg or Lys, for example, Q249K or Q249R, especially by Arg; and

[0129] The modification at position 251 is replaced by Arg or Lys, for example, N251K or N251R, especially by Arg.

[0130] The modifier at position 28 (as in az.) is replaced by Arg, for example, A28R.

[0131] The modification at position 29 is replaced by Arg, for example, P29R;

[0132] The modification at position 30 is replaced by Arg, for example, A30R;

[0133] bc. The modification at position 31 is replaced by Arg, for example, G31R;

[0134] bd. The modification at position 33 is replaced by Arg, for example, N33R;

[0135] The modifier at position 99 of be is replaced by Arg, for example, E99R;

[0136] bf. The modification at the position corresponding to position 101 is replaced by Arg, for example, N101R;

[0137] bg. The modification at position 102 is replaced by Arg, for example, D102R;

[0138] bh. The modification at the position corresponding to position 105 is replaced by Arg, such as S105R, and / or Arg is inserted, such as S105SR or S105RR;

[0139] The modification at position bi. corresponding to position 178 is replaced by Arg, for example, N178R.

[0140] bj. The modifier at position 180 is replaced by Arg, for example, A180R;

[0141] bk. The modification at the position corresponding to position 211 is replaced by Arg, for example, F211R;

[0142] In bl., the modification at position 250 is replaced by Arg, for example, P250R;

[0143] The modification at position 252 is replaced by Arg, for example, I252R;

[0144] bn. The modification at position 254 is replaced by Arg, for example, D254R;

[0145] The modification at position 256 of bo. is replaced by Arg, for example, P256R.

[0146] In another embodiment, the lipase variant is contained in the positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 30, 31, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 167, 178, 180, 192, 193, 194, 199, 2 Modifications at three or more positions of 00, 202, 210, 211, 217, 218, 220, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254, and 256. On the other hand, this lipase variant is contained in the lipases corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 30, 31, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 167, 178, 180, 192, 193, 194, 199, 20. Modifications at four or more positions of 0, 202, 210, 211, 217, 218, 220, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254, and 256. On the other hand, this lipase variant is contained in the lipases corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 30, 31, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 167, 178, 180, 192, 193, 194, 199, 20. Modifications at five or more positions of 0, 202, 210, 211, 217, 218, 220, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254, and 256.On the other hand, this lipase variant contains modifications at six or more positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 30, 31, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 167, 178, 180, 192, 193, 194, 199, 200, 202, 217, 218, 220, 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254, and 256.

[0147] In one embodiment, the variant includes a deletion at the position corresponding to position 1, such as E1*, an Arg insertion at the position corresponding to position 1, such as E1ER, or a substitution at the position corresponding to position 1 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, particularly by Arg, Gln, Lys, or Pro, and most particularly by Gln or Pro. In another embodiment, the variant is a variant of SEQ ID NO:1 that includes substitutions for E1P or E1Q.

[0148] In another embodiment, the variant includes a deletion at the position corresponding to position 2, such as V2*, or a substitution of Arg or Lys at the position corresponding to position 2. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of V2K or V2R.

[0149] In another embodiment, the variant includes a deletion at the position corresponding to position 3, such as S3*, or a substitution of Arg or Lys at the position corresponding to position 3, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of S3K or S3R.

[0150] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 4, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of Q4K or Q4R.

[0151] In another embodiment, the variant includes a deletion at the position corresponding to position 5, such as D5*, or a substitution at the position corresponding to position 5 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, particularly Arg or Lys, and most particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution for D5K or D5R.

[0152] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 6, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of L6K or L6R.

[0153] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 7, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of F7K or F7R.

[0154] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 8, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N8K or N8R.

[0155] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 9, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of Q9K or Q9R.

[0156] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 11, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N11K or N11R.

[0157] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 12, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of L12K or L12R.

[0158] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 15, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of Q15K or Q15R.

[0159] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 28, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of A28K or A28R.

[0160] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 29, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of P29K or P29R.

[0161] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 30, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of A30K or A30R.

[0162] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 31, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of G31K or G31R.

[0163] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 33, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N33K or N33R.

[0164] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 37, particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of T37K or T37R. In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 38, particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of Q38K or Q38R.

[0165] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 39, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N39K or N39R.

[0166] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 40, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of A40K or A40R.

[0167] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 42, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of P42K or P42R.

[0168] In another embodiment, the variant includes a deletion at the position corresponding to position 43, such as E43*, or a substitution at the position corresponding to position 43 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, particularly by Arg or Lys, and most particularly by Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes substitutions for E43K or E43R.

[0169] In another embodiment, the variant includes a deletion at the position corresponding to position 45, such as E45*, or a substitution at the position corresponding to position 45 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, particularly Arg or Lys, and most particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes substitutions for E45K or E45R.

[0170] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 73, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N73K or N73R.

[0171] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 99, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of E99K or E99R.

[0172] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 105, particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of S105K or S105R. In another embodiment, the variant includes a deletion at the position corresponding to position 167, such as D167*, or a substitution of Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the position corresponding to position 167, particularly Arg or Lys, most particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of D167K or D167R.

[0173] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 178, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N178K or N178R.

[0174] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 180, particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of A180K or A180R. In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 192, particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of T192K or T192R.

[0175] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 193, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of L193K or L193R.

[0176] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 194, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of Y194K or Y194R.

[0177] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 199, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of T199K or T199R.

[0178] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 200, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N200K or N200R.

[0179] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 202, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of I202K or I202R.

[0180] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 211, particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of F211K or F211R. In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 217, particularly Arg. In another aspect, the variant is a variant of SEQ ID NO:1 that includes a substitution of S217K or S217R.

[0181] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 218, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of P218K or P218R.

[0182] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 220, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of Y220K or Y220R.

[0183] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 221, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of W221K or W221R.

[0184] In another embodiment, the variant includes a deletion at the position corresponding to position 224, such as S224*, or a substitution of Arg or Lys at the position corresponding to position 224, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of S224K or S224R.

[0185] In another embodiment, the variant includes a deletion at the position corresponding to position 225, such as G225*, or a substitution of Arg or Lys at the position corresponding to position 225, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of G225K or G225R.

[0186] In another embodiment, the variant includes a deletion at the position corresponding to position 226, such as T226*, or a substitution of Arg or Lys at the position corresponding to position 226, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of T226K or T226R.

[0187] In another embodiment, the variant includes a deletion at the position corresponding to position 227, such as L227*, or a substitution of Arg or Lys at the position corresponding to position 227, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of L227K or L227R.

[0188] In another embodiment, the variant includes a deletion at the position corresponding to position 228, such as V228*, or a substitution of Arg or Lys at the position corresponding to position 228, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of V228K or V228R.

[0189] In another embodiment, the variant includes a deletion at the position corresponding to position 229, such as P229*, or a substitution of Arg or Lys at the position corresponding to position 229, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of P229K or P229R.

[0190] In another embodiment, the variant includes a deletion at the position corresponding to position 230, such as V230*, or a substitution of Arg or Lys at the position corresponding to position 230, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of V230K or V230R.

[0191] In one embodiment, the variant includes one or both Arg insertions, such as T231TR or T231TRR, at the position corresponding to position 231, or is replaced by Arg or Lys at the position corresponding to position 231, particularly by Arg. In another embodiment, the variant is a variant of SEQ ID NO:1, which includes a substitution for T231R.

[0192] In one embodiment, the variant includes an Arg insert at the position corresponding to position 232, such as R232RR.

[0193] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 233, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N233K or N233R.

[0194] In another embodiment, the variant includes a deletion at the position corresponding to position 234, such as D234*, or a substitution at the position corresponding to position 234 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, particularly Arg or Lys, and most particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution for D234K or D234R.

[0195] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 236, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of V236K or V236R.

[0196] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 237, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of I238K or I238R.

[0197] In another embodiment, the variant includes a deletion at the position corresponding to position 239, such as E239*, or a substitution at the position corresponding to position 239 by Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, particularly Arg or Lys, and most particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution for E239K or E239R.

[0198] In another embodiment, the variant includes an Arg insertion at the position corresponding to position 243, such as A243AR. In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 243, particularly a substitution of Arg. In another aspect, the variant is a variant of SEQ ID NO:1, which includes a substitution of A243K or A243R.

[0199] In another embodiment, the variant includes one or both Arg insertions, such as T244TR or T244TRR, at the position corresponding to position 244. In one embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 244, particularly a substitution of Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of T244K or T244R.

[0200] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 245, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of G245K or G245R.

[0201] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 248, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N248K or N248R.

[0202] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 249, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of Q249K or Q249R.

[0203] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 250, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of P250K or P250R.

[0204] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 251, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of N251K or N251R.

[0205] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 252, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of I252K or I252R.

[0206] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 254, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1, which includes a substitution of D254K or D254R.

[0207] In another embodiment, the variant includes a substitution of Arg or Lys at the position corresponding to position 256, particularly Arg. On the other hand, the variant is a variant of SEQ ID NO:1 that includes a substitution of P256K or P256R. In yet another embodiment, the variant includes or consists of modifications at positions corresponding to positions 1 and 2, such as those described above.

[0208] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 1 and 3, such as those described above.

[0209] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 1 and 4, such as those described above.

[0210] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 2 and 3, such as those described above.

[0211] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 2 and 4, such as those described above.

[0212] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 4 and 8, such as those described above.

[0213] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 5 and 8, such as those described above.

[0214] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 8 and 11, such as those described above.

[0215] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 8 and 43, such as those described above.

[0216] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 30 and 31, such as those described above.

[0217] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 30 and 33, such as those described above.

[0218] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 30 and 34, such as those described above. In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 37 and 38, such as those described above.

[0219] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 37 and 39, such as those described above.

[0220] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 38 and 39, such as those described above.

[0221] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 38 and 229, such as those described above.

[0222] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 39 and 229, such as those described above.

[0223] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 102 and 105, such as those described above.

[0224] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 105 and 106, such as those described above.

[0225] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 106 and 178, such as those described above.

[0226] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 199 and 200, such as those described above.

[0227] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 200 and 225, such as those described above.

[0228] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 200 and 227, such as those described above.

[0229] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 200 and 231, such as those described above.

[0230] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 210 and 211, such as those described above.

[0231] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 225, such as those described above.

[0232] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 226, such as those described above.

[0233] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 227, such as those described above.

[0234] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 228, such as those described above.

[0235] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 229, such as those described above.

[0236] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 230, such as those described above.

[0237] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 231, such as those described above.

[0238] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 232, such as those described above.

[0239] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 233, such as those described above.

[0240] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224 and 234, such as those described above.

[0241] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 226, such as those described above.

[0242] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 227, such as those described above.

[0243] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 228, such as those described above.

[0244] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 229, such as those described above.

[0245] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 230, such as those described above.

[0246] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 231, such as those described above.

[0247] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 232, such as those described above.

[0248] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 233, such as those described above.

[0249] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225 and 234, such as those described above.

[0250] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 227, such as those described above.

[0251] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 228, such as those described above.

[0252] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 229, such as those described above.

[0253] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 230, such as those described above.

[0254] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 231, such as those described above.

[0255] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 232, such as those described above.

[0256] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 233, such as those described above.

[0257] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 234, such as those described above.

[0258] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226 and 249, such as those described above.

[0259] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 228, such as those described above.

[0260] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 229, such as those described above.

[0261] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 230, such as those described above.

[0262] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 231, such as those described above.

[0263] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 232, such as those described above.

[0264] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 233, such as those described above.

[0265] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 234, such as those described above.

[0266] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227 and 249, such as those described above.

[0267] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 229 and 230, such as those described above.

[0268] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 229 and 231, such as those described above.

[0269] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 233 and 236, such as those described above.

[0270] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 233 and 243, such as those described above.

[0271] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 233 and 249, such as those described above.

[0272] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 234 and 236, such as those described above.

[0273] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 236 and 239, such as those described above.

[0274] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 243 and 245, such as those described above.

[0275] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 245 and 249, such as those described above.

[0276] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 249 and 250, such as those described above.

[0277] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 250 and 252, such as those described above. In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 1, 2, and 3, such as those described above.

[0278] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 1, 2, and 4, such as those described above.

[0279] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 37, 38, and 39, such as those described above.

[0280] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, and 226, such as those described above.

[0281] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, and 227, such as those described above.

[0282] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, and 228, such as those described above.

[0283] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, and 229, such as those described above.

[0284] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, and 230, such as those described above.

[0285] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, and 227, such as those described above.

[0286] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, and 228, such as those described above.

[0287] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, and 229, such as those described above.

[0288] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, and 230, such as those described above.

[0289] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 227, and 228, such as those described above.

[0290] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 227, and 229, such as those described above.

[0291] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 227, and 230, such as those described above.

[0292] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 228, and 229, such as those described above.

[0293] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 228, and 230, such as those described above.

[0294] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 229, and 230, such as those described above.

[0295] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, and 227, such as those described above.

[0296] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, and 228, such as those described above.

[0297] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, and 229, such as those described above.

[0298] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, and 230, such as those described above.

[0299] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 227, and 228, such as those described above.

[0300] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 227, and 229, such as those described above.

[0301] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 227, and 230, such as those described above.

[0302] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 228, and 229, such as those described above.

[0303] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 228, and 230, such as those described above.

[0304] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 229, and 230, such as those described above.

[0305] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 227, and 228, such as those described above.

[0306] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 227, and 229, such as those described above.

[0307] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 227, and 230, such as those described above.

[0308] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 228, and 229, such as those described above.

[0309] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 228, and 230, such as those described above.

[0310] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 229, and 230, such as those described above.

[0311] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227, 228, and 229, such as those described above.

[0312] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227, 228, and 230, such as those described above.

[0313] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227, 229, and 230, such as those described above.

[0314] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 228, 229, and 230, such as those described above.

[0315] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 231, 233, and 244, such as those described above.

[0316] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 231, 239, and 249, such as those described above.

[0317] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226, and 227, such as those described above.

[0318] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226 and 228, such as those described above.

[0319] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226 and 229, such as those described above.

[0320] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226, and 230, such as those described above.

[0321] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 227 and 228, such as those described above.

[0322] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 227 and 229, such as those described above.

[0323] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 227, and 230, such as those described above.

[0324] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 228, and 229, such as those described above.

[0325] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 228, and 230, such as those described above.

[0326] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 229, and 230, such as those described above.

[0327] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 227 and 228, such as those described above.

[0328] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 227 and 229, such as those described above.

[0329] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 227 and 230, such as those described above.

[0330] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 228, and 229, such as those described above.

[0331] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 228, and 230, such as those described above.

[0332] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 229 and 230, such as those described above.

[0333] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 227, 228, and 229, such as those described above.

[0334] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 227, 228, and 230, such as those described above.

[0335] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 227, 229 and 230, such as those described above.

[0336] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 228, 229 and 230, such as those described above.

[0337] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 227 and 228, such as those described above.

[0338] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 227 and 229, such as those described above.

[0339] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 227 and 230, such as those described above.

[0340] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 228, and 229, such as those described above.

[0341] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 228, and 230, such as those described above.

[0342] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 229 and 230, such as those described above.

[0343] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 227, 228, and 229, such as those described above.

[0344] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 227, 228, and 230, such as those described above.

[0345] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 228, 229 and 230, such as those described above.

[0346] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 227, 228, and 229, such as those described above.

[0347] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 227, 228, and 230, such as those described above.

[0348] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 228, 229 and 230, such as those described above.

[0349] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 227, 228, 229 and 230, such as those described above.

[0350] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226, 227, and 228, such as those described above.

[0351] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226, 227, and 229, such as those described above.

[0352] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226, 227, and 230, such as those described above.

[0353] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 227, 228, and 229, such as those described above.

[0354] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 227, 228, and 230, such as those described above.

[0355] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 227, 228, 229, and 230, such as those described above.

[0356] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 227, 228, and 229, such as those described above.

[0357] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 227, 228, and 230, such as those described above.

[0358] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 228, 229, and 230, such as those described above.

[0359] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 227, 228, 229, and 230, such as those described above.

[0360] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 226, 227, 228, 229, and 230, such as those described above.

[0361] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226, 227, 228, and 229, such as those described above.

[0362] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 225, 226, 227, 228, and 230, such as those described above.

[0363] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 224, 226, 227, 228, 229 and 230, such as those described above.

[0364] In another embodiment, the variant includes or consists of modifications at positions corresponding to positions 225, 226, 227, 228, 229 and 230, such as those described above.

[0365] In another embodiment, the variant includes a modification at a position corresponding to a position selected from the group consisting of: SEQ ID E1*+V2* of NO:1; E1*+S3*; E1*+D5*; E1*+N8R; E1*+N8K; E1*+E43*; E1*+E 45*;E1*+D167*;E1*+S224*;E1*+S225*;E1*+T226*;E1*+L227*;E1*+V2 28*;E1*+P229*;E1*+V230*;E1*+V230K;E1*+V230R;E1*+D234*;E1*+V2 36K; E1*+V236R; E1*+E239*; E1ER+V2*; E1ER+S3*; E1ER+D5*; E1ER+N8R; E1ER+N8K; E1ER+E43*; E1ER+E45*; E1ER+D167*; E1ER+S224*; E1ER+S225*; E1ER+T226*; ER+V230K; E1ER+V230R; E1ER+D234*; E1ER+V236K; E1ER+V236R; E1ER+E239*; E1Q+V2K; V2*+S3*; *;V2*+D167*;V2*+S224*;V2*+S225*;V2*+T226*;V2*+L227*;V2*+V22 8*; V2*+P229*; V2*+V230*; V2*+V230K; V2*+V230R; V2*+D234*; V2*+V23 6K; V2*+V236R; V2*+E239*; S3*+D5*; S3*+N8R; S3*+N8K; S3*+E43*; S3*+ E45*; S3*+D167*; S3*+S224*; S3*+S225*; S3*+T226*; S3*+L227*; S3*+V 228*; S3*+P229*; S3*+V230*; S3*+V230K; S3*+V230R; S3*+D234*; S3*+ V236K; S3*+V236R; S3*+E239*; D5*+N8R; D5*+N8K; D5*+E43*; D5*+E45*; D5*+D167*; D5*+S224*; D5*+S225*; D5*+T226*; D5*+L227*; D5*+V228*; D5*+P229*; D5*+V230*; D5*+V230K; D5*+V230R; D5*+D234*; D5*+V236K;D5*+V236R;D5*+E239*;N8R+E43*;N8R+E45*;N8R+D167*;N8R+S224*;N8R+S225*;N8R+T226*;N8R+L227*;N8R+V228*;N8R+P229*;N8R+V230*;N8R+V230K;N8R+V230R;N8R+D234*;N8R+V236K;N8R+V236R;N8R+E239*;N8K+E43*;N8K+E45*;N8K+D167*;N8K+S224*;N8K+S225*;N8K+T226*;N8K+L227*;N8K+V228*;N8K+P229*;N8K+V230*;N8K+V230K;N8K+V230R;N8K+D234*;N8K+V236K;N8K+V236R;N8K+E239*;E43*+E45*;E43*+D167*;E43*+S224*;E43*+S225*;E43*+T226*;E43*+L227*;E43*+V228*;E43*+P229*;E43*+V230*;E43*+V230K;E43*+V230R;E43*+D234*;E43*+V236K;E43*+V236R;E43*+E239*;E45*+D167*;E45*+S224*;E45*+S225*;E45*+T226*;E45*+L227*;E45*+V228*;E45*+P229*;E45*+V230*;E45*+V230K;E45*+V230R;E45*+D234*;E45*+V236K;E45*+V236R;E45*+E239*;D167*+S224*;D167*+S225*;D167*+T226*;D167*+L227*;D167*+V228*;D167*+P229*;D167*+V230*;D167*+V230K;D167*+V230R;D167*+D234*;D167*+V236K;D167*+V236R;D167*+E239*;S224*+S225*;S224*+T226*;S224*+L227*;S224*+V228*;S224*+P229*;S224*+V230*;S224*+V230K;S224*+V230R;S224*+D234*;S224*+V236K;S224*+V236R;S224*+E239*;S225*+T226*;S225*+L227*;S225*+V228*;S225*+P229*; S225*+V230*; S225*+V230K; S225*+V230R; S225*+D234*; S225*+V236K; S225*+V236R; S225*+E 239*; T226*+L227*; T226*+V228*; T226*+P229*; T226*+V230*; T226*+V230K; T226*+V230R; T226*+D234*; T22 6*+V236K; T226*+V236R; T226*+E239*; L227*+V228*; L227*+P229*; L227*+V230*; L227*+V230K; L227*+V230 R;L227*+D234*;L227*+V236K;L227*+V236R;L227*+E239*;V228*+P229*;V228*+V230*;V228*+V230K;V228*+ V230R; V228*+D234*; V228*+V236K; V228*+V236R; V228*+E239*; P229*+V230*; P229*+V230K; P229*+V230R; P 229*+D234*; P229*+V236K; P229*+V236R; P229*+E239*; V230*+V230R; V230*+D234*; V230*+V236K; V230*+V23 6R; V230*+E239*; V230K+V230R; V230K+D234*; V230K+V236K; V230K+V236R; V230K+E239*; V230R+D234*; V230R +V236K; V230R+V236R; V230R+E239*; D234*+V236K; D234*+V236R; D234*+E239*; V236K+E239*; and V236R+E239*. ;

[0366] In another embodiment, the variant comprises (a) E1ER, E1P or E1Q+ (b) V2*, V2K or V2R or composed thereof.

[0367] In another embodiment, the variant comprises (a) E1ER, E1P or E1Q+ (b) S3* or S3R or composed thereof.

[0368] In another embodiment, the variant comprises (a) E1ER, E1P, or E1Q+(b) Q4R or is composed of therewith.

[0369] In another embodiment, the variant comprises (a) V2*, V2K or V2R+(b) S3* or S3R or composed thereof.

[0370] In another embodiment, the variant comprises (a) V2*, V2K, or V2R+(b) Q4R or composed thereof.

[0371] In another embodiment, the variant comprises (a) Q4R+(b) N8R or is composed of therewith.

[0372] In another embodiment, the variant comprises (a) D5R+(b) N8R or is composed of therewith.

[0373] In another embodiment, the variant comprises (a) N8R + (b) N11R or consists of therewith.

[0374] In another embodiment, the variant comprises (a) N8R+(b) E43R or is composed of therewith.

[0375] In another embodiment, the variant comprises (a) T37R+(b) G38R or is composed of therewith.

[0376] In another embodiment, the variant comprises (a) T37R + (b) N39R or is composed of therewith.

[0377] In another embodiment, the variant comprises (a) G38R+(b) N39R or is composed of therewith.

[0378] In another embodiment, the variant comprises (a) T199R+(b) N200R or is composed of therewith.

[0379] In another embodiment, the variant comprises (a) N200R+(b) G225R or is composed of therewith.

[0380] In another embodiment, the variant comprises (a) N200R+(b) L227R or is composed of therewith.

[0381] In another embodiment, the variant comprises (a) N200R+(b) T231R or is composed of therewith.

[0382] In another embodiment, the variant comprises (a) S224* or S224R+(b) G225* or G225R or composed thereof.

[0383] In another embodiment, the variant comprises (a) S224* or S224R+(b) T226* or T226R or composed thereof.

[0384] In another embodiment, the variant comprises (a) S224* or S224R+(b) L227* or L227R or composed thereof.

[0385] In another embodiment, the variant comprises (a) S224* or S224R+(b) V228* or V228R or composed thereof.

[0386] In another embodiment, the variant comprises (a) S224* or S224R+(b) P229* or P229R or composed thereof.

[0387] In another embodiment, the variant comprises (a) S224* or S224R+(b) V230* or V230R or thereof.

[0388] In another embodiment, the variant comprises (a) S224* or S224R+(b) T231TR or T231R or composed thereof.

[0389] In another embodiment, the variant comprises (a) S224* or S224R+(b) R232RR or is composed of therewith.

[0390] In another embodiment, the variant comprises (a) S224* or S224R+(b) N233R or composed thereof.

[0391] In another embodiment, the variant comprises (a) S224* or S224R+(b) D234* or D234R or composed thereof.

[0392] In another embodiment, the variant comprises (a) G225* or G225R+(b) T226* or T226R or composed thereof.

[0393] In another embodiment, the variant comprises (a) G225* or G225R+(b) L227*+L227R or is composed of therewith.

[0394] In another embodiment, the variant comprises (a) G225* or G225R+(b) V228* or V228R or composed thereof.

[0395] In another embodiment, the variant comprises (a) G225* or G225R+(b) P229* or P229R or composed thereof.

[0396] In another embodiment, the variant comprises (a) G225* or G225R+(b) V230* or V230R or composed thereof.

[0397] In another embodiment, the variant comprises (a) G225* or G225R+(b) T231TR or T231R or composed thereof.

[0398] In another embodiment, the variant comprises (a) G225* or G225R+(b) R232RR or is composed of therewith.

[0399] In another embodiment, the variant comprises (a) G225* or G225R+(b) N233R or composed thereof.

[0400] In another embodiment, the variant comprises (a) G225* or G225R+(b) D234* or D234R or composed thereof.

[0401] In another embodiment, the variant comprises (a) T226* or T226R + (b) L227* or L227R or composed thereof.

[0402] In another embodiment, the variant comprises (a) T226* or T226R+(b) V228* or V228R or composed thereof.

[0403] In another embodiment, the variant comprises (a) T226* or T226R+(b) P229* or P229R or composed thereof.

[0404] In another embodiment, the variant comprises (a) T226* or T226R+(b) V230* or V230R or composed thereof.

[0405] In another embodiment, the variant comprises (a) T226* or T226R+(b) T231TR or T231R or composed thereof.

[0406] In another embodiment, the variant comprises (a) T226* or T226R+(b) R232RR or is composed of therewith.

[0407] In another embodiment, the variant comprises (a) T226* or T226R+(b) N233R or composed thereof.

[0408] In another embodiment, the variant comprises (a) T226* or T226R+(b) D234* or D234R or composed thereof.

[0409] In another embodiment, the variant comprises (a) T226* or T226R+(b) Q249R or composed thereof.

[0410] In another embodiment, the variant comprises (a) L227* or L227R+(b) V228* or V228R or composed thereof.

[0411] In another embodiment, the variant comprises (a) L227* or L227R+(b) P229* or P229R or composed thereof.

[0412] In another embodiment, the variant comprises (a) L227* or L227R+(b) V230* or V230R or composed thereof.

[0413] In another embodiment, the variant comprises (a) L227* or L227R+(b) T231TR or T231R or composed thereof.

[0414] In another embodiment, the variant comprises (a) L227* or L227R+(b) R232RR or is composed of therewith.

[0415] In another embodiment, the variant comprises (a) L227* or L227R+(b) N233R or is composed of therewith.

[0416] In another embodiment, the variant comprises (a) L227* or L227R+(b) D234* or D234R or composed thereof.

[0417] In another embodiment, the variant comprises (a) L227* or L227R+(b) Q249R or is composed of therewith.

[0418] In another embodiment, the variant comprises (a) T231TR or T231R+(b) V236R or composed thereof.

[0419] In another embodiment, the variant comprises (a) T231TR or T231R+(b) G239* or G239E or composed thereof.

[0420] In another embodiment, the variant comprises (a) T231TR or T231R+(b) Q249R or composed thereof.

[0421] In another embodiment, the variant comprises (a) N233R+(b) V236R or is composed of therewith.

[0422] In another embodiment, the variant comprises (a) N233R + (b) Q249R or is composed of therewith.

[0423] In another embodiment, the variant comprises (a) V236R+(b) G239* or G239E or is composed of therewith.

[0424] In another embodiment, the variant includes modifications at positions corresponding to the groups selected from the following: E1*+V2*; E1*+V2R; E1*+V2*; E1*+V2K; E1*+S3*; E1*+V3R; E1*+V3K; E1*+Q4R; E1*+Q4K; E1*+D5*; E1*+D5R; E1*+D5K; E1*+L6R; E1*+L6K; E1*+F7R; E1*+F7K; E1*+N8R; E1*+N8K; E1*+Q9R; E1*+Q9K; E1*+N11R; E1*+N11K; E1*+L12R; E1*+L12K; E1*+Q15R; E1*+Q 15K; E1*+T37R; E1*+T37K; E1*+Q38R; E1*+Q38K; E1*+N39R; E1*+N39K; E1*+ A40R;E1*+A40K;E1*+P42R;E1*+P42K;E1*+E43*;E1*+E43R;E1*+E43K;E1* +E45*;E1*+E45R;E1*+E45K;E1*+N73R;E1*+N73K;E1*+D167*;E1*+D167R; ;1*+D167K;E1*+T192R;E1*+T192K;E1*+L193R;E1*+L193K;E1*+Y194R;E1* +Y194K;E1*+T199R;E1*+T199K;E1*+N200R;E1*+N200K;E1*+I202R;E1*+I 202K; E1*+S217R; E1*+S217K; E1*+P218R; E1*+P218K; E1*+Y220R; E1*+Y22 0K;E1*+W221R;E1*+W221K;E1*+S224*;E1*+S224R;E1*+S224K;E1*+S225* ;E1*+S225R;E1*+S225K;E1*+T226*;E1*+T226R;E1*+T226K;E1*+L227*;E1 *+L227R;E1*+L227K;E1*+V228*;E1*+V228R;E1*+V228K;E1*+P229*;E1*+ P229R;E1*+P229K;E1*+V230*;E1*+V230K;E1*+V230R;E1*+T231R;E1*+T2 31K; E1*+R232RR; E1*+N233R; E1*+N233K; E1*+D234*; E1*+D234R; E1*+D23 4K;E1*+V236K;E1*+V236R;E1*+I238R;E1*+I238K;E1*+E239*;E1*+E239R;E1*+E239K;E1*+A243R;E1*+A243K;E1*+T244R;E1*+T244K;E1*+G245R;E1 *+G245K;E1*+N248R;E1*+N248K;E1*+Q249R;E1*+Q249K;E1*+N251R;E1*+ N251K;E1ER+V2*;E1ER+V2R;E1ER+V2K;E1ER+S3*;E1ER+V3R;E1ER+V3K;E1 ER+Q4R;E1ER+Q4K;E1ER+D5*;E1ER+D5R;E1ER+D5K;E1ER+L6R;E1ER+L6K;E 1ER+F7R;E1ER+F7K;E1ER+N8R;E1ER+N8K;E1ER+Q9R;E1ER+Q9K;E1ER+N11R ;E1ER+N11K;E1ER+L12R;E1ER+L12K;E1ER+Q15R;E1ER+Q15K;E1ER+T37R;E 1ER+T37K;E1ER+Q38R;E1ER+Q38K;E1ER+N39R;E1ER+N39K;E1ER+A40R;E1E R+A40K;E1ER+P42R;E1ER+P42K;E1ER+E43*;E1ER+E43R;E1ER+E43K;E1ER+ E45*;E1ER+E45R;E1ER+E45K;E1ER+N73R;E1ER+N73K;E1ER+D167*;E1ER+D 167R;E1ER+D167K;E1ER+T192R;E1ER+T192K;E1ER+L193R;E1ER+L193K;E1 ER+Y194R;E1ER+Y194K;E1ER+T199R;E1ER+T199K;E1ER+N200R;E1ER+N200 K;E1ER+I202R;E1ER+I202K;E1ER+S217R;E1ER+S217K;E1ER+P218R;E1ER+ P218K;E1ER+Y220R;E1ER+Y220K;E1ER+W221R;E1ER+W221K;E1ER+S224*;E 1ER+S224R;E1ER+S224K;E1ER+S225*;E1ER+S225R;E1ER+S225K;E1ER+T22 6*;E1ER+T226R;E1ER+T226K;E1ER+L227*;E1ER+L227R;E1ER+L227K;E1ER +V228*;E1ER+V228R;E1ER+V228K;E1ER+P229*;E1ER+P229R;E1ER+P229K;E1ER+V230*;E1ER+V230K;E1ER+V230R;E1ER+T231R;E1ER+T231K;E1ER+R2 32RR;E1ER+N233R;E1ER+N233K;E1ER+D234*;E1ER+D234R;E1ER+D234K;E1 ER+V236K;E1ER+V236R;E1ER+I238R;E1ER+I238K;E1ER+E239*;E1ER+E239 R;E1ER+E239K;E1ER+A243R;E1ER+A243K;E1ER+T244R;E1ER+T244K;E1ER+G 245R; E1ER+G245K; E1ER+N248R; E1ER+N248K; E1ER+Q249R; E1ER+Q249K; E1ER+N251R; E1ER+N251K; E1R+V2*; E1R+V2R; E1R+V2K; E1R+S3*; E1R+V3R; E1R +V3K;E1R+Q4R;E1R+Q4K;E1R+D5*;E1R+D5R;E1R+D5K;E1R+L6R;E1R+L6K;E 1R+F7R;E1R+F7K;E1R+N8R;E1R+N8K;E1R+Q9R;E1R+Q9K;E1R+N11R;E1R+N11 K;E1R+L12R;E1R+L12K;E1R+Q15R;E1R+Q15K;E1R+T37R;E1R+T37K;E1R+Q3 8R;E1R+Q38K;E1R+N39R;E1R+N39K;E1R+A40R;E1R+A40K;E1R+P42R;E1R+P4 2K;E1R+E43*;E1R+E43R;E1R+E43K;E1R+E45*;E1R+E45R;E1R+E45K;E1R+N 73R;E1R+N73K;E1R+D167*;E1R+D167R;E1R+D167K;E1R+T192R;E1R+T192K; E1R+L193R;E1R+L193K;E1R+Y194R;E1R+Y194K;E1R+T199R;E1R+T199K;E1 R+N200R;E1R+N200K;E1R+I202R;E1R+I202K;E1R+S217R;E1R+S217K;E1R+P 218R;E1R+P218K;E1R+Y220R;E1R+Y220K;E1R+W221R;E1R+W221K;E1R+S22 4*;E1R+S224R;E1R+S224K;E1R+S225*;E1R+S225R;E1R+S225K;E1R+T226*;E1R+T226R;E1R+T226K;E1R+L227*;E1R+L227R;E1R+L227K;E1R+V228*;E1 R+V228R;E1R+V228K;E1R+P229*;E1R+P229R;E1R+P229K;E1R+V230*;E1R+V 230K;E1R+V230R;E1R+T231R;E1R+T231K;E1R+R232RR;E1R+N233R;E1R+N2 33K;E1R+D234*;E1R+D234R;E1R+D234K;E1R+V236K;E1R+V236R;E1R+I238R ;E1R+I238K;E1R+E239*;E1R+E239R;E1R+E239K;E1R+A243R;E1R+A243K;E 1R+T244R;E1R+T244K;E1R+G245R;E1R+G245K;E1R+N248R;E1R+N248K;E1R+ Q249R; E1R+Q249K; E1R+N251R; E1R+N251K; E1L+V2*; E1L+V2R; E1L+V2K; E1L+S3*; E1L+V3R; E1L+V3K; E1L+Q4R; E1L+Q4K; E1L+D5*; E1L+D5R; E1L+D5K; E 1L+L6R;E1L+L6K;E1L+F7R;E1L+F7K;E1L+N8R;E1L+N8K;E1L+Q9R;E1L+Q9K ;E1L+N11R;E1L+N11K;E1L+L12R;E1L+L12K;E1L+Q15R;E1L+Q15K;E1L+T37R ;E1L+T37K;E1L+Q38R;E1L+Q38K;E1L+N39R;E1L+N39K;E1L+A40R;E1L+A40 K;E1L+P42R;E1L+P42K;E1L+E43*;E1L+E43R;E1L+E43K;E1L+E45*;E1L+E45 R;E1L+E45K;E1L+N73R;E1L+N73K;E1L+D167*;E1L+D167R;E1L+D167K;E1L +T192R;E1L+T192K;E1L+L193R;E1L+L193K;E1L+Y194R;E1L+Y194K;E1L+T1 99R′;E1L+T199K;E1L+N200R;E1L+N200K;E1L+I202R;E1L+I202K;E1L+S21 7R;E1L+S217K;E1L+P218R;E1L+P218K;E1L+Y220R;E1L+Y220K;E1L+W221R;E1L+W221K;E1L+S224*;E1L+S224R;E1L+S224K;E1L+S225*;E1L+S225R;E1 L+S225K;E1L+T226*;E1L+T226R;E1L+T226K;E1L+L227*;E1L+L227R;E1L+ L227K;E1L+V228*;E1L+V228R;E1L+V228K;E1L+P229*;E1L+P229R;E1L+P2 29K;E1L+V230*;E1L+V230K;E1L+V230R;E1L+T231R;E1L+T231K;E1L+R232 RR;E1L+N233R;E1L+N233K;E1L+D234*;E1L+D234R;E1L+D234K;E1L+V236K ;E1L+V236R;E1L+I238R;E1L+I238K;E1L+E239*;E1L+E239R;E1L+E239K;E 1L+A243R; E1L+A243K; E1L+T244R; E1L+T244K; E1L+G245R; E1L+G245K; E1L+N248R; E1L+N248K; E1L+Q249R; E1L+Q249K; E1L+N251R; E1L+N251K; V2*+S3 *;V2*+V3R;V2*+V3K;V2*+Q4R;V2*+Q4K;V2*+D5*;V2*+D5R;V2*+D5K;V2*+L6R;V2*+L6K;V2*+F7R;V2*+F7K;V2*+N8R;V2*+N8K;V2*+Q9R;V2*+Q9K;V2 *+N11R; V2*+N11K; V2*+L12R; V2*+L12K; V2*+Q15R; V2*+Q15K; V2*+T37R; V2*+T37K; V2*+Q38R; V2*+Q38K; V2*+N39R; V2*+N39K; V2*+A40R; V2*+A40K; V 2*+P42R; V2*+P42K; V2*+E43*; V2*+E43R; V2*+E43K; V2*+E45*; V2*+E45R; V2*+E45K; V2*+N73R; V2*+N73K; V2*+D167*; V2*+D167R; V2*+D167K; V2*+T 192R; V2*+T192K; V2*+L193R; V2*+L193K; V2*+Y194R; V2*+Y194K; V2*+T199R; V2*+T199K; V2*+N200R; V2*+N200K; V2*+I202R; V2*+I202K; V2*+S217R;V2*+S217K; V2*+P218R; V2*+P218K; V2*+Y220R; V2*+Y220K; V2*+W221R; V2*+W221K; V2*+S224*; V2*+S224R; V2*+S224K; V2*+S225*; V2*+S225R; V2*+ S225K; V2*+T226*; V2*+T226R; V2*+T226K; V2*+L227*; V2*+L227R; V2*+L227K; V2*+V228*; V2*+V228R; V2*+V228K; V2*+P229*; V2*+P229R; V2*+P229 K; V2*+V230*; V2*+V230K; V2*+V230R; V2*+T231R; V2*+T231K; V2*+R232RR; V2*+N233R; V2*+N233K; V2*+D234*; V2*+D234R; V2*+D234K; V2*+V236K; V 2*+V236R; V2*+I238R; V2*+I238K; V2*+E239*; V2*+E239R; V2*+E239K; V2*+A243R; V2*+A243K; V2*+T244R; V2*+T244K; V2*+G245R; V2*+G245K; V2*+N2 48R; V2*+N248K; V2*+Q249R; V2*+Q249K; V2*+N251R; V2*+N251K; V2R+S3*; V2R+V3R; V2R+V3K; V2R+Q4R; V2R+Q4K; V2R+D5*; V2R+D5R; V2R+D5K; V2R+L6 R;V2R+L6K;V2R+F7R;V2R+F7K;V2R+N8R;V2R+N8K;V2R+Q9R;V2R+Q9K;V2R+ N11R;V2R+N11K;V2R+L12R;V2R+L12K;V2R+Q15R;V2R+Q15K;V2R+T37R;V2R+ T37K; V2R+Q38R; V2R+Q38K; V2R+N39R; V2R+N39K; V2R+A40R; V2R+A40K; V2R+P42R; V2R+P42K; V2R+E43*; V2R+E43R; V2R+E43K; V2R+E45*; V2R+E45R; V2 R+E45K;V2R+N73R;V2R+N73K;V2R+D167*;V2R+D167R;V2R+D167K;V2R+T19 2R;V2R+T192K;V2R+L193R;V2R+L193K;V2R+Y194R;V2R+Y194K;V2R+T199R;V2R+T199K;V2R+N200R;V2R+N200K;V2R+I202R;V2R+I202K;V2R+S217R;V2 R+S217K;V2R+P218R;V2R+P218K;V2R+Y220R;V2R+Y220K;V2R+W221R;V2R+ W221K;V2R+S224*;V2R+S224R;V2R+S224K;V2R+S225*;V2R+S225R;V2R+S2 25K;V2R+T226*;V2R+T226R;V2R+T226K;V2R+L227*;V2R+L227R;V2R+L227 K;V2R+V228*;V2R+V228R;V2R+V228K;V2R+P229*;V2R+P229R;V2R+P229K;V2R+V230*;V2R+V230K;V2R+V230R;V2R+T231R;V2R+T231K;V2R+R232RR;V 2R+N233R;V2R+N233K;V2R+D234*;V2R+D234R;V2R+D234K;V2R+V236K;V2R +V236R;V2R+I238R;V2R+I238K;V2R+E239*;V2R+E239R;V2R+E239K;V2R+A2 43R;V2R+A243K;V2R+T244R;V2R+T244K;V2R+G245R;V2R+G245K;V2R+N248 R;V2R+N248K;V2R+Q249R;V2R+Q249K;V2R+N251R;V2R+N251K;V2K+S3*;V2 K+V3R;V2K+V3K;V2K+Q4R;V2K+Q4K;V2K+D5*;V2K+D5R;V2K+D5K;V2K+L6R; V2K+L6K;V2K+F7R;V2K+F7K;V2K+N8R;V2K+N8K;V2K+Q9R;V2K+Q9K;V2K+N11 R;V2K+N11K;V2K+L12R;V2K+L12K;V2K+Q15R;V2K+Q15K;V2K+T37R;V2K+T3 7K;V2K+Q38R;V2K+Q38K;V2K+N39R;V2K+N39K;V2K+A40R;V2K+A40K;V2K+P 42R;V2K+P42K;V2K+E43*;V2K+E43R;V2K+E43K;V2K+E45*;V2K+E45R;V2K+ E45K;V2K+N73R;V2K+N73K;V2K+D167*;V2K+D167R;V2K+D167K;V2K+T192R;V2K+T192K;V2K+L193R;V2K+L193K;V2K+Y194R;V2K+Y194K;V2K+T199R;V2 K+T199K;V2K+N200R;V2K+N200K;V2K+I202R;V2K+I202K;V2K+S217R;V2K+S 217K;V2K+P218R;V2K+P218K;V2K+Y220R;V2K+Y220K;V2K+W221R;V2K+W22 1K;V2K+S224*;V2K+S224R;V2K+S224K;V2K+S225*;V2K+S225R;V2K+S225K; V2K+T226*;V2K+T226R;V2K+T226K;V2K+L227*;V2K+L227R;V2K+L227K;V2 K+V228*;V2K+V228R;V2K+V228K;V2K+P229*;V2K+P229R;V2K+P229K;V2K+V 230*;V2K+V230K;V2K+V230R;V2K+T231R;V2K+T231K;V2K+R232RR;V2K+N2 33R;V2K+N233K;V2K+D234*;V2K+D234R;V2K+D234K;V2K+V236K;V2K+V236R ;V2K+I238R;V2K+I238K;V2K+E239*;V2K+E239R;V2K+E239K;V2K+A243R;V 2K+A243K;V2K+T244R;V2K+T244K;V2K+G245R;V2K+G245K;V2K+N248R;V2K+ N248K; V2K+Q249R; V2K+Q249K; V2K+N251R; V2K+N251K; S3*+Q4R; S3*+Q4K; S3*+D5*; S3*+D5R; S3*+D5K; S3*+L6R; S3*+L6K; S3*+F7R; S3*+F7K; S3*+N8R S3*+N8K; S3*+Q9R; S3*+Q9K; S3*+N11R; S3*+N11K; S3*+L12R; S3*+L12K; S3*+Q15R; S3*+Q15K; S3*+T37R; S3*+T37K; S3*+Q38R; S3*+Q38K; S3*+N39R; S 3*+N39K; S3*+A40R; S3*+A40K; S3*+P42R; S3*+P42K; S3*+E43*; S3*+E43R; S3*+E43K; S3*+E45*; S3*+E45R; S3*+E45K; S3*+N73R; S3*+N73K; S3*+D167*;S3*+D167R; S3*+D167K; S3*+T192R; S3*+T192K; S3*+L193R; S3*+L193K; S3*+Y194R; S3*+Y194K; S3*+T199R; S3*+T199K; S3*+N200R; S3*+N200K; S3*+ I202R; S3*+I202K; S3*+S217R; S3*+S217K; S3*+P218R; S3*+P218K; S3*+Y220R; S3*+Y220K; S3*+W221R; S3*+W221K; S3*+S224*; S3*+S224R; S3*+S224 K; S3*+S225*; S3*+S225R; S3*+S225K; S3*+T226*; S3*+T226R; S3*+T226K; S3*+L227*; S3*+L227R; S3*+L227K; S3*+V228*; S3*+V228R; S3*+V228K; S3 *+P229*; S3*+P229R; S3*+P229K; S3*+V230*; S3*+V230K; S3*+V230R; S3*+T231R; S3*+T231K; S3*+R232RR; S3*+N233R; S3*+N233K; S3*+D234*; S3*+D2 34R; S3*+D234K; S3*+V236K; S3*+V236R; S3*+I238R; S3*+I238K; S3*+E239*; S3*+E239R; S3*+E239K; S3*+A243R; S3*+A243K; S3*+T244R; S3*+T244K; S3*+G245R; S3*+G245K; S3*+N248R; S3*+N248K; S3*+Q249R; S3*+Q249K; S3*+N251R; S3*+N251K; V3R+Q4R; V3R+Q4K; V3R+D5*; V3R+D5R; V3R+D5K; V3R+ L6R;V3R+L6K;V3R+F7R;V3R+F7K;V3R+N8R;V3R+N8K;V3R+Q9R;V3R+Q9K;V3 R+N11R;V3R+N11K;V3R+L12R;V3R+L12K;V3R+Q15R;V3R+Q15K;V3R+T37R;V 3R+T37K; V3R+Q38R; V3R+Q38K; V3R+N39R; V3R+N39K; V3R+A40R; V3R+A40K; V3R+P42R; V3R+P42K; V3R+E43*; V3R+E43R; V3R+E43K; V3R+E45*; V3R+E45R;V3R+E45K; V3R+N73R; V3R+N73K; V3R+D167*; V3R+D167R; V3R+D167K; V3R+T192R; V3R+T192K; V3R+L193R; V3R+L193K; V3R+Y194R; V3R+Y194K; V3R+T19 9R;V3R+T199K;V3R+N200R;V3R+N200K;V3R+I202R;V3R+I202K;V3R+S217R ;V3R+S217K;V3R+P218R;V3R+P218K;V3R+Y220R;V3R+Y220K;V3R+W221R;V 3R+W221K; V3R+S224*; V3R+S224R; V3R+S224K; V3R+S225*; V3R+S225R; V3R+S225K; V3R+T226*; V3R+T226R; V3R+T226K; V3R+L227*; V3R+L227R; V3R+L 227K; V3R+V228*; V3R+V228R; V3R+V228K; V3R+P229*; V3R+P229R; V3R+P229K; V3R+V230*; V3R+V230K; V3R+V230R; V3R+T231R; V3R+T231K; V3R+R232RR V3R+N233R; V3R+N233K; V3R+D234*; V3R+D234R; V3R+D234K; V3R+V236K; V3R+V236R; V3R+I238R; V3R+I238K; V3R+E239*; V3R+E239R; V3R+E239K; V3R +A243R; V3R+A243K; V3R+T244R; V3R+T244K; V3R+G245R; V3R+G245K; V3R+N248R; V3R+N248K; V3R+Q249R; V3R+Q249K; V3R+N251R; V3R+N251K; V3K+Q4R; V3K+Q4K;V3K+D5*;V3K+D5R;V3K+D5K;V3K+L6R;V3K+L6K;V3K+F7R;V3K+F7 K;V3K+N8R;V3K+N8K;V3K+Q9R;V3K+Q9K;V3K+N11R;V3K+N11K;V3K+L12R;V 3K+L12K; V3K+Q15R; V3K+Q15K; V3K+T37R; V3K+T37K; V3K+Q38R; V3K+Q38K; V3K+N39R; V3K+N39K; V3K+A40R; V3K+A40K; V3K+P42R; V3K+P42K; V3K+E43*;V3K+E43R;V3K+E43K;V3K+E45*;V3K+E45R;V3K+E45K;V3K+N73R;V3K+N73K;V3K+D167*;V3K+D167R;V3K+D167K;V3K+T192R;V3K+T192K;V3K+L193R;V3K+L193K;V3K+Y194R;V3K+Y194K;V3K+T199R;V3K+T199K;V3K+N200R;V3K+N200K;V3K+I202R;V3K+I202K;V3K+S217R;V3K+S217K;V3K+P218R;V3K+P218K;V3K+Y220R;V3K+Y220K;V3K+W221R;V3K+W221K;V3K+S224*;V3K+S224R;V3K+S224K;V3K+S225*;V3K+S225R;V3K+S225K;V3K+T226*;V3K+T226R;V3K+T226K;V3K+L227*;V3K+L227R;V3K+L227K;V3K+V228*;V3K+V228R;V3K+V228K;V3K+P229*;V3K+P229R;V3K+P229K;V3K+V230*;V3K+V230K;V3K+V230R;V3K+T231R;V3K+T231K;V3K+R232RR;V3K+N233R;V3K+N233K;V3K+D234*;V3K+D234R;V3K+D234K;V3K+V236K;V3K+V236R;V3K+I238R;V3K+I238K;V3K+E239*;V3K+E239R;V3K+E239K;V3K+A243R;V3K+A243K;V3K+T244R;V3K+T244K;V3K+G245R;V3K+G245K;V3K+N248R;V3K+N248K;V3K+Q249R;V3K+Q249K;V3K+N251R;V3K+N251K;Q4R+D5*;Q4R+D5R;Q4R+D5K;Q4R+L6R;Q4R+L6K;Q4R+F7R;Q4R+F7K;Q4R+N8R;Q4R+N8K;Q4R+Q9R;Q4R+Q9K;Q4R+N11R;Q4R+N11K;Q4R+L12R;Q4R+L12K;Q4R+Q15R;Q4R+Q15K;Q4R+T37R;Q4R+T37K;Q4R+Q38R;Q4R+Q38K;Q4R+N39R;Q4R+N39K;Q4R+A40R;Q4R+A40K;Q4R+P42R;Q4R+P42K;Q4R+E43*;Q4R+E43R;Q4R+E43K;Q4R+E45*;Q4R+E45R;Q4R+E45K;Q4R+N73R;Q4R+N73K;Q4R+D167*;Q4R+D167R;Q4R+D167K;Q4R+T192R;Q4R+T192K;Q4R+L193R;Q4R+L193K;Q4R+Y194R;Q4R+Y194K;Q4R+T199R;Q4R+T199K;Q4R+N200R;Q4R+N200K;Q4R+I202R;Q4R+I202K;Q4R+S217R;Q4R+S217K;Q4R+P218R;Q4R+P218K;Q4R+Y220R;Q4R+Y220K;Q4R+W221R;Q4R+W221K;Q4R+S224*;Q4R+S224R;Q4R+S224K;Q4R+S225*;Q4R+S225R;Q4R+S225K;Q4R+T226*;Q4R+T226R;Q4R+T226K;Q4R+L227*;Q4R+L227R;Q4R+L227K;Q4R+V228*;Q4R+V228R;Q4R+V228K;Q4R+P229*;Q4R+P229R;Q4R+P229K;Q4R+V230*;Q4R+V230K;Q4R+V230R;Q4R+T231R;Q4R+T231K;Q4R+R232RR;Q4R+N233R;Q4R+N233K;Q4R+D234*;Q4R+D234R;Q4R+D234K;Q4R+V236K;Q4R+V236R;Q4R+I238R;Q4R+I238K;Q4R+E239*;Q4R+E239R;Q4R+E239K;Q4R+A243R;Q4R+A243K;Q4R+T244R;Q4R+T244K;Q4R+G245R;Q4R+G245K;Q4R+N248R;Q4R+N248K;Q4R+Q249R;Q4R+Q249K;Q4R+N251R;Q4R+N251K;Q4K+D5*;Q4K+D5R;Q4K+D5K;Q4K+L6R;Q4K+L6K;Q4K+F7R;Q4K+F7K;Q4K+N8R;Q4K+N8K;Q4K+Q9R;Q4K+Q9K;Q4K+N11R;Q4K+N11K;Q4K+L12R;Q4K+L12K;Q4K+Q15R;Q4K+Q15K;Q4K+T37R;Q4K+T37K;Q4K+Q38R;Q4K+Q38K;Q4K+N39R;Q4K+N39K;Q4K+A40R;Q4K+A40K;Q4K+P42R;Q4K+P42K;Q4K+E43*;Q4K+E43R;Q4K+E43K;Q4K+E45*;Q4K+E45R;Q4K+E45K;Q4K+N73R;Q4K+N73K;Q4K+D167*;Q4K+D167R;Q4K+D167K;Q4K+T192R;Q4K+T192K;Q4K+L193R;Q4K+L193K;Q4K+Y194R;Q4K+Y194K;Q4K+T199R;Q4K+T199K;Q4K+N200R;Q4K+N200K;Q4K+I202R;Q4K+I202K;Q4K+S217R;Q4K+S217K;Q4K+P218R;Q4K+P218K;Q4K+Y220R;Q4K+Y220K;Q4K+W221R;Q4K+W221K;Q4K+S224*;Q4K+S224R;Q4K+S224K;Q4K+S225*;Q4K+S225R;Q4K+S225K;Q4K+T226*;Q4K+T226R;Q4K+T226K;Q4K+L227*;Q4K+L227R;Q4K+L227K;Q4K+V228*;Q4K+V228R;Q4K+V228K;Q4K+P229*;Q4K+P229R;Q4K+P229K;Q4K+V230*;Q4K+V230K;Q4K+V230R;Q4K+T231R;Q4K+T231K;Q4K+R232RR;Q4K+N233R;Q4K+N233K;Q4K+D234*;Q4K+D234R;Q4K+D234K;Q4K+V236K;Q4K+V236R;Q4K+I238R;Q4K+I238K;Q4K+E239*;Q4K+E239R;Q4K+E239K;Q4K+A243R;Q4K+A243K;Q4K+T244R;Q4K+T244K;Q4K+G245R;Q4K+G245K;Q4K+N248R;Q4K+N248K;Q4K+Q249R;Q4K+Q249K;Q4K+N251R;Q4K+N251K;D5*+L6R;D5*+L6K;D5*+F7R;D5*+F7K;D5*+N8R;D5*+N8K;D5*+Q9R;D5*+Q9K;D5*+N11R;D5*+N11K;D5*+L12R;D5*+L12K;D5*+Q15R;D5*+Q15K;D5*+T37R;D5*+T37K;D5*+Q38R;D5*+Q38K;D5*+N39R;D5*+N39K;D5*+A40R;D5*+A40K;D5*+P42R;D5*+P42K;D5*+E43*;D5*+E43R;D5*+E43K;D5*+E45*;D5*+E45R;D5*+E45K;D5*+N73R;D5*+N73K;D5*+D167*;D5*+D167R;D5*+D167K;D5*+T192R;D5*+T192K;D5*+L193R;D5*+L193K;D5*+Y194R;D5*+Y194K;D5*+T199R;D5*+T199K;D5*+N200R;D5*+N200K;D5*+I202R;D5*+I202K;D5*+S217R;D5*+S217K;D5*+P218R;D5*+P218K;D5*+Y220R;D5*+Y220K;D5*+W221R;D5*+W221K;D5*+S224*;D5*+S224R;D5*+S224K;D5*+S225*;D5*+S225R;D5*+S225K;D5*+T226*;D5*+T226R;D5*+T226K;D5*+L227*;D5*+L227R;D5*+L227K;D5*+V228*;D5*+V228R;D5*+V228K;D5*+P229*;D5*+P229R;D5*+P229K;D5*+V230*;D5*+V230K;D5*+V230R;D5*+T231R;D5*+T231K;D5*+R232RR;D5*+N233R;D5*+N233K;D5*+D234*;D5*+D234R;D5*+D234K;D5*+V236K;D5*+V236R;D5*+I238R;D5*+I238K;D5*+E239*;D5*+E239R;D5*+E239K;D5*+A243R;D5*+A243K′;D5*+T244R;D5*+T244K;D5*+G245R;D5*+G245K;D5*+N248R;D5*+N248K;D5*+Q249R;D5*+Q249K;D5*+N251R;D5*+N251K;D5R+L6R;D5R+L6K;D5R+F7R;D5R+F7K;D5R+N8R;D5R+N8K;D5R+Q9R;D5R+Q9K;D5R+N11R;D5R+N11K;D5R+L12R;D5R+L12K;D5R+Q15R;D5R+Q15K;D5R+T37R;D5R+T37K;D5R+Q38R;D5R+Q38K;D5R+N39R;D5R+N39K;D5R+A40R;D5R+A40K;D5R+P42R;D5R+P42K;D5R+E43*;D5R+E43R;D5R+E43K;D5R+E45*;D5R+E45R;D5R+E45K;D5R+N73R;D5R+N73K;D5R+D167*;D5R+D167R;D5R+D167K;D5R+T192R;D5R+T192K;D5R+L193R;D5R+L193K;D5R+Y194R;D5R+Y194K;D5R+T199R;D5R+T199K;D5R+N200R;D5R+N200K;D5R+I202R;D5R+I202K;D5R+S217R;D5R+S217K;D5R+P218R;D5R+P218K;D5R+Y220R;D5R+Y220K;D5R+W221R;D5R+W221K;D5R+S224*;D5R+S224R;D5R+S224K;D5R+S225*;D5R+S225R;D5R+S225K;D5R+T226*;D5R+T226R;D5R+T226K;D5R+L227*;D5R+L227R;D5R+L227K;D5R+V228*;D5R+V228R;D5R+V228K;D5R+P229*;D5R+P229R;D5R+P229K;D5R+V230*;D5R+V230K;D5R+V230R;D5R+T231R;D5R+T231K;D5R+R232RR;D5R+N233R;D5R+N233K;D5R+D234*;D5R+D234R;D5R+D234K;D5R+V236K;D5R+V236R;D5R+I238R;D5R+I238K;D5R+E239*;D5R+E239R;D5R+E239K;D5R+A243R;D5R+A243K;D5R+T244R;D5R+T244K;D5R+G245R;D5R+G245K;D5R+N248R;D5R+N248K;D5R+Q249R;D5R+Q249K;D5R+N251R;D5R+N251K;D5K+L6R;D5K+L6K;D5K+F7R;D5K+F7K;D5K+N8R;D5K+N8K;D5K+Q9R;D5K+Q9K;D5K+N11R;D5K+N11K;D5K+L12R;D5K+L12K;D5K+Q15R;D5K+Q15K;D5K+T37R;D5K+T37K;D5K+Q38R;D5K+Q38K;D5K+N39R;D5K+N39K;D5K+A40R;D5K+A40K;D5K+P42R;D5K+P42K;D5K+E43*;D5K+E43R;D5K+E43K;D5K+E45*;D5K+E45R;D5K+E45K;D5K+N73R;D5K+N73K;D5K+D167*;D5K+D167R;D5K+D167K;D5K+T192R;D5K+T192K;D5K+L193R;D5K+L193K;D5K+Y194R;D5K+Y194K;D5K+T199R;D5K+T199K;D5K+N200R;D5K+N200K;D5K+I202R;D5K+I202K;D5K+S217R;D5K+S217K;D5K+P218R;D5K+P218K;D5K+Y220R;D5K+Y220K;D5K+W221R;D5K+W221K;D5K+S224*;D5K+S224R;D5K+S224K;D5K+S225*;D5K+S225R;D5K+S225K;D5K+T226*;D5K+T226R;D5K+T226K;D5K+L227*;D5K+L227R;D5K+L227K;D5K+V228*;D5K+V228R;D5K+V228K;D5K+P229*;D5K+P229R;D5K+P229K;D5K+V230*;D5K+V230K;D5K+V230R;D5K+T231R;D5K+T231K;D5K+R232RR;D5K+N233R;D5K+N233K;D5K+D234*;D5K+D234R;D5K+D234K;D5K+V236K;D5K+V236R;D5K+I238R;D5K+I238K;D5K+E239*;D5K+E239R;D5K+E239K;D5K+A243R;D5K+A243K;D5K+T244R;D5K+T244K;D5K+G245R;D5K+G245K;D5K+N248R;D5K+N248K;D5K+Q249R;D5K+Q249K;D5K+N251R;D5K+N251K;L6R+F7R;L6R+F7K;L6R+N8R;L6R+N8K;L6R+Q9R;L6R+Q9K;L6R+N11R;L6R+N11K;L6R+L12R;L6R+L12K;L6R+Q15R;L6R+Q15K;L6R+T37R;L6R+T37K;L6R+Q38R;L6R+Q38K;L6R+N39R;L6R+N39K;L6R+A40R;L6R+A40K;L6R+P42R;L6R+P42K;L6R+E43*;L6R+E43R;L6R+E43K;L6R+E45*;L6R+E45R;L6R+E45K;L6R+N73R;L6R+N73K;L6R+D167*;L6R+D167R;L6R+D167K;L6R+T192R;L6R+T192K;L6R+L193R;L6R+L193K;L6R+Y194R;L6R+Y194K;L6R+T199R;L6R+T199K;L6R+N200R;L6R+N200K;L6R+I202R;L6R+I202K;L6R+S217R;L6R+S217K;L6R+P218R;L6R+P218K;L6R+Y220R;L6R+Y220K;L6R+W221R;L6R+W221K;L6R+S224*;L6R+S224R;L6R+S224K;L6R+S225*;L6R+S225R;L6R+S225K;L6R+T226*;L6R+T226R;L6R+T226K;L6R+L227*;L6R+L227R;L6R+L227K;L6R+V228*;L6R+V228R;L6R+V228K;L6R+P229*;L6R+P229R;L6R+P229K;L6R+V230*;L6R+V230K;L6R+V230R;L6R+T231R;L6R+T231K;L6R+R232RR;L6R+N233R;L6R+N233K;L6R+D234*;L6R+D234R;L6R+D234K;L6R+V236K;L6R+V236R;L6R+I238R;L6R+I238K;L6R+E239*;L6R+E239R;L6R+E239K;L6R+A243R;L6R+A243K;L6R+T244R;L6R+T244K;L6R+G245R;L6R+G245K;L6R+N248R;L6R+N248K;L6R+Q249R;L6R+Q249K′;L6R+N251R;L6R+N251K;L6K+F7R;L6K+F7K;L6K+N8R;L6K+N8K;L6K+Q9R;L6K+Q9K;L6K+N11R;L6K+N11K;L6K+L12R;L6K+L12K;L6K+Q15R;L6K+Q15K;L6K+T37R;L6K+T37K;L6K+Q38R;L6K+Q38K;L6K+N39R;L6K+N39K;L6K+A40R;L6K+A40K;L6K+P42R;L6K+P42K;L6K+E43*;L6K+E43R;L6K+E43K;L6K+E45*;L6K+E45R;L6K+E45K;L6K+N73R;L6K+N73K;L6K+D167*;L6K+D167R;L6K+D1 67K;L6K+T192R;L6K+T192K;L6K+L193R;L6K+L193K;L6K+Y194R;L6K+Y194 K; L6K+T199R; L6K+T199K; L6K+N200R; L6K+N200K; L6K+I202R; L6K+I202K; L6K+S217R; L6K+S217K; L6K+P218R; L6K+P218K; L6K+Y220R; L6K+Y220K; L6 K+W221R; L6K+W221K; L6K+S224*; L6K+S224R; L6K+S224K; L6K+S225*; L6K+S225R; L6K+S225K; L6K+T226*; L6K+T226R; L6K+T226K; L6K+L227*; L6K+L2 27R;L6K+L227K;L6K+V228*;L6K+V228R;L6K+V228K;L6K+P229*;L6K+P229 R;L6K+P229K;L6K+V230*;L6K+V230K;L6K+V230R;L6K+T231R;L6K+T231K;L 6K+R232RR; L6K+N233R; L6K+N233K; L6K+D234*; L6K+D234R; L6K+D234K; L6K+V236K; L6K+V236R; L6K+I238R; L6K+I238K; L6K+E239*; L6K+E239R; L6K+ E239K; L6K+A243R; L6K+A243K; L6K+T244R; L6K+T244K; L6K+G245R; L6K+G245K; L6K+N248R; 6K+N248K; L6K+Q249R; L6K+Q249K; L6K+N251R; L6K+N251K ;F7R+N8R;F7R+N8K;F7R+Q9R;F7R+Q9K;F7R+N11R;F7R+N11K;F7R+L12R;F7 R+L12K;F7R+Q15R;F7R+Q15K;F7R+T37R;F7R+T37K;F7R+Q38R;F7R+Q38K;F 7R+N39R; F7R+N39K; F7R+A40R; F7R+A40K; F7R+P42R; F7R+P42K; F7R+E43*; F7R+E43R; F7R+E43K; F7R+E45*; F7R+E45R; F7R+E45K; F7R+N73R; F7R+N73K;F7R+D167*;F7R+D167R;F7R+D167K;F7R+T192R;F7R+T192K;F7R+L193R;F7 R+L193K;F7R+Y194R;F7R+Y194K;F7R+T199R;F7R+T199K;F7R+N200R;F7R+ N200K;F7R+I202R;F7R+I202K;F7R+S217R;F7R+S217K;F7R+P218R;F7R+P2 18K;F7R+Y220R;F7R+Y220K;F7R+W221R;F7R+W221K;F7R+S224*;F7R+S224R F7R+S224K; F7R+S225*; F7R+S225R; F7R+S225K; F7R+T226*; F7R+T226R; F7R+T226K; F7R+L227*; F7R+L227R; F7R+L227K; F7R+V228*; F7R+V228R; F7R+ V228K; F7R+P229*; F7R+P229R; F7R+P229K; F7R+V230*; F7R+V230K; F7R+V230R; F7R+T231R; F7R+T231K; F7R+R232RR; F7R+N233R; F7R+N233K; F7R+D234 *; F7R+D234R; F7R+D234K; F7R+V236K; F7R+V236R; F7R+I238R; F7R+I238K; F7R+E239*; F7R+E239R; F7R+E239K; F7R+A243R; F7R+A243K; F7R+T244R; F7R +T244K;F7R+G245R;F7R+G245K;F7R+N248R;F7R+N248K;F7R+Q249R;F7R+Q 249K;F7R+N251R;F7R+N251K;F7K+N8R;F7K+N8K;F7K+Q9R;F7K+Q9K;F7K+N1 1R;F7K+N11K;F7K+L12R;F7K+L12K;F7K+Q15R;F7K+Q15K;F7K+T37R;F7K+T 37K;F7K+Q38R;F7K+Q38K;F7K+N39R;F7K+N39K;F7K+A40R;F7K+A40K;F7K+P 42R;F7K+P42K;F7K+E43*;F7K+E43R;F7K+E43K;F7K+E45*;F7K+E45R;F7K+ 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1R;Q9K+W221K;Q9K+S224*;Q9K+S224R;Q9K+S224K;Q9K+S225*;Q9K+S225R;Q9K+S225K;Q9K+T226*;Q9K+T226R;Q9K+T226K;Q9K+L227*;Q9K+L227R;Q9K+L227K;Q9K+V228*;Q9K+V228R;Q9K+V228K;Q9K+P229*;Q9K+P229R;Q9K+P229K;Q9K+V230*;Q9K+V230K;Q9K+V230R;Q9K+T231R;Q9K+T231K;Q9K+R232RR;Q9K+N233R;Q9K+N233K;Q9K+D234*;Q9K+D234R;Q9K+D234K;Q9K+V236K;Q9K+V236R;Q9K+I238R;Q9K+I238K;Q9K+E239*;Q9K+E239R;Q9K+E239K;Q9K+A243R;Q9K+A243K;Q9K+T244R;Q9K+T244K;Q9K+G245R;Q9K+G245K;Q9K+N248R;Q9K+N248K;Q9K+Q249R;Q9K+Q249K;Q9K+N251R;Q9K+N251K;N11R+L12R;N11R+L12K;N11R+Q15R;N11R+Q15K;N11R+T37R;N11R+T37K;N11R+Q38R;N11R+Q38K;N11R+N39R;N11R+N39K;N11R+A40R;N11R+A40K;N11R+P42R;N11R+P42K;N11R+E43*;N11R+E43R;N11R+E43K;N11R+E45*;N11R+E45R;N11R+E45K;N11R+N73R;N11R+N73K;N11R+D167*;N11R+D167R;N11R+D167K;N11R+T192R;N11R+T192K;N11R+L193R;N11R+L193K;N11R+Y194R;N11R+Y194K;N11R+T199R;N11R+T199K;N11R+N200R;N11R+N200K;N11R+I202R;N11R+I202K;N11R+S217R;N11R+S217K;N11R+P218R;N11R+P218K;N11R+Y220R;N11R+Y220K;N11R+W221R;N11R+W221K;N11R+S224*;N11R+S224R;N11R+S224K;N11R+S225*;N11R+S225R;N11R+S225K;N11R+T226*;N11R+T226R;N11R+T226K;N11R+L227*;N11R+L227R;N11R+L227K;N11R+V228*;N11R+V228R;N11R+V228K;N11R+P229*;N11R+P229R;N11R+P229K;N11R+V230*;N11R+V230K;N11R+V230R;N11R+T231R;N11R+T231K;N11R+R232RR;N11R+N233R;N11R+N233K;N11R+D234*;N11R+D234R;N11R+D234K;N11R+V236K;N11R+V236R;N11R+I238R;N11R+I238K;N11R+E239*;N11R+E239R;N11R+E239K;N11R+A243R;N11R+A243K;N11R+T244R;N11R+T244K;N11R+G245R;N11R+G245K;N11R+N248R;N11R+N248K;N11R+Q249R;N11R+Q249K;N11R+N251R;N11R+N251K;N11K+L12R;N11K+L12K;N11K+Q15R;N11K+Q15K;N11K+T37R;N11K+T37K;N11K+Q38R;N11K+Q38K;N11K+N39R;N11K+N39K;N11K+A40R;N11K+A40K;N11K+P42R;N11K+P42K;N11K+E43*;N11K+E43R;N11K+E43K;N11K+E45*;N11K+E45R;N11K+E45K;N11K+N73R;N11K+N73K;N11K+D167*;N11K+D167R;N11K+D167K;N11K+T192R;N11K+T192K;N11K+L193R;N11K+L193K;N11K+Y194R;N11K+Y194K;N11K+T199R;N11K+T199K;N11K+N200R;N11K+N200K;N11K+I202R;N11K+I202K;N11K+S217R;N11K+S217K;N11K+P218R;N11K+P218K;N11K+Y220R;N11K+Y220K;N11K+W221R;N11K+W221K;N11K+S224*;N11K+S224R;N11K+S224K;N11K+S225*;N11K+S225R;N11K+S225K;N11K+T226*;N11K+T226R;11K+T226K;N11K+L227*;N11K+L227R;N11K+L227K;N11K+V228*;N11K+V228R;N11K+V228K;N11K+P229*;N11K+P229R;N11K+P229K;N11K+V230*;N11K+V230K;N11K+V230R;N11K+T231R;N11K+T231K;N11K+R232RR;N11K+N233R;N11K+N233K;N11K+D234*;N11K+D234R;N11K+D234K;N11K+V236K;N11K+V236R;N11K+I238R;N11K+I238K;N11K+E239*;N11K+E239R;N11K+E239K;N11K+A243R;N11K+A243K;N11K+T244R;N11K+T244K;N11K+G245R;N11K+G245K;N11K+N248R;N11K+N248K;N11K+Q249R;N11K+Q249K;N11K+N251R;N11K+N251K;L12R+Q15R;L12R+Q15K;L12R+T37R;L12R+T37K;L12R+Q38R;L12R+Q38K;L12R+N39R;L12R+N39K;L12R+A40R;L12R+A40K;L12R+P42R;L12R+P42K;L12R+E43*;L12R+E43R;L12R+E43K;L12R+E45*;L12R+E45R;L12R+E45K;L12R+N73R;L12R+N73K;L12R+D167*;L12R+D167R;L12R+D167K;L12R+T192R;L12R+T192K;L12R+L193R;L12R+L193K;L12R+Y194R;L12R+Y194K;L12R+T199R;L12R+T199K;L12R+N200R;L12R+N200K;L12R+I202R;L12R+I202K;L12R+S217R;L12R+S217K;L12R+P218R;L12R+P218K;L12R+Y220R;L12R+Y220K;L12R+W221R;L12R+W221K;L12R+S224*;L12R+S224R;L12R+S224K;L12R+S225*;L12R+S225R;L12R+S225K;L12R+T226*;L12R+T226R;L12R+T226K;L12R+L227*;L12R+L227R;L12R+L227K;L12R+V228*;L12R+V228R;L12R+V228K;L12R+P229*;L12R+P229R;L12R+P229K;L12R+V230*;L12R+V230K;L12R+V230R;L12R+T231R;L12R+T231K;L12R+R232RR;L12R+N233R;L12R+N233K;L12R+D234*;L12R+D234R;L12R+D234K;L12R+V236K;L12R+V236R;L12R+I238R;L12R+I238K;L12R+E239*;L12R+E239R;L12R+E239K;L12R+A243R;L12R+A243K;L12R+T244R;L12R+T244K;L12R+G245R;L12R+G245K;L12R+N248R;L12R+N248K;L12R+Q249R;L12R+Q249K;12R+N251R;L12R+N251K;L12K+Q15R;L12K+Q15K;L12K+T37R;L12K+T37K;L12K+Q38R;L12K+Q38K;L12K+N39R;L12K+N39K;L12K+A40R;L12K+A40K;L12K+P42R;L12K+P42K;L12K+E43*;L12K+E43R;L12K+E43K;L12K+E45*;L12K+E45R;L12K+E45K;L12K+N73R;L12K+N73K;L12K+D167*;L12K+D167R;L12K+D167K;L12K+T192R;L12K+T192K;L12K+L193R;L12K+L193K;L12K+Y194R;L12K+Y194K;L12K+T199R;L12K+T199K;L12K+N200R;L12K+N200K;L12K+I202R;L12K+I202K;L12K+S217R;L12K+S217K;L12K+P218R;L12K+P218K;L12K+Y220R;L12K+Y220K;L12K+W221R;L12K+W221K;L12K+S224*;L12K+S224R;L12K+S224K;L12K+S225*;L12K+S225R;L12K+S225K;L12K+T226*;L12K+T226R;L12K+T226K;L12K+L227*;L12K+L227R;L12K+L227K;L12K+V228*;L12K+V228R;L12K+V228K;L12K+P229*;L12K+P229R;L12K+P229K;L12K+V230*;L12K+V230K;L12K+V230R;L12K+T231R;L12K+T231K;L12K+R232RR;L12K+N233R;L12K+N233K;L12K+D234*;L12K+D234R;L12K+D234K;L12K+V236K;L12K+V236R;L12K+I238R;L12K+I238K;L12K+E239*;L12K+E239R;L12K+E239K;L12K+A243R;L12K+A243K;L12K+T244R;L12K+T244K;L12K+G245R;L12K+G245K;L12K+N248R;L12K+N248K;L12K+Q249R;L12K+Q249K;L12K+N251R;L12K+N251K;Q15R+T37R;Q15R+T37K;Q15R+Q38R;Q15R+Q38K;Q15R+N39R;Q15R+N39K;Q15R+A40R;Q15R+A40K;Q15R+P42R;Q15R+P42K;Q15R+E43*;Q15R+E43R;Q15R+E43K;Q15R+E45*;Q15R+E45R;Q15R+E45K;Q15R+N73R;Q15R+N73K;Q15R+D167*;Q15R+D167R;Q15R+D167K;Q15R+T192R;Q15R+T192K;Q15R+L193R;Q15R+L193K;Q15R+Y194R;Q15R+Y194K;Q15R+T199R;Q15R+T199K;Q15R+N200R;Q15R+N200K;Q15R+I202R;Q15R+I202K;Q15R+S217R;Q15R+S217K;Q15R+P218R;Q15R+P218K;Q15R+Y220R;Q15R+Y220K;Q15R+W221R;Q15R+W221K;Q15R+S224*;Q15R+S224R;Q15R+S224K;Q15R+S225*;Q15R+S225R;Q15R+S225K;Q15R+T226*;Q15R+T226R;Q15R+T226K;Q15R+L227*;Q15R+L227R;Q15R+L227K;Q15R+V228*;Q15R+V228R;Q15R+V228K;Q15R+P229*;Q15R+P229R;Q15R+P229K;Q15R+V230*;Q15R+V230K;Q15R+V230R;Q15R+T231R;Q15R+T231K;Q15R+R232RR;Q15R+N233R;Q15R+N233K;Q15R+D234*;Q15R+D234R;Q15R+D234K;Q15R+V236K;Q15R+V236R;Q15R+I238R;Q15R+I238K;Q15R+E239*;Q15R+E239R;Q15R+E239K;Q15R+A243R;Q15R+A243K;Q15R+T244R;Q15R+T244K;Q15R+G245R;Q15R+G245K;Q15R+N248R;Q15R+N248K;Q15R+Q249R;Q15R+Q249K;Q15R+N251R;Q15R+N251K;Q15K+T37R;Q15K+T37K;Q15K+Q38R;Q15K+Q38K;Q15K+N39R Q15K+N39K;Q15K+A40R;Q15K+A40K;Q15K+P42R;Q15K+P42K;Q15K+E43*;Q15K+E43R;Q15K+E43K;Q15K+E45*;Q15K+E45R;Q15K+E45K;Q15K+N73R;Q15K+N73K;Q15K+D167*;Q15K+D167R;Q15K+D167K;Q15K+T192R;Q15K+T192K;Q15K+L193R;Q15K+L193K;Q15K+Y194R;Q15K+Y194K;Q15K+T199R;Q15K+T199K;Q15K+N200R;Q15K+N200K;Q15K+I202R;Q15K+I202K;Q15K+S217R;Q15K+S217K;Q15K+P218R;Q15K+P218K;Q15K+Y220R;Q15K+Y220K;Q15K+W221R;Q15K+W221K;Q15K+S224*;Q15K+S224R;Q15K+S224K;Q15K+S225*;Q15K+S225R;Q15K+S225K;Q15K+T226*;Q15K+T226R;Q15K+T226K;Q15K+L227*;Q15K+L227R;Q15K+L227K;Q15K+V228*;Q15K+V228R;Q15K+V228K;Q15K+P229*;Q15K+P229R;Q15K+P229K;Q15K+V230*;Q15K+V230K;Q15K+V2 30R;Q15K+T231R;Q15K+T231K;Q15K+R232RR;Q15K+N233R;Q15K+N233K;Q1 5K+D234*;Q15K+D234R;Q15K+D234K;Q15K+V236K;Q15K+V236R;Q15K+I238 R;Q15K+I238K;Q15K+E239*;Q15K+E239R;Q15K+E239K;Q15K+A243R;Q15K+A 243K;Q15K+T244R;Q15K+T244K;Q15K+G245R;Q15K+G245K;Q15K+N248R;Q1 5K+N248K;Q15K+Q249R;Q15K+Q249K;Q15K+N251R;Q15K+N251K;T37R+Q38R T37R+Q38K; T37R+N39R; T37R+N39K; T37R+A40R; T37R+A40K; T37R+P42R; T37R+P42K; T37R+E43*; T37R+E43R; T37R+E43K; T37R+E45*; T37R+E45R; T37R +E45K; T37R+N73R; T37R+N73K; T37R+D167*; T37R+D167R; T37R+D167K; T37R+T192R; T37R+T192K; T37R+L193R; T37R+L193K; T37R+Y194R; T37R+Y194K T37R+T199R; T37R+T199K; T37R+N200R; T37R+N200K; T37R+I202R; T37R+I202K; T37R+S217R; T37R+S217K; T37R+P218R; T37R+P218K; T37R+Y220R; T37 R+Y220K; T37R+W221R; T37R+W221K; T37R+S224*; T37R+S224R; T37R+S224K; T37R+S225*; T37R+S225R; T37R+S225K; T37R+T226*; T37R+T226R; T37R+T2 26K; T37R+L227*; T37R+L227R; T37R+L227K; T37R+V228*; T37R+V228R; T37R+V228K; T37R+P229*; T37R+P229R; T37R+P229K; T37R+V230*; T37R+V230K;T37R+V230R; T37R+T231R; T37R+T231K; T37R+R232RR; T37R+N233R; T37R+N233K; T37R+D234*; T37R+D234R; T37R+D234K; T37R+V236K; T37R+V236R; T3 7R+I238R; T37R+I238K; T37R+E239*; T37R+E239R; T37R+E239K; T37R+A243R; T37R+A243K; T37R+T244R; T37R+T244K; T37R+G245R; T37R+G245K; T37R+N 248R; T37R+N248K; T37R+Q249R; T37R+Q249K; T37R+N251R; T37R+N251K; T37K+Q38R; T37K+Q38K; T37K+N39R; T37K+N39K; T37K+A40R; T37K+A40K; T37K +P42R; T37K+P42K; T37K+E43*; T37K+E43R; T37K+E43K; T37K+E45*; T37K+E45R; T37K+E45K; T37K+N73R; T37K+N73K; T37K+D167*; T37K+D167R; 37K+D16 7K; T37K+T192R; T37K+T192K; T37K+L193R; T37K+L193K; T37K+Y194R; T37K+Y194K; T37K+T199R; T37K+T199K; T37K+N200R; T37K+N200K; T37K+I202R; T37K+I202K; T37K+S217R; T37K+S217K; T37K+P218R; T37K+P218K; T37K+Y220R; T37K+Y220K; T37K+W221R; T37K+W221K; T37K+S224*; T37K+S224R; T37K +S224K; T37K+S225*; T37K+S225R; T37K+S225K; T37K+T226*; T37K+T226R; T37K+T226K; T37K+L227*; T37K+L227R; T37K+L227K; T37K+V228*; T37K+V22 8R; T37K+V228K; T37K+P229*; T37K+P229R; T37K+P229K; T37K+V230*; T37K+V230K; T37K+V230R; T37K+T231R; T37K+T231K; T37K+R232RR; T37K+N233R;T37K+N233K; T37K+D234*; T37K+D234R; T37K+D234K; T37K+V236K; T37K+V236R; T37K+I238R; T37K+I238K; T37K+E239*; T37K+E239R; T37K+E239K; T37 K+A243R; T37K+A243K; T37K+T244R; T37K+T244K; T37K+G245R; T37K+G245K; T37K+N248R; T37K+N248K; T37K+Q249R; T37K+Q249K; T37K+N251R; T37K+N2 51K;Q38R+N39R;Q38R+N39K;Q38R+A40R;Q38R+A40K;Q38R+P42R;Q38R+P42 K;Q38R+E43*;Q38R+E43R;Q38R+E43K;Q38R+E45*;Q38R+E45R;Q38R+E45K;Q 38R+N73R;Q38R+N73K;Q38R+D167*;Q38R+D167R;Q38R+D167K;Q38R+T192R ;Q38R+T192K;Q38R+L193R;Q38R+L193K;Q38R+Y194R;Q38R+Y194K;Q38R+T1 99R;Q38R+T199K;Q38R+N200R;Q38R+N200K;Q38R+I202R;Q38R+I202K;Q38 R+S217R;Q38R+S217K;Q38R+P218R;Q38R+P218K;Q38R+Y220R;Q38R+Y220K; Q38R+W221R; Q38R+W221K; Q38R+S224*; Q38R+S224R; Q38R+S224K; Q38R+S225*; Q38R+S225R; Q38R+S225K; Q38R+T226*; Q38R+T226R; Q38R+T226K; Q38R +L227*; Q38R+L227R; Q38R+L227K; Q38R+V228*; Q38R+V228R; Q38R+V228K; Q38R+P229*; Q38R+P229R; Q38R+P229K; Q38R+V230*; Q38R+V230K; Q38R+V23 0R; Q38R+T231R; Q38R+T231K; Q38R+R232RR; Q38R+N233R; Q38R+N233K; Q38R+D234*; Q38R+D234R; Q38R+D234K; Q38R+V236K; Q38R+V236R; Q38R+I238R;Q38R+I238K;Q38R+E239*;Q38R+E239R;Q38R+E239K;Q38R+A243R;Q38R+A243K;Q38R+T244R;Q38R+T244K;Q38R+G245R;Q38R+G245K;Q38R+N248R;Q38R+N248K;Q38R+Q249R;Q38R+Q249K;Q38R+N251R;Q38R+N251K;Q38K+N39R;Q38K+N39K;Q38K+A40R;Q38K+A40K;Q38K+P42R;Q38K+P42K;Q38K+E43*;Q38K+E43R;Q38K+E43K;Q38K+E45*;Q38K+E45R;Q38K+E45K;Q38K+N73R;Q38K+N73K;Q38K+D167*;Q38K+D167R;Q38K+D167K;Q38K+T192R;Q38K+T192K;Q38K+L193R;Q38K+L193K;Q38K+Y194R;Q38K+Y194K;Q38K+T199R;Q38K+T199K;Q38K+N200R;Q38K+N200K;Q38K+I202R;Q38K+I202K;Q38K+S217R;Q38K+S217K;Q38K+P218R;Q38K+P218K;Q38K+Y220R;Q38K+Y220K;Q38K+W221R;Q38K+W221K;Q38K+S224*;Q38K+S224R;Q38K+S224K;Q38K+S225*;Q38K+S225R;Q38K+S225K;Q38K+T226*;Q38K+T226R;Q38K+T226K;Q38K+L227*;Q38K+L227R;Q38K+L227K;Q38K+V228*;Q38K+V228R;Q38K+V228K;Q38K+P229*;Q38K+P229R;Q38K+P229K;Q38K+V230*;Q38K+V230K;Q38K+V230R;Q38K+T231R;Q38K+T231K;Q38K+R232RR;Q38K+N233R;Q38K+N233K;Q38K+D234*;Q38K+D234R;Q38K+D234K;Q38K+V236K;Q38K+V236R;Q38K+I238R;Q38K+I238K;Q38K+E239*;Q38K+E239R;Q38K+E239K;Q38K+A243R;Q38K+A243K;Q38K+T244R;Q38K+T244K;Q38K+G245R;Q38K+G245K;Q38K+N248R;Q38K+N248K;Q38K+Q2 49R;Q38K+Q249K;Q38K+N251R;Q38K+N251K;N39R+A40R;N39R+A40K;N39R+P 42R; N39R+P42K; N39R+E43*; N39R+E43R; N39R+E43K; N39R+E45*; N39R+E45R; N39R+E45K; N39R+N73R; N39R+N73K; N39R+D167*; N39R+D167R; N39R+D167 K; N39R+T192R; N39R+T192K; N39R+L193R; N39R+L193K; N39R+Y194R; N39R+Y194K; N39R+T199R; N39R+T199K; N39R+N200R; N39R+N200K; N39R+I202R; N3 9R+I202K;N39R+S217R;N39R+S217K;N39R+P218R;N39R+P218K;N39R+Y220 R;N39R+Y220K;N39R+W221R;N39R+W221K;N39R+S224*;N39R+S224R;N39R+S 224K; N39R+S225*; N39R+S225R; N39R+S225K; N39R+T226*; N39R+T226R; N39R+T226K; N39R+L227*; N39R+L227R; N39R+L227K; N39R+V228*; N39R+V228R N39R+V228K; N39R+P229*; N39R+P229R; N39R+P229K; N39R+V230*; N39R+V230K; N39R+V230R; N39R+T231R; N39R+T231K; N39R+R232RR; N39R+N233R; N3 9R+N233K;N39R+D234*;N39R+D234R;N39R+D234K;N39R+V236K;N39R+V236 R;N39R+I238R;N39R+I238K;N39R+E239*;N39R+E239R;N39R+E239K;N39R+A 243R; N39R+A243K; N39R+T244R; N39R+T244K; N39R+G245R; N39R+G245K; N39R+N248R; N39R+N248K; N39R+Q249R; N39R+Q249K; N39R+N251R; N39R+N251K;N39K+A40R; N39K+A40K; N39K+P42R; N39K+P42K; N39K+E43*; N39K+E43R; N39K+E43K; N39K+E45*; N39K+E45R; N39K+E45K; N39K+N73R; N39K+N73K; N39K+ D167*; N39K+D167R; N39K+D167K; N39K+T192R; N39K+T192K; N39K+L193R; N39K+L193K; N39K+Y194R; N39K+Y194K; N39K+T199R; N39K+T199K; N39K+N200 R; N39K+N200K; N39K+I202R; N39K+I202K; N39K+S217R; N39K+S217K; N39K+P218R; N39K+P218K; N39K+Y220R; N39K+Y220K; N39K+W221R; N39K+W221K; N3 9K+S224*; N39K+S224R; N39K+S224K; N39K+S225*; N39K+S225R; N39K+S225K; N39K+T226*; N39K+T226R; 39K+T226K; N39K+L227*; N39K+L227R; N39K+L2 27K; N39K+V228*; N39K+V228R; N39K+V228K; N39K+P229*; N39K+P229R; N39K+P229K; N39K+V230*; N39K+V230K; N39K+V230R; N39K+T231R; N39K+T231K; N39K+R232RR; N39K+N233R; N39K+N233K; N39K+D234*; N39K+D234R; N39K+D234K; N39K+V236K; N39K+V236R; N39K+I238R; N39K+I238K; N39K+E239*; N39 K+E239R; N39K+E239K; N39K+A243R; N39K+A243K; N39K+T244R; N39K+T244K; N39K+G245R; N39K+G245K; N39K+N248R; N39K+N248K; N39K+Q249R; N39K+Q2 49K;N39K+N251R;N39K+N251K;A40R+P42R;A40R+P42K;A40R+E43*;A40R+E4 3R;A40R+E43K;A40R+E45*;A40R+E45R;A40R+E45K;A40R+N73R;A40R+N73K;A40R+D167*;A40R+D167R;A40R+D167K;A40R+T192R;A40R+T192K;A40R+L193R;A40R+L193K;A40R+Y194R;A40R+Y194K;A40R+T199R;A40R+T199K;A40R+N200R;A40R+N200K;A40R+I202R;A40R+I202K;A40R+S217R;A40R+S217K;A40R+P218R;A40R+P218K;A40R+Y220R;A40R+Y220K;A40R+W221R;A40R+W221K;A40R+S224*;A40R+S224R;A40R+S224K;A40R+S225*;A40R+S225R;A40R+S225K;A40R+T226*;A40R+T226R;A40R+T226K;A40R+L227*;A40R+L227R;A40R+L227K;A40R+V228*;A40R+V228R;A40R+V228K;A40R+P229*;A40R+P229R;A40R+P229K;A40R+V230*;A40R+V230K;A40R+V230R;A40R+T231R;A40R+T231K;A40R+R232RR;A40R+N233R;A40R+N233K;A40R+D234*;A40R+D234R;A40R+D234K;A40R+V236K;A40R+V236R;A40R+I238R;A40R+I238K;A40R+E239*;A40R+E239R;A40R+E239K;A40R+A243R;A40R+A243K;A40R+T244R;A40R+T244K;A40R+G245R;A40R+G245K;A40R+N248R;A40R+N248K;A40R+Q249R;A40R+Q249K;A40R+N251R;A40R+N251K;A40K+P42R;A40K+P42K;A40K+E43*;A40K+E43R;A40K+E43K;A40K+E45*;A40K+E45R;A40K+E45K;A40K+N73R;A40K+N73K;A40K+D167*;A40K+D167R;A40K+D167K;A40K+T192R;A40K+T192K;A40K+L193R;A40K+L193K;A40K+Y194R;A40K+Y194K;A40K+T199R;A40K+T199K;A40K+N200R;A40K+N200K;A40K+I202R;A40K+I202K;A40K+S217R;A40K+S217K;A40K+P218R;A40K+P218K;A40K+Y220R;A40K+Y220K;A40K+W221R;A40K+W221K;A40K+S224*;A40K+S224R;A40K+S224K;A40K+S225*;A40K+S225R;A40K+S225K;A40K+T226*;A40K+T226R;A40K+T226K;A40K+L227*;A40K+L227R;A40K+L227K;A40K+V228*;A40K+V228R;A40K+V228K;A40K+P229*;A40K+P229R;A40K+P229K;A40K+V230*;A40K+V230K;A40K+V230R;A40K+T231R;A40K+T231K;A40K+R232RR;A40K+N233R;A40K+N233K;A40K+D234*;A40K+D234R;A40K+D234K;A40K+V236K;A40K+V236R;A40K+I238R;A40K+I238K;A40K+E239*;A40K+E239R;A40K+E239K;A40K+A243R;A40K+A243K;A40K+T244R;A40K+T244K;A40K+G245R;A40K+G245K;A40K+N248R;A40K+N248K;A40K+Q249R;A40K+Q249K;A40K+N251R;A40K+N251K;P42R+E43*;P42R+E43R;P42R+E43K;P42R+E45*;P42R+E45R;P42R+E45K;P42R+N73R;P42R+N73K;P42R+D167*;P42R+D167R;P42R+D167K;P42R+T192R;P42R+T192K;P42R+L193R;P42R+L193K;P42R+Y194R;P42R+Y194K;P42R+T199R;P42R+T199K;P42R+N200R;P42R+N200K;P42R+I202R;P42R+I202K;P42R+S217R;P42R+S217K;P42R+P218R;P42R+P218K;P42R+Y220R;P42R+Y220K;P42R+W221R;P42R+W221K;P42R+S224*;P42R+S224R;P42R+S224K;P42R+S225*;P42R+S225R;P42R+S225K;P42R+T226*;P42R+T226R;P42R+T226K;P42R+L227*;P42R+L227R;P42R+L227K;P42R+V228*;P42R+V228R;P42R+V228K;P42R+P229*;P42R+P229R;P42R+P229K;P42R+V230*;P42R+V230K;P42R+V230R;P42R+T231R;P42R+T231K;P42R+R232RR;P42R+N233R;P42R+N233K;P42R+D234*;P42R+D234R;P42R+D234K;P42R+V236K;P42R+V236R;P42R+I238R;P42R+I238K;P42R+E239*;P42R+E239R;P42R+E239K;P42R+A243R;P42R+A243K;P42R+T244R;P42R+T244K;P42R+G245R;P42R+G245K;P42R+N248R;P42R+N248K;P42R+Q249R;P42R+Q249K;P42R+N251R;P42R+N251K;P42K+E43*;P42K+E43R;P42K+E43K;P42K+E45*;P42K+E45R;P42K+E45K;P42K+N73R;P42K+N73K;P42K+D167*;P42K+D167R;P42K+D167K;P42K+T192R;P42K+T192K;P42K+L193R;P42K+L193K;P42K+Y194R;P42K+Y194K;P42K+T199R;P42K+T199K;P42K+N200R;P42K+N200K;P42K+I202R;P42K+I202K;P42K+S217R;P42K+S217K;P42K+P218R;P42K+P218K;P42K+Y220R;P42K+Y220K;P42K+W221R;P42K+W221K;P42K+S224*;P42K+S224R;P42K+S224K;P42K+S225*;P42K+S225R;P42K+S225K;P42K+T226*;P42K+T226R;P42K+T226K;P42K+L227*;P42K+L227R;P42K+L227K;P42K+V228*;P42K+V228R;P42K+V228K;P42K+P229*;P42K+P229R;P42K+P229K;P42K+V230*;P42K+V230K;P42K+V230R;P42K+T231R;P42K+T231K;P42K+R232RR;P42K+N233R;P42K+N233K;P42K+D234*;P42K+D234R;P42K+D234K;P42K+V236K;P42K+V236R;P42K+I238R;P42K+I238K;P42K+E239*;P42K+E239R;P42K+E239K;P42K+A243R;P42K+A243K;P42K+T244R;P42K+T244K;P42K+G245R;P42K+G245K;P42K+N248R;P42K+N248K;P42K+Q249R;P42K+Q249K;P42K+N251R;P42K+N251K;E43*+E45*;E43*+E45R;E43*+E45K;E43*+N73R;E43*+N73K;E43*+D167*;E43*+D167R;E43*+D167K;E43*+T192R;E43*+T192K;E43*+L193R;E43*+L193K;E43*+Y194R;E43*+Y194K;E43*+T199R;E43*+T199K;E43*+N200R;E43*+N200K;E43*+I202R;E43*+I202K;E43*+S217R;E43*+S217K;E43*+P218R;E43*+P218K;E43*+Y220R;E43*+Y220K;E43*+W221R;E43*+W221K;E43*+S224*;E43*+S224R;E43*+S224K;E43*+S225*;E43*+S225R;E43*+S225K;E43*+T226*;E43*+T226R;E43*+T226K;E43*+L227*;E43*+L227R;E43*+L227K;E43*+V228*;E43*+V228R;E43*+V228K;E43*+P229*;E43*+P229R;E43*+P229K;E43*+V230*;E43*+V230K;E43*+V230R;E43*+T231R;E43*+T231K;E43*+R232RR;E43*+N233R;E43*+N233K;E43*+D234*; E43*+D234R; E43*+D234K; E43*+V236K; E43*+V236R; E43*+I238R; E43*+I238K; E43*+E239*; E43*+E239R; E43*+E239K; E43*+A243R; E43 *+A243K;E43*+T244R;E43*+T244K;E43*+G245R;E43*+G245K;E43*+N248R ;E43*+N248K;E43*+Q249R;E43*+Q249K;E43*+N251R;E43*+N251K;E43R+E4 5*;E43R+E45R;E43R+E45K;E43R+N73R;E43R+N73K;E43R+D167*;E43R+D16 7R;E43R+D167K;E43R+T192R;E43R+T192K;E43R+L193R;E43R+L193K;E43R +Y194R; E43R+Y194K; E43R+T199R; E43R+T199K; E43R+N200R; E43R+N200K; E43R+I202R; E43R+I202K; E43R+S217R; E43R+S217K; E43R+P218R; E43R+P21 8K; E43R+Y220R; E43R+Y220K; E43R+W221R; E43R+W221K; E43R+S224*; E43R+S224R; E43R+S224K; E43R+S225*; E43R+S225R; E43R+S225K; E43R+T226*; E43R+T226R; E43R+T226K; E43R+L227*; E43R+L227R; E43R+L227K; E43R+V228*; E43R+V228R; E43R+V228K; E43R+P229*; E43R+P229R; E43R+P229K; E43R +V230*; E43R+V230K; E43R+V230R; E43R+T231R; E43R+T231K; E43R+R232RR; E43R+N233R; E43R+N233K; E43R+D234*; E43R+D234R; E43R+D234K; E43R+V2 36K; E43R+V236R; E43R+I238R; E43R+I238K; E43R+E239*; E43R+E239R; E43R+E239K; E43R+A243R; E43R+A243K; E43R+T244R; E43R+T244K; E43R+G245R;E43R+G245K;E43R+N248R;E43R+N248K;E43R+Q249R;E43R+Q249K;E43R+N251R;E43R+N251K;E43K+E45*;E43K+E45R;E43K+E45K;E43K+N73R;E43K+N73K;E43K+D167*;E43K+D167R;E43K+D167K;E43K+T192R;E43K+T192K;E43K+L193R;E43K+L193K;E43K+Y194R;E43K+Y194K;E43K+T199R;E43K+T199K;E43K+N200R;E43K+N200K;E43K+I202R;E43K+I202K;E43K+S217R;E43K+S217K;E43K+P218R;E43K+P218K;E43K+Y220R;E43K+Y220K;E43K+W221R;E43K+W221K;E43K+S224*;E43K+S224R;E43K+S224K;E43K+S225*;E43K+S225R;E43K+S225K;E43K+T226*;E43K+T226R;E43K+T226K;E43K+L227*;E43K+L227R;E43K+L227K;E43K+V228*;E43K+V228R;E43K+V228K;E43K+P229*;E43K+P229R;E43K+P229K;E43K+V230*;E43K+V230K;E43K+V230R;E43K+T231R;E43K+T231K;E43K+R232RR;E43K+N233R;E43K+N233K;E43K+D234*;E43K+D234R;E43K+D234K;E43K+V236K;E43K+V236R;E43K+I238R;E43K+I238K;E43K+E239*;E43K+E239R;E43K+E239K;E43K+A243R;E43K+A243K;E43K+T244R;E43K+T244K;E43K+G245R;E43K+G245K;E43K+N248R;E43K+N248K;E43K+Q249R;E43K+Q249K;E43K+N251R;E43K+N251K;E45*+N73R;E45*+N73K;E45*+D167*;E45*+D167R;E45*+D167K;E45*+T192R;E45*+T192K;E45*+L193R;E45*+L193K;E45*+Y194R;E45*+Y194K;E45*+T199R;E45*+T199K;E45*+N200R;E45*+N200K;E45*+I202R;E45*+I202K;E45*+S217R;E45*+S217K;E45*+P218R;E45*+P218K;E45*+Y220R;E45*+Y220K;E45*+W221R;E45*+W221K;E45*+S224*;E45*+S224R;E45*+S224K;E45*+S225*;E45*+S225R;E45*+S225K;E45*+T226*;E45*+T226R;E45*+T226K;E45*+L227*;E45*+L227R;E45*+L227K;E45*+V228*;E45*+V228R;E45*+V228K;E45*+P229*;E45*+P229R;E45*+P229K;E45*+V230*;E45*+V230K;E45*+V230R;E45*+T231R;E45*+T231K;E45*+R232RR;E45*+N233R;E45*+N233K;E45*+D234*;E45*+D234R;E45*+D234K;E45*+V236K;E45*+V236R;E45*+I238R;E45*+I238K;E45*+E239*;E45*+E239R;E45*+E239K;E45*+A243R;E45*+A243K;E45*+T244R;E45*+T244K;E45*+G245R;E45*+G245K;E45*+N248R;E45*+N248K;E45*+Q249R;E45*+Q249K;E45*+N251R;E45*+N251K;E45R+N73R;E45R+N73K;E45R+D167*;E45R+D167R;E45R+D167K;E45R+T192R;E45R+T192K;E45R+L193R;E45R+L193K;E45R+Y194R;E45R+Y194K;E45R+T199R;E45R+T199K;E45R+N200R;E45R+N200K;E45R+I202R;E45R+I202K;E45R+S217R;E45R+S217K;E45R+P218R;E45R+P218K;E45R+Y220R;E45R+Y220K;E45R+W221R;E45R+W221K;E45R+S224*;E45R+S224R;E45R+S224K;E45R+S225*;E45R+S225R;E45R+S225K;E45R+T226*;E45R+T226R;E45R+T226K;E45R+L227*;E45R+L227R;E45R+L227K;E45R+V228*;E45R+V228R;E45R+V228K;E45R+P229*;E45R+P229R;E45R+P229K;E45R+V230*;E45R+V230K;E45R+V230R;E45R+T231R;E45R+T231K;E45R+R232RR;E45R+N233R;E45R+N233K;E45R+D234*;E45R+D234R;E45R+D234K;E45R+V236K;E45R+V236R;E45R+I238R;E45R+I238K;E45R+E239*;E45R+E239R;E45R+E239K;E45R+A243R;E45R+A243K;E45R+T244R;E45R+T244K;E45R+G245R;E45R+G245K;E45R+N248R;E45R+N248K;E45R+Q249R;E45R+Q249K;E45R+N251R;E45R+N251K;E45K+N73R;E45K+N73K;E45K+D167*;E45K+D167R;E45K+D167K;E45K+T192R;E45K+T192K;E45K+L193R;E45K+L193K;E45K+Y194R;E45K+Y194K;E45K+T199R;E45K+T199K;E45K+N200R;E45K+N200K;E45K+I202R;E45K+I202K;E45K+S217R;E45K+S217K;E45K+P218R;E45K+P218K;E45K+Y220R;E45K+Y220K;E45K+W221R;E45K+W221K;E45K+S224*;E45K+S224R;E45K+S224K;E45K+S225*;E45K+S225R;E45K+S225K;E45K+T226*;E45K+T226R;E45K+T226K;E45K+L227*;E45K+L227R;E45K+L227K;E45K+V228*;E45K+V228R;E45K+V228K;E45K+P229*;E45K+P229R;E45K+P229K;E45K+V230*;E45K+V230K;E45K+V230R;E45K+T231R;E45K+T231K;E45K+R232RR;E45K+N 233R;E45K+N233K;E45K+D234*;E45K+D234R;E45K+D234K;E45K+V236K;E4 5K+V236R;E45K+I238R;E45K+I238K;E45K+E239*;E45K+E239R;E45K+E239 K;E45K+A243R;E45K+A243K;E45K+T244R;E45K+T244K;E45K+G245R;E45K+ G245K;E45K+N248R;E45K+N248K;E45K+Q249R;E45K+Q249K;E45K+N251R;E 45K+N251K;N73R+D167*;N73R+D167R;N73R+D167K;N73R+T192R;N73R+T19 2K; N73R+L193R; N73R+L193K; N73R+Y194R; N73R+Y194K; N73R+T199R; N73R+T199K; N73R+N200R; N73R+N200K; N73R+I202R; N73R+I202K; N73R+S217R; N73R+S217K;N73R+P218R;N73R+P218K;N73R+Y220R;N73R+Y220K;N73R+W2 21R;N73R+W221K;N73R+S224*;N73R+S224R;N73R+S224K;N73R+S225*;N73 R+S225R; N73R+S225K; N73R+T226*; N73R+T226R; N73R+T226K; N73R+L227*; N73R+L227R; N73R+L227K; N73R+V228*; N73R+V228R; N73R+V228K; N73R+P 229*; N73R+P229R; N73R+P229K; N73R+V230*; N73R+V230K; N73R+V230R; N73R+T231R; N73R+T231K; N73R+R232RR; N73R+N233R; N73R+N233K; N73R+D23 4*; N73R+D234R; N73R+D234K; N73R+V236K; N73R+V236R; N73R+I238R; N73R+I238K; N73R+E239*; N73R+E239R; N73R+E239K; N73R+A243R; N73R+A243K;N73R+T244R;N73R+T244K;N73R+G245R;N73R+G245K;N73R+N248R;N73R+N248K;N73R+Q249R;N73R+Q249K;N73R+N251R;N73R+N251K;N73K+D167*;N73K+D167R;N73K+D167K;N73K+T192R;N73K+T192K;N73K+L193R;N73K+L193K;N73K+Y194R;N73K+Y194K;N73K+T199R;N73K+T199K;N73K+N200R;N73K+N200K;N73K+I202R;N73K+I202K;N73K+S217R;N73K+S217K;N73K+P218R;N73K+P218K;N73K+Y220R;N73K+Y220K;N73K+W221R;N73K+W221K;N73K+S224*;N73K+S224R;N73K+S224K;N73K+S225*;N73K+S225R;N73K+S225K;N73K+T226*;N73K+T226R;N73K+T226K;N73K+L227*;N73K+L227R;N73K+L227K;N73K+V228*;N73K+V228R;N73K+V228K;N73K+P229*;N73K+P229R;N73K+P229K;N73K+V230*;N73K+V230K;N73K+V230R;N73K+T231R;N73K+T231K;N73K+R232RR;N73K+N233R;N73K+N233K;N73K+D234*;N73K+D234R;N73K+D234K;N73K+V236K;N73K+V236R;N73K+I238R;N73K+I238K;N73K+E239*;N73K+E239R;N73K+E239K;N73K+A243R;N73K+A243K;N73K+T244R;N73K+T244K;N73K+G245R;N73K+G245K;N73K+N248R;N73K+N248K;N73K+Q249R;N73K+Q249K;N73K+N251R;N73K+N251K;D167*+T192R;D167*+T192K;D167*+L193R;D167*+L193K;D167*+Y194R;D167*+Y194K;D167*+T199R;D167*+T199K;D167*+N200R;D167*+N200K;D167*+I202R;D167*+I202K;D167*+S217R;D167*+S217K;D167*+P218R;D167*+P218K;D167*+Y220R;D167*+Y220K;D167*+W221R;D167*+W221K;D167*+S224*;D167*+S224R;D167*+S224K;D167*+S225*;D167*+S225R;D167*+S225K;D167*+T226*;D167*+T226R;D167*+T226K;D167*+L227*;D167*+L227R;D167*+L227K;D167*+V228*;D167*+V228R;D167*+V228K;D167*+P229*;D167*+P229R;D167*+P229K;D167*+V230*;D167*+V230K;D167*+V230R;D167*+T231R;D167*+T231K;D167*+R232RR;D167*+N233R;D167*+N233K;D167*+D234*;D167*+D234R;D167*+D234K;D167*+V236K;D167*+V236R;D167*+I238R;D167*+I238K;D167*+E239*;D167*+E239R;D167*+E239K;D167*+A243R;D167*+A243K;D167*+T244R;D167*+T244K;D167*+G245R;D167*+G245K;D167*+N248R;D167*+N248K;D167*+Q249R;D167*+Q249K;D167*+N251R;D167*+N251K;D167R+T192R;D167R+T192K;D167R+L193R;D167R+L193K;D167R+Y194R;D167R+Y194K;D167R+T199R;167R+T199K;D167R+N200R;D167R+N200K;D167R+I202R;D167R+I202K;D167R+S217R;D167R+S217K;D167R+P218R;D167R+P218K;D167R+Y220R;D167R+Y220K;D167R+W221R D167R+W221K;D167R+S224*;D167R+S224R;D167R+S224K;D167R+S225*;D167R+S225R;D167R+S225K;D167R+T226*;D167R+T226R;D167R+T226K;D167R+L227*;D167R+L227R;D167R+L227K;D167R+V228*;D167R+V228R;D167R+V228K;D167R+P229*;D167R+P229R;D167R+P229K;D167R+V230*;D167R+V230K;D167R+V230R;D167R+T231R;D167R+T231K;D167R+R232RR;D167R+N233R;D167R+N233K;D167R+D234*;D167R+D234R;D167R+D234K;D167R+V236K;D167R+V236R;D167R+I238R;D167R+I238K;D167R+E239*;D167R+E239R;D167R+E239K;D167R+A243R;D167R+A243K;D167R+T244R;D167R+T244K;D167R+G245R;D167R+G245K;D167R+N248R;D167R+N248K;D167R+Q249R;D167R+Q249K;D167R+N251R;D167R+N251K;D167K+T192R;D167K+T192K;D167K+L193R;D167K+L193K;D167K+Y194R;D167K+Y194K;D167K+T199R;D167K+T199K;D167K+N200R;D167K+N200K;D167K+I202R;D167K+I202K;D167K+S217R;D167K+S217K;D167K+P218R;D167K+P218K;D167K+Y220R;D167K+Y220K;D167K+W221R;D167K+W221K;D167K+S224*;D167K+S224R;D167K+S224K;D167K+S225*;D167K+S225R;D167K+S225K;D167K+T226*;D167K+T226R;D167K+T226K;D167K+L227*;D167K+L227R;D167K+L227K;D167K+V228*;D167K+V228R;D167K+V228K;D167K+P229*;D167K+P229R;D167K+P229K;D167K+V230*;D167K+V230K;D167K+V230R;D167K+T231R;D167K+T231K;D167K+R232RR;D167K+N233R;D167K+N233K;D167K+D234*;D167K+D234R;D167K+D234K;D167K+V236K;D167K+V236R;D167K+I238R;D167K+I238K;D167K+E239*;D167K+E239R;D167K+E239K;D167K+A243R;D167K+A243K;D167K+T244R;D167K+T244K;D167K+G245R;D167K+G245K;D167K+N248R;D167K+N248K;D167K+Q249R;D167K+Q249K;D167K+N251R;D167K+N251K;T192R+L193R;T192R+L193K;T192R+Y194R;T192R+Y194K;T192R+T199R;T192R+T199K;T192R+N200R;T192R+N200K;T192R+I202R;T192R+I202K;T192R+S217R;T192R+S217K;T192R+P218R;T192R+P218K;T192R+Y220R;T192R+Y220K;T192R+W221R;T192R+W221K;T192R+S224*;T192R+S224R;T192R+S224K;T192R+S225*;T192R+S225R;T192R+S225K;T192R+T226*;T192R+T226R;T192R+T226K;T192R+L227*;T192R+L227R;T192R+L227K;T192R+V228*;T192R+V228R;T192R+V228K;T192R+P229*;T192R+P229R;T192R+P229K;T192R+V230*;T192R+V230K;T192R+V230R;T192R+T231R;T192R+T231K;T192R+R232RR;T192R+N233R;T192R+N233K;T192R+D234*;T192R+D234R;T192R+D234K;T192R+V236K;T192R+V236R;T192R+I238R;T192R+I238K;T192R+E239*;T192R+E239R;T192R+E239K;T192R+A243R;T192R+A243K;T192R+T244R;T192R+T244K;T192R+G245R;T192R+G245K;T192R+N248R;T192R+N248K;T192R+Q249R;T192R+Q249K;T192R+N251R;T192R+N251K;T192K+L193R;T192K+L193K;T192K+Y194R;T192K+Y194K;T192K+T199R;T192K+T199K;T192K+N200R;T192K+N200K;T192K+I202R;T192K+I202K;T192K+S217R;T192K+S217K;T192K+P218R;T192K+P218K;T192K+Y220R;T192K+Y220K;T192K+W221R;T192K+W221K;T192K+S224*;T192K+S224R;T192K+S224K;T192K+S225*;T192K+S225R;T192K+S225K;T192K+T226*;T192K+T226R;T192K+T226K;T192K+L227*;T192K+L227R;T192K+L227K;T192K+V228*;T192K+V228R;T192K+V228K;;T192K+P229*;T192K+P229R;T192K+P229K;T192K+V230*;T192K+V230K;T192K+V230R;T192K+T231R;T192K+T231K;T192K+R232RR;T192K+N233R;T192K+N233K;T192K+D234*;T192K+D234R;T192K+D234K;T192K+V236K;T192K+V236R;T192K+I238R;T192K+I238K;T192K+E239*;T192K+E239R;T192K+E239K;T192K+A243R;T192K+A243K;T192K+T244R;T192K+T244K;T192K+G245R;T192K+G245K;T192K+N248R;T192K+N248K;T192K+Q249R;T192K+Q249K;T192K+N251R;T192K+N251K;L193R+Y194R;L193R+Y194K;L193R+T199R;L193R+T199K;L193R+N200R;L193R+N200K; L193R+I202R; L193R+I202K; L193R+S217R; L193R+S217K; L193R+P218R; L193R+P218K; L193R+Y220R; L193R+Y220K; L193R+W221R; L193 R+W221K;L193R+S224*;L193R+S224R;L193R+S224K;L193R+S225*;L193R+ S225R;L193R+S225K;L193R+T226*;L193R+T226R;L193R+T226K;L193R+L22 7*; L193R+L227R; L193R+L227K; L193R+V228*; L193R+V228R; L193R+V228K; L193R+P229*; L193R+P229R; L193R+P229K; L193R+V230*; L193R+V230K; L 193R+V230R; L193R+T231R; L193R+T231K; L193R+R232RR; L193R+N233R; L193R+N233K; L193R+D234*; L193R+D234R; L193R+D234K; L193R+V236K; L193R +V236R; L193R+I238R; L193R+I238K; L193R+E239*; L193R+E239R; L193R+E239K; L193R+A243R; L193R+A243K; L193R+T244R; L193R+T244K; L193R+G24 5R;L193R+G245K;L193R+N248R;L193R+N248K;L193R+Q249R;L193R+Q249K ;L193R+N251R;L193R+N251K;L193K+Y194R;L193K+Y194K;L193K+T199R;L1 93K+T199K; L193K+N200R; L193K+N200K; L193K+I202R; L193K+I202K; L193K+S217R; L193K+S217K; L193K+P218R; L193K+P218K; L193K+Y220R; L193K+Y 220K; L193K+W221R; L193K+W221K; L193K+S224*; L193K+S224R; L193K+S224K; L193K+S225*; L193K+S225R; L193K+S225K; L193K+T226*; L193K+T226R;L193K+T226K; L193K+L227*; L193K+L227R; L193K+L227K; L193K+V228*; L193K+V228R; L193K+V228K; L193K+P229*; L193K+P229R; L193K+P229K; L193 K+V230*; L193K+V230K; L193K+V230R; L193K+T231R; L193K+T231K; L193K+R232RR; L193K+N233R; L193K+N233K; L193K+D234*; L193K+D234R; L193K+D2 34K; L193K+V236K; L193K+V236R; L193K+I238R; L193K+I238K; L193K+E239*; L193K+E239R; L193K+E239K; L193K+A243R; L193K+A243K; L193K+T244R; L 193K+T244K;L193K+G245R;L193K+G245K;L193K+N248R;L193K+N248K;L19 3K+Q249R;L193K+Q249K;L193K+N251R;L193K+N251K;Y194R+T199R;Y194R+ T199K;Y194R+N200R;Y194R+N200K;Y194R+I202R;Y194R+I202K;Y194R+S2 17R;Y194R+S217K;Y194R+P218R;Y194R+P218K;Y194R+Y220R;Y194R+Y220 K;Y194R+W221R;Y194R+W221K;Y194R+S224*;Y194R+S224R;Y194R+S224K; Y194R+S225*;Y194R+S225R;Y194R+S225K;Y194R+T226*;Y194R+T226R;Y19 4R+T226K; Y194R+L227*; Y194R+L227R; Y194R+L227K; Y194R+V228*; Y194R+V228R; Y194R+V228K; Y194R+P229*; Y194R+P229R; Y194R+P229K; Y194R+V2 30*;Y194R+V230K;Y194R+V230R;Y194R+T231R;Y194R+T231K;Y194R+R232 RR;Y194R+N233R;Y194R+N233K;Y194R+D234*;Y194R+D234R;Y194R+D234K;Y194R+V236K;Y194R+V236R;Y194R+I238R;Y194R+I238K;Y194R+E239*;Y194R+E239R;Y194R+E239K;Y194R+A243R;Y194R+A243K;Y194R+T244R;Y194R+T244K;Y194R+G245R;Y194R+G245K;Y194R+N248R;Y194R+N248K;Y194R+Q249R;Y194R+Q249K;Y194R+N251R;Y194R+N251K;Y194K+T199R;Y194K+T199K;Y194K+N200R;Y194K+N200K;Y194K+I202R;Y194K+I202K;Y194K+S217R;Y194K+S217K;Y194K+P218R;Y194K+P218K;Y194K+Y220R;Y194K+Y220K;Y194K+W221R;Y194K+W221K;Y194K+S224*;Y194K+S224R;Y194K+S224K;Y194K+S225*;Y194K+S225R;Y194K+S225K;Y194K+T226*;Y194K+T226R;Y194K+T226K;Y194K+L227*;Y194K+L227R;Y194K+L227K;Y194K+V228*;Y194K+V228R;Y194K+V228K;Y194K+P229*;Y194K+P229R;Y194K+P229K;Y194K+V230*;Y194K+V230K;Y194K+V230R;Y194K+T231R;Y194K+T231K;Y194K+R232RR;Y194K+N233R;Y194K+N233K;Y194K+D234*;Y194K+D234R;Y194K+D234K;Y194K+V236K;Y194K+V236R;Y194K+I238R;Y194K+I238K;Y194K+E239*;Y194K+E239R;Y194K+E239K;Y194K+A243R;Y194K+A243K;Y194K+T244R;Y194K+T244K;Y194K+G245R;Y194K+G245K;Y194K+N248R;Y194K+N248K;Y194K+Q249R;Y194K+Q249K;Y194K+N251R;Y194K+N251K;T199R+N200R;T199R+N200K;T199R+I202R;T199R+I202K;T199R+S217R;T199R+S217K;T199R+P218R;T199R+P218K;T199R+Y220R;T199R+Y220K;T199R+W221R;T199R+W221K;T199R+S224*;T199R+S224R;T199R+S224K;T199R+S225*;T199R+S225R;T199R+S225K;T199R+T226*;T199R+T226R;T199R+T226K;T199R+L227*;T199R+L227R;T199R+L227K;T199R+V228*;T199R+V228R;T199R+V228K;T199R+P229*;T199R+P229R;T199R+P229K;T199R+V230*;T199R+V230K;T199R+V230R;T199R+T231R;T199R+T231K;T199R+R232RR;T199R+N233R;T199R+N233K;T199R+D234*;T199R+D234R;T199R+D234K;T199R+V236K;T199R+V236R;T199R+I238R;T199R+I238K;T199R+E239*;T199R+E239R;T199R+E239K;T199R+A243R;T199R+A243K;T199R+T244R;T199R+T244K;T199R+G245R;T199R+G245K;T199R+N248R;T199R+N248K;T199R+Q249R;T199R+Q249K;T199R+N251R;T199R+N251K;T199K+N200R;T199K+N200K;T199K+I202R;T199K+I202K;T199K+S217R;T199K+S217K;T199K+P218R;T199K+P218K;T199K+Y220R;T199K+Y220K;T199K+W221R;T199K+W221K;T199K+S224*;T199K+S224R;T199K+S224K;T199K+S225*;T199K+S225R;T199K+S225K;T199K+T226*;T199K+T226R;T199K+T226K;T199K+L227*;T199K+L227R;T199K+L227K;T199K+V228*;T199K+V228R; T199K+V228K; T199K+P229*; T199K+P229R; T199K+P229K; T199K+V230*; T199K+V230K; T199K+V230R; T199K+T231R; T199K+T231K; T199 K+R232RR; T199K+N233R; T199K+N233K; T199K+D234*; T199K+D234R; T199K+D234K; T199K+V236K; T199K+V236R; T199K+I238R; T199K+I238K; T199K+E2 39*;T199K+E239R;T199K+E239K;T199K+A243R;T199K+A243K;T199K+T244 R;T199K+T244K;T199K+G245R;T199K+G245K;T199K+N248R;T199K+N248K;T 199K+Q249R;T199K+Q249K;T199K+N251R;T199K+N251K;N200R+I202R;N20 0R+I202K;N200R+S217R;N200R+S217K;N200R+P218R;N200R+P218K;N200R+ Y220R;N200R+Y220K;N200R+W221R;N200R+W221K;N200R+S224*;N200R+S2 24R;N200R+S224K;N200R+S225*;N200R+S225R;N200R+S225K;N200R+T226 *;N200R+T226R;N200R+T226K;N200R+L227*;N200R+L227R;N200R+L227K; N200R+V228*;N200R+V228R;N200R+V228K;N200R+P229*;N200R+P229R;N20 0R+P229K;N200R+V230*;N200R+V230K;N200R+V230R;N200R+T231R;N200R +T231K;N200R+R232RR;N200R+N233R;N200R+N233K;N200R+D234*;N200R+D 234R;N200R+D234K;N200R+V236K;N200R+V236R;N200R+I238R;N200R+I23 8K;N200R+E239*;N200R+E239R;N200R+E239K;N200R+A243R;N200R+A243K;N200R+T244R;N200R+T244K;N200R+G245R;N200R+G245K;N200R+N248R;N200R+N248K;N200R+Q249R;N200R+Q249K;N200R+N251R;N200R+N251K;N200K+I202R;N200K+I202K;N200K+S217R;N200K+S217K;N200K+P218R;N200K+P218K;N200K+Y220R;N200K+Y220K;N200K+W221R;N200K+W221K;N200K+S224*;N200K+S224R;N200K+S224K;N200K+S225*;N200K+S225R;N200K+S225K;N200K+T226*;N200K+T226R;N200K+T226K;N200K+L227*;N200K+L227R;N200K+L227K;N200K+V228*;N200K+V228R;N200K+V228K;N200K+P229*;N200K+P229R;N200K+P229K;N200K+V230*;N200K+V230K;N200K+V230R;N200K+T231R;N200K+T231K;N200K+R232RR;N200K+N233R;N200K+N233K;N200K+D234*;N200K+D234R;N200K+D234K;N200K+V236K;N200K+V236R;N200K+I238R;N200K+I238K;N200K+E239*;N200K+E239R;N200K+E239K;N200K+A243R;N200K+A243K;N200K+T244R;N200K+T244K;N200K+G245R;N200K+G245K;N200K+N248R;N200K+N248K;N200K+Q249R;N200K+Q249K;N200K+N251R;N200K+N251K;I202R+S217R;I202R+S217K;I202R+P218R;I202R+P218K;I202R+Y220R;I202R+Y220K;I202R+W221R;I202R+W221K;I202R+S224*;I202R+S224R;I202R+S224K;I202R+S225*;I202R+S225R;I202R+S225K;I202R+T226*;I202R+T226R; I202R+T226K; I202R+L227*; I202R+L227R; I202R+L227K; I202R+V228*; I202R+V228R; I202R+V228K; I202R+P229*; I202R+P229R; I202 R+P229K; I202R+V230*; I202R+V230K; I202R+V230R; I202R+T231R; I202R+T231K; I202R+R232RR; I202R+N233R; I202R+N233K; I202R+D234*; I202R+D2 34R;I202R+D234K;I202R+V236K;I202R+V236R;I202R+I238R;I202R+I238 K;I202R+E239*;I202R+E239R;I202R+E239K;I202R+A243R;I202R+A243K;I 202R+T244R;I202R+T244K;I202R+G245R;I202R+G245K;I202R+N248R;I20 2R+N248K;I202R+Q249R;I202R+Q249K;I202R+N251R;I202R+N251K;I202K+ S217R;I202K+S217K;I202K+P218R;I202K+P218K;I202K+Y220R;I202K+Y2 20K;I202K+W221R;I202K+W221K;I202K+S224*;I202K+S224R;I202K+S224 K;I202K+S225*;I202K+S225R;I202K+S225K;I202K+T226*;I202K+T226R; I202K+T226K;I202K+L227*;I202K+L227R;I202K+L227K;I202K+V228*;I20 2K+V228R;I202K+V228K;I202K+P229*;I202K+P229R;I202K+P229K;I202K +V230*;I202K+V230K;I202K+V230R;I202K+T231R;I202K+T231K;I202K+R2 32RR; I202K+N233R; I202K+N233K; I202K+D234*; I202K+D234R; I202K+D234K; I202K+V236K; I202K+V236R; I202K+I238R; I202K+I238K; I202K+E239*;I202K+E239R;I202K+E239K;I202K+A243R;I202K+A243K;I202K+T244R;I2 02K+T244K;I202K+G245R;I202K+G245K;I202K+N248R;I202K+N248K;I202 K+Q249R;I202K+Q249K;I202K+N251R;I202K+N251K;S217R+P218R;S217R+ P218K;S217R+Y220R;S217R+Y220K;S217R+W221R;S217R+W221K;S217R+S22 4*; S217R+S224R; S217R+S224K; S217R+S225*; S217R+S225R; S217R+S225K; S217R+T226*; S217R+T226R; S217R+T226K; S217R+L227*; S217R+L227R; S 217R+L227K; S217R+V228*; S217R+V228R; S217R+V228K; S217R+P229*; S217R+P229R; S217R+P229K; S217R+V230*; S217R+V230K; S217R+V230R; S217R+ T231R; S217R+T231K; S217R+R232RR; S217R+N233R; S217R+N233K; S217R+D234*; S217R+D234R; S217R+D234K; S217R+V236K; S217R+V236R; S217R+I23 8R; S217R+I238K; S217R+E239*; S217R+E239R; S217R+E239K; S217R+A243R; S217R+A243K; S217R+T244R; S217R+T244K; S217R+G245R; S217R+G245K; S2 17R+N248R; S217R+N248K; S217R+Q249R; S217R+Q249K; S217R+N251R; S217R+N251K; S217K+P218R; S217K+P218K; S217K+Y220R; S217K+Y220K; S217K+W 221R; S217K+W221K; S217K+S224*; S217K+S224R; S217K+S224K; S217K+S225*; S217K+S225R; S217K+S225K; S217K+T226*; S217K+T226R; S217K+T226K;217K+L227*;S217K+L227R;S217K+L227K;S217K+V228*;S217K+V228R;S217K+V228K;S217K+P229*;S217K+P229R;S217K+P229K;S217K+V230*;S217K+V230K;S217K+V230R;S217K+T231R;S217K+T231K;S217K+R232RR;S217K+N233R;S217K+N233K;S217K+D234*;S217K+D234R;S217K+D234K;S217K+V236K;S217K+V236R;S217K+I238R;S217K+I238K;S217K+E239*;S217K+E239R;S217K+E239K;S217K+A243R;S217K+A243K;S217K+T244R;S217K+T244K;S217K+G245R;S217K+G245K;S217K+N248R;S217K+N248K;S217K+Q249R;S217K+Q249K;S217K+N251R;S217K+N251K;P218R+Y220R;P218R+Y220K;P218R+W221R;P218R+W221K;P218R+S224*;P218R+S224R;P218R+S224K;P218R+S225*;P218R+S225R;P218R+S225K;P218R+T226*;P218R+T226R;P218R+T226K;P218R+L227*;P218R+L227R;P218R+L227K;P218R+V228*;P218R+V228R;P218R+V228K;P218R+P229*;P218R+P229R;P218R+P229K;P218R+V230*;P218R+V230K;P218R+V230R;P218R+T231R;P218R+T231K;P218R+R232RR;P218R+N233R;P218R+N233K;P218R+D234*;P218R+D234R;P218R+D234K;P218R+V236K;P218R+V236R;P218R+I238R;P218R+I238K;P218R+E239*;P218R+E239R;P218R+E239K;P218R+A243R;P218R+A243K;P218R+T244R;P218R+T244K;P218R+G245R;P218R+G245K;P218R+N248R;P218R+N248K;P218R+Q249R;P218R+Q249K;P218R+N251R;P218R+N251K;P218K+Y220R;P218K+Y220K;P218K+W221R;P218K+W221K;P218K+S224*;P218K+S224R;P218K+S224K;P218K+S225*;P218K+S225R;P218K+S225K;P218K+T226*;P218K+T226R;P218K+T226K;P218K+L227*;P218K+L227R;P218K+L227K;P218K+V228*;P218K+V228R;P218K+V228K;P218K+P229*;P218K+P229R;P218K+P229K;P218K+V230*;P218K+V230K;P218K+V230R;P218K+T231R;P218K+T231K;P218K+R232RR;P218K+N233R;P218K+N233K;P218K+D234*;P218K+D234R;P218K+D234K;P218K+V236K;P218K+V236R;P218K+I238R;P218K+I238K;P218K+E239*;P218K+E239R;P218K+E239K;P218K+A243R;P218K+A243K;P218K+T244R;P218K+T244K;P218K+G245R;P218K+G245K;P218K+N248R;P218K+N248K;P218K+Q249R;P218K+Q249K;P218K+N251R;P218K+N251K;Y220R+W221R;Y220R+W221K;Y220R+S224*;Y220R+S224R;Y220R+S224K;Y220R+S225*;Y220R+S225R;Y220R+S225K;Y220R+T226*;Y220R+T226R;Y220R+T226K;Y220R+L227*;Y220R+L227R;Y220R+L227K;Y220R+V228*;Y220R+V228R;Y220R+V228K;Y220R+P229*;Y220R+P229R;Y220R+P229K;Y220R+V230*;Y220R+V230K;Y220R+V230R;Y220R+T231R;Y220R+T231K;Y220R+R232RR;Y220R+N233R;Y220R+N233K;Y220R+D234*;Y220R+D234R;Y220R+D234K;Y220R+V236K;Y220R+V236R;Y220R+I238R;Y220R+I238K;Y220R+E239*;Y220R+E239R;Y220R+E239K;Y220R+A243R;Y220R+A243K;Y220R+T244R;Y220R+T244K;Y220R+G245R;Y220R+G245K;Y220R+N248R;Y220R+N248K;Y220R+Q249R;Y220R+Q249K;Y220R+N251R;Y220R+N251K;Y220K+W221R;Y220K+W221K;Y220K+S224*;Y220K+S224R;Y220K+S224K;Y220K+S225*;Y220K+S225R;Y220K+S225K;Y220K+T226*;Y220K+T226R;Y220K+T226K;Y220K+L227*;Y220K+L227R;Y220K+L227K;Y220K+V228*;Y220K+V228R;Y220K+V228K;Y220K+P229*;Y220K+P229R;Y220K+P229K;Y220K+V230*;Y220K+V230K;Y220K+V230R;Y220K+T231R;Y220K+T231K;Y220K+R232RR;Y220K+N233R;Y220K+N233K;Y220K+D234*;Y220K+D234R;Y220K+D234K;Y220K+V236K;Y220K+V236R;Y220K+I238R;Y220K+I238K;Y220K+E239*;Y220K+E239R;Y220K+E239K;Y220K+A243R;Y220K+A243K;Y220K+T244R;Y220K+T244K;Y220K+G245R;Y220K+G245K;Y220K+N248R;Y220K+N248K;Y220K+Q249R;Y220K+Q249K;Y220K+N251R;Y220K+N251K;W221R+S224*;W221R+S224R;W221R+S224K;W221R+S225*;W221R+S225R;W221R+S225K;W221R+T226*; W221R+T226R; W221R+T226K; W221R+L227*; W221R+L227R; W221R+L227K; W221R+V228*; W221R+V228R; W221R+V228K; W221R+P229*; W221 R+P229R; W221R+P229K; W221R+V230*; W221R+V230K; W221R+V230R; W221R+T231R; W221R+T231K; W221R+R232RR; W221R+N233R; W221R+N233K; W221R+D2 34*;W221R+D234R;W221R+D234K;W221R+V236K;W221R+V236R;W221R+I238 R;W221R+I238K;W221R+E239*;W221R+E239R;W221R+E239K;W221R+A243R;W 221R+A243K;W221R+T244R;W221R+T244K;W221R+G245R;W221R+G245K;W22 1R+N248R;W221R+N248K;W221R+Q249R;W221R+Q249K;W221R+N251R;W221R+ N251K; W221K+S224*; W221K+S224R; W221K+S224K; W221K+S225*; W221K+S225R; W221K+S225K; W221K+T226*; W221K+T226R; W221K+T226K; W221K+L227 *; W221K+L227R; W221K+L227K; W221K+V228*; W221K+V228R; W221K+V228K; W221K+P229*; W221K+P229R; W221K+P229K; W221K+V230*; W221K+V230K; W22 1K+V230R;W221K+T231R;W221K+T231K;W221K+R232RR;W221K+N233R;W221 K+N233K;W221K+D234*;W221K+D234R;W221K+D234K;W221K+V236K;W221K+V 236R; W221K+I238R; W221K+I238K; W221K+E239*; W221K+E239R; W221K+E239K; W221K+A243R; W221K+A243K; W221K+T244R; W221K+T244K; W221K+G245R;W221K+G245K;W221K+N248R;W221K+N248K;W221K+Q249R;W221K+Q249K;W2 21K+N251R;W221K+N251K;S224*+S225*;S224*+S225R;S224*+S225K;S224 *+T226*; S224*+T226R; S224*+T226K; S224*+L227*; S224*+L227R; S224*+L227K; S224*+V228*; S224*+V228R; S224*+V228K; S224*+P229*; S224*+P22 9R;S224*+P229K;S224*+V230*;S224*+V230K;S224*+V230R;S224*+T231R ;S224*+T231K;S224*+R232RR;S224*+N233R;S224*+N233K;S224*+D234*;S 224*+D234R;S224*+D234K;S224*+V236K;S224*+V236R;S224*+I238R;S22 4*+I238K;S224*+E239*;S224*+E239R;S224*+E239K;S224*+A243R;S224*+ A243K;S224*+T244R;S224*+T244K;S224*+G245R;S224*+G245K;S224*+N2 48R;S224*+N248K;S224*+Q249R;S224*+Q249K;S224*+N251R;S224*+N251 K;S224R+S225*;S224R+S225R;S224R+S225K;S224R+T226*;S224R+T226R; S224R+T226K;S224R+L227*;S224R+L227R;S224R+L227K;S224R+V228*;S22 4R+V228R;S224R+V228K;S224R+P229*;S224R+P229R;S224R+P229K;S224R +V230*;S224R+V230K;S224R+V230R;S224R+T231R;S224R+T231K;S224R+R2 32RR; S224R+N233R; S224R+N233K; S224R+D234*; S224R+D234R; S224R+D234K; S224R+V236K; S224R+V236R; S224R+I238R; S224R+I238K; S224R+E239*;S224R+E239R;S224R+E239K;S224R+A243R;S224R+A243K;S224R+T244R;S224R+T244K;S224R+G245R;S224R+G245K;S224R+N248R;S224R+N248K;S224R+Q249R;S224R+Q249K;S224R+N251R;S224R+N251K;S224K+S225*;S224K+S225R;S224K+S225K;S224K+T226*;S224K+T226R;S224K+T226K;S224K+L227*;S224K+L227R;S224K+L227K′;S224K+V228*;S224K+V228R;S224K+V228K;S224K+P229*;S224K+P229R;S224K+P229K;S224K+V230*;S224K+V230K;S224K+V230R;S224K+T231R;S224K+T231K;S224K+R232RR;S224K+N233R;S224K+N233K;S224K+D234*;S224K+D234R;S224K+D234K;S224K+V236K;S224K+V236R;S224K+I238R;S224K+I238K;S224K+E239*;S224K+E239R;S224K+E239K;S224K+A243R;S224K+A243K;S224K+T244R;S224K+T244K;S224K+G245R;S224K+G245K;S224K+N248R;S224K+N248K;S224K+Q249R;S224K+Q249K;S224K+N251R;S224K+N251K;S225*+T226*;S225*+T226R;S225*+T226K;S225*+L227*;S225*+L227R;S225*+L227K;S225*+V228*;S225*+V228R;S225*+V228K;S225*+P229*;S225*+P229R;S225*+P229K;S225*+V230*;S225*+V230K;S225*+V230R;S225*+T231R;S225*+T231K;S225*+R232RR;S225*+N233R;S225*+N233K;S225*+D234*;S225*+D234R;S225*+D234K;S225*+V236K;S225*+V236R;S225*+I238R;S225*+I238K;S225*+E239*;S225*+E239R;S225*+E239K;S225*+A243R;S225*+A243K;S225*+T244R;S225*+T244K;S225*+G245R;S225*+G245K;S225*+N248R;S225*+N248K;S225*+Q249R;S225*+Q249K;S225*+N251R;S225*+N251K;S225R+T226*;S225R+T226R;S225R+T226K;S225R+L227*;S225R+L227R;S225R+L227K;S225R+V228*;S225R+V228R;S225R+V228K;S225R+P229*;S225R+P229R;S225R+P229K;S225R+V230*;S225R+V230K;S225R+V230R;S225R+T231R;S225R+T231K;S225R+R232RR;S225R+N233R;S225R+N233K;S225R+D234*;S225R+D234R;S225R+D234K;S225R+V236K;S225R+V236R;S225R+I238R;S225R+I238K;S225R+E239*;S225R+E239R;S225R+E239K;S225R+A243R;S225R+A243K;S225R+T244R;S225R+T244K;S225R+G245R;S225R+G245K;S225R+N248R;S225R+N248K;S225R+Q249R;S225R+Q249K;S225R+N251R;S225R+N251K;S225K+T226*;S225K+T226R;S225K+T226K;S225K+L227*;S225K+L227R;S225K+L227K;S225K+V228*;S225K+V228R;S225K+V228K;S225K+P229*;S225K+P229R;S225K+P229K;S225K+V230*;S225K+V230K;S225K+V230R;S225K+T231R;S225K+T231K;S225K+R232RR;S225K+N233R;S225K+N233K;S225K+D234*;S225K+D234R;S225K+D234K;S225K+V236K; S225K+V236R; S225K+I238R; S225K+I238K; S225K+E239*; S225K+E239R; S225K+E239K; S225K+A243R; S225K+A243K; S225K+T244R; S225 K+T244K;S225K+G245R;S225K+G245K;S225K+N248R;S225K+N248K;S225K+ Q249R;S225K+Q249K;S225K+N251R;S225K+N251K;T226*+L227*;T226*+L22 7R; T226*+L227K; T226*+V228*; T226*+V228R; T226*+V228K; T226*+P229*; T226*+P229R; T226*+P229K; T226*+V230*; T226*+V230K; T226*+V230R; T2 26*+T231R; T226*+T231K; T226*+R232RR; T226*+N233R; T226*+N233K; T226*+D234*; T226*+D234R; T226*+D234K; T226*+V236K; T226*+V236R; T226*+ I238R; T226*+I238K; T226*+E239*; T226*+E239R; T226*+E239K; T226*+A243R; T226*+A243K; T226*+T244R; T226*+T244K; T226*+G245R; T226*+G245 K;T226*+N248R;T226*+N248K;T226*+Q249R;T226*+Q249K;T226*+N251R; T226*+N251K;T226R+L227*;T226R+L227R;T226R+L227K;T226R+V228*;T22 6R+V228R; T226R+V228K; T226R+P229*; T226R+P229R; T226R+P229K; T226R+V230*; T226R+V230K; T226R+V230R; T226R+T231R; T226R+T231K; T226R+R2 32RR; T226R+N233R; T226R+N233K; T226R+D234*; T226R+D234R; T226R+D234K; T226R+V236K; T226R+V236R; T226R+I238R; T226R+I238K; T226R+E239*;T226R+E239R; T226R+E239K; T226R+A243R; T226R+A243K; T226R+T244R; T226R+T244K; T226R+G245R; T226R+G245K; T226R+N248R; T226R+N248K; T226R+Q249R; T226R+Q249K; T226R+N251R; T22 6R+N251K; T226K+L227*; T226K+L227R; T226K+L227K; T226K+V228*; T226K+V228R; T226K+V228K; T226K+P229*; T226K+P229R; T226K+P229K; T226K+V230*; T226K+V230K; T226K+V230R; T226K+ T231R; T226K+T231K; T226K+R232RR; T226K+N233R; T226K+N233K; T226K+D234*; T226K+D234R; T226K+D234K; T226K+V236K; T226K+V236R; T226K+I238R; T226K+I238K; T226K+E239*; T226K+E2 39R; T226K+E239K; T226K+A243R; T226K+A243K; T226K+T244R; T226K+T244K; T226K+G245R; T226K+G245K; T226K+N248R; T226K+N248K; T226K+Q249R; T226K+Q249K; T226K+N251R; T226K+N251K L227*+V228*; L227*+V228R; L227*+V228K; L227*+P229*; L227*+P229R; L227*+P229K; L227*+V230*; L227*+V230K; L227*+V230R; L227*+T231R; L227*+T231K; L227*+R232RR; L227*+N233R; L227*+N233K; L227*+D234* L227*+D234R; L227*+D234K; L227*+V236K; L227*+V236R; L227*+I238R; L227*+I238K; L227*+E239*; L227*+E239R; L227*+E239K; L227*+A243R; L227*+A243K; L227*+T244R; L227*+T244K; L227*+G245R; L227*+G245K;L227*+N248R; L227*+N248K; L227*+Q249R; L227*+Q249K; L227*+N251R; L227*+N251K; L227R+V228*; L227R+V228R; L227R+V228K; L227R+P229*; L227 R+P229R; L227R+P229K; L227R+V230*; L227R+V230K; L227R+V230R; L227R+T231R; L227R+T231K; L227R+R232RR; L227R+N233R; L227R+N233K; L227R+D2 34*; L227R+D234R; L227R+D234K; L227R+V236K; L227R+V236R; L227R+I238R; L227R+I238K; L227R+E239*; L227R+E239R; L227R+E239K; L227R+A243R; L 227R+A243K; L227R+T244R; L227R+T244K; L227R+G245R; L227R+G245K; L227R+N248R; L227R+N248K; L227R+Q249R; L227R+Q249K; L227R+N251R; L227R+ N251K; L227K+V228*; L227K+V228R; L227K+V228K; L227K+P229*; L227K+P229R; L227K+P229K; L227K+V230*; L227K+V230K; L227K+V230R; L227K+T231 R; L227K+T231K; L227K+R232RR; L227K+N233R; L227K+N233K; L227K+D234*; L227K+D234R; L227K+D234K; L227K+V236K; L227K+V236R; L227K+I238R; L2 27K+I238K; L227K+E239*; L227K+E239R; L227K+E239K; L227K+A243R; L227K+A243K; L227K+T244R; L227K+T244K; L227K+G245R; L227K+G245K; L227K+N 248R; L227K+N248K; L227K+Q249R; L227K+Q249K; L227K+N251R; L227K+N251K; V228*+P229*; V228*+P229R; V228*+P229K; V228*+V230*; V228*+V230K;V228*+V230R;V228*+T231R;V228*+T231K;V228*+R232RR;V228*+N233R;V228*+N233K;V228*+D234*;V228*+D234R;V228*+D234K;V228*+V236K;V228*+V236R;V228*+I238R;V228*+I238K;V228*+E239*;V228*+E239R;V228*+E239K;V228*+A243R;V228*+A243K;V228*+T244R;V228*+T244K;V228*+G245R;V228*+G245K;V228*+N248R;V228*+N248K;V228*+Q249R;V228*+Q249K;V228*+N251R;V228*+N251K;V228R+P229*;V228R+P229R;V228R+P229K;V228R+V230*;V228R+V230K;V228R+V230R;V228R+T231R;V228R+T231K;V228R+R232RR;V228R+N233R;V228R+N233K;V228R+D234*;V228R+D234R;V228R+D234K;V228R+V236K;V228R+V236R;V228R+I238R;V228R+I238K;V228R+E239*;V228R+E239R;V228R+E239K;V228R+A243R;V228R+A243K;V228R+T244R;V228R+T244K;V228R+G245R;V228R+G245K;V228R+N248R;V228R+N248K;V228R+Q249R;V228R+Q249K;V228R+N251R;V228R+N251K;V228K+P229*;V228K+P229R;V228K+P229K;V228K+V230*;V228K+V230K;V228K+V230R;V228K+T231R;V228K+T231K;V228K+R232RR;V228K+N233R;V228K+N233K;V228K+D234*;V228K+D234R;V228K+D234K;V228K+V236K;V228K+V236R;V228K+I238R;V228K+I238K;V228K+E239*;V228K+E239R;V228K+E239K;V228K+A243R;V228K+A243K;V228K+T244R;V228K+T244K;V228K+G245R;V228K+G245K;V228K+N248R;V228K+N248K;V228K+Q249R;V228K+Q249K;V228K+N251R;V228K+N251K;P229*+V230*;P229*+V230K;P229*+V230R;P229*+T231R;P229*+T231K;P229*+R232RR;P229*+N233R;P229*+N233K;P229*+D234*;P229*+D234R;P229*+D234K;P229*+V236K;P229*+V236R;P229*+I238R;P229*+I238K;P229*+E239*;P229*+E239R;P229*+E239K;P229*+A243R;P229*+A243K;P229*+T244R;P229*+T244K;P229*+G245R;P229*+G245K;P229*+N248R;P229*+N248K;P229*+Q249R;P229*+Q249K;P229*+N251R;P229*+N251K;P229R+V230*;P229R+V230K;P229R+V230R;P229R+T231R;P229R+T231K;P229R+R232RR;P229R+N233R;P229R+N233K;P229R+D234*;P229R+D234R;P229R+D234K;P229R+V236K;P229R+V236R;P229R+I238R;P229R+I238K;P229R+E239*;P229R+E239R;P229R+E239K;P229R+A243R;P229R+A243K;P229R+T244R;P229R+T244K;P229R+G245R;P229R+G245K;P229R+N248R;P229R+N248K;P229R+Q249R;P229R+Q249K;P229R+N251R;P229R+N251K;P229K+V230*;P229K+V230K;P229K+V230R;P229K+T231R;P229K+T231K;P229K+R232RR;P229K+N233R;P229K+N233K;P229K+D234*;P229K+D234R;P229K+D234K;P229K+V236K;P229K+V236R;P229K+I238R;P229K+I238K;P229K+E239*;P229K+E239R;P229K+E239K;P229K+A243R;P229K+A243K;P229K+T244R;P229K+T244K;P229K+G245R;P229K+G245K;P229K+N248R;P229K+N248K;P229K+Q249R;P229K+Q249K;P229K+N251R;P229K+N251K;V230*+T231R;V230*+T231K;V230*+R232RR;V230*+N233R;V230*+N233K;V230*+D234*;V230*+D234R;V230*+D234K;V230*+V236K;V230*+V236R;V230*+I238R;V230*+I238K;V230*+E239*;V230*+E239R;V230*+E239K;V230*+A243R;V230*+A243K;V230*+T244R;V230*+T244K;V230*+G245R;V230*+G245K;V230*+N248R;V230*+N248K;V230*+Q249R;V230*+Q249K;V230*+N251R;V230*+N251K;V230K+T231R;V230K+T231K;V230K+R232RR;V230K+N233R;V230K+N233K;V230K+D234*;V230K+D234R;V230K+D234K;V230K+V236K;V230K+V236R;V230K+I238R;V230K+I238K;V230K+E239*;V230K+E239R;V230K+E239K;V230K+A243R;V230K+A243K;V230K+T244R;V230K+T244K;V230K+G245R;V230K+G245K;V230K+N248R;V230K+N248K;V230K+Q249R;V230K+Q249K;V230K+N251R;V230K+N251K;V230R+T231R;V230R+T231K;V230R+R232RR;V230R+N233R;V230R+N233K;V230R+D234*;V230R+D234R;V230R+D234K;V230R+V236K;V230R+V236R;V230R+I238R;V230R+I238K;V230R+E239*;V230R+E239R;V230R+E239K;V230R+A243R;V 230R+A243K;V230R+T244R;V230R+T244K;V230R+G245R;V230R+G245K;V23 0R+N248R;V230R+N248K;V230R+Q249R;V230R+Q249K;V230R+N251R;V230R +N251K;T231R+R232RR;T231R+N233R;T231R+N233K;T231R+D234*;T231R+ D234R; T231R+D234K; T231R+V236K; T231R+V236R; T231R+I238R; T231R+I238K; T231R+E239*; T231R+E239R; T231R+E239K; T231R+A243R; T231R+A24 3K;T231R+T244R;T231R+T244K;T231R+G245R;T231R+G245K;T231R+N248R ;T231R+N248K;T231R+Q249R;T231R+Q249K;T231R+N251R;T231R+N251K;T 231K+R232RR; T231K+N233R; T231K+N233K; T231K+D234*; T231K+D234R; T231K+D234K; T231K+V236K; T231K+V236R; T231K+I238R; T231K+I238K; T23 1K+E239*; T231K+E239R; T231K+E239K; T231K+A243R; T231K+A243K; T231K+T244R; T231K+T244K; T231K+G245R; T231K+G245K; T231K+N248R; T231K+N 248K; T231K+Q249R; T231K+Q249K; T231K+N251R; T231K+N251K; R232RR+N233R; R232RR+N233K; R232RR+D234*; R232RR+D234R; R232RR+D234K; R232RR +V236K;R232RR+V236R;R232RR+I238R;R232RR+I238K;R232RR+E239*;R23 2RR+E239R;R232RR+E239K;R232RR+A243R;R232RR+A243K;R232RR+T244R;R232RR+T244K;R232RR+G245R;R232RR+G245K;R232RR+N248R;R232RR+N24 8K;R232RR+Q249R;R232RR+Q249K;R232RR+N251R;R232RR+N251K;N233R+D 234*; N233R+D234R; N233R+D234K; N233R+V236K; N233R+V236R; 33R+I238R; N233R+I238K; N233R+E239*; N233R+E239R; N233R+E239K; N233R+A243R; N233R+A243K; N233R+T244R; N233R+T244K; N233R+G245R; N233R+G245K; N233R+N248R; N233R+N248K; N233R+Q249R; N233R+Q249K; N233R+N251R; N233 R+N251K; N233K+D234*; N233K+D234R; N233K+D234K; N233K+V236K; 233K+V236R; N233K+I238R; N233K+I238K; N233K+E239*; N233K+E239R; N233K+E2 39K; N233K+A243R; N233K+A243K; N233K+T244R; N233K+T244K; N233K+G245R; N233K+G245K; N233K+N248R; N233K+N248K; N233K+Q249R; N233K+Q249K; N233K+N251R; N233K+N251K; D234*+V236K; D234*+V236R; D234*+I238R; D234*+I238K; D234*+E239*; D234*+E239R; D234*+E239K; D234*+A243R; D234 *+A243K;D234*+T244R;D234*+T244K;D234*+G245R;D234*+G245K;D234*+ N248R;D234*+N248K;D234*+Q249R;D234*+Q249K;D234*+N251R;D234*+N2 51K;D234R+D234K;D234R+V236K;D234R+V236R;D234R+I238R;D234R+I238 K;D234R+E239*;D234R+E239R;D234R+E239K;D234R+A243R;D234R+A243K;D234R+T244R;D234R+T244K;D234R+G245R;D234R+G245K;D234R+N248R;D234R+N248K;D234R+Q249R;D234R+Q249K;D234R+N251R;D234R+N251K;D234K+V236K;D234K+V236R;;D234K+I238R;D234K+I238KD234K+E239*;D234K+E239R;D234K+E239K;D234K+A243R;D234K+A243K;D234K+T244R;D234K+T244K;D234K+G245R;D234K+G245K;D234K+N248R;D234K+N248K;D234K+Q249R;D234K+Q249K;D234K+N251R;D234K+N251K;V236K+I238R;V236K+I238K;V236K+E239*;V236K+E239R;V236K+E239K;V236K+A243R;V236K+A243K;V236K+T244R;V236K+T244K;V236K+G245R;V236K+G245K;V236K+N248R;V236K+N248K;V236K+Q249R;V236K+Q249K;V236K+N251R;V236K+N251K;V236R+I238R;V236R+I238K;V236R+E239*;V236R+E239R;V236R+E239K;V236R+A243R;V236R+A243K;V236R+T244R;V236R+T244K;V236R+G245R;V236R+G245K;V236R+N248R;V236R+N248K;V236R+Q249R;V236R+Q249K;V236R+N251R;V236R+N251K;I238R+E239*;I238R+E239R;I238R+E239K;I238R+A243R;I238R+A243K;I238R+T244R;I238R+T244K;I238R+G245R;I238R+G245K;I238R+N248R;I238R+N248K;I238R+Q249R;I238R+Q249K;I238R+N251R;I238R+N251K;I238K+E239*;I238K+E239R;I238K+E239K;I238K+A243R;I238K+A243K;I238K+T244R;I238K+T244K;I238K+G245R;I238K+G245K;I238K+N248R;I238K+N248K;I238K+Q249R;I238K+Q249K;I238K+N251R;I238K+N251K;E239*+A243R;E239*+A243K;E239*+T244R;E239*+T244K;E239*+G245R;E239*+G245K;E239*+N248R;E239*+N248K;E239*+Q249R;E239*+Q249K;E239*+N251R;E239*+N251K;E239R+A243R;E239R+A243K;E239R+T244R;E239R+T244K;E239R+G245R;E239R+G245K;E239R+N248R;E239R+N248K;E239R+Q249R;E239R+Q249K;E239R+N251R;E239R+N251K;E239K+A243R;E239K+A243K;E239K+T244R;E239K+T244K;E239K+G245R;E239K+G245K;E239K+N248R;E239K+N248K;E239K+Q249R;E239K+Q249K;E239K+N251R;E239K+N251K;A243R+T244R;A243R+T244K;A243R+G245R;A243R+G245K;A243R+N248R;A243R+N248K;A243R+Q249R;A243R+Q249K;A243R+N251R;A243R+N251K;A243K+T244R;A243K+T244K;A243K+G245R;A243K+G245K;A243K+N248R;A243K+N248K;A243K+Q249R;A243K+Q249K;A243K+N251R;A243K+N251K;T244R+G245R;T244R+G245K;T244R+N248R;T244R+N248K;T244R+Q249R;T244R+Q249K;T244R+N251R;T244R+N251K;T244K+G245R;T244K+G245K;T244K+N248R;T244K+N248K;T244K+Q249R;T244K+Q249K;T244K+N251R;T244K+N251K;G245R+N248R;G245R+N248K; G245R+Q249R; G245R+Q249K; G245R+N251R; G245R+N251K; G245K+N24 8R; G245K+N248K; G245K+Q249R; G245K+Q249K; G245K+N251R; G245K+N251K; N248R+Q 249R; N248R+Q249K; N248R+N251R; N248R+N251K; N248K+Q249R; N248K+Q249K; N248K+N251R; N248K+N251K; Q249R+N251R; Q249R+N251K; Q249K+N251R; and Q249K+N251K.

[0425] In another embodiment, the variant comprises (a) E1ER, E1P, or E1Q, (b) V2*, V2K, or V2R, and (c) S3* or S3R, or is composed of the latter.

[0426] In another embodiment, the variant comprises (a) E1ER, E1P, or E1Q, (b) V2*, V2K, or V2R, + (c) Q4R or a combination thereof.

[0427] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, + (c) T226* or T226R, or composed thereof.

[0428] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, + (c) L227* or L227R, or composed thereof.

[0429] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, + (c) V228* or V228R, or composed thereof.

[0430] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, + (c) P229* or P229R, or composed thereof.

[0431] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, + (c) V230* or V230R, or a combination thereof.

[0432] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, + (c) L227* or L227R, or composed thereof.

[0433] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R+(c) V228* or V228R or composed thereof.

[0434] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, + (c) P229* or P229R, or composed thereof.

[0435] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, + (c) V230* or V230R, or a combination thereof.

[0436] In another embodiment, the variant comprises (a) S224* or S224R, (b) L227* or L227R, + (c) V228* or V228R, or composed thereof.

[0437] In another embodiment, the variant comprises (a) S224* or S224R, (b) L227* or L227R, + (c) P229* or P229R, or composed thereof.

[0438] In another embodiment, the variant comprises (a) S224* or S224R, (b) L227* or L227R, + (c) V230* or V230R, or a combination thereof.

[0439] In another embodiment, the variant comprises (a) S224* or S224R, (b) V228* or V228R, + (c) P229* or P229R, or is composed of therewith.

[0440] In another embodiment, the variant comprises (a) S224* or S224R, (b) V228* or V228R, + (c) V230* or V230R, or a combination thereof.

[0441] In another embodiment, the variant comprises (a) S224* or S224R, (b) P229* or P229R, + (c) V230* or V230R, or composed thereof.

[0442] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, + (b) L227* or L227R, or composed thereof.

[0443] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, + (c) V228* or V228R, or a combination thereof.

[0444] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, + (c) P229* or P229R, or is composed of the latter.

[0445] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, + (c) V230* or V230R, or a combination thereof.

[0446] In another embodiment, the variant comprises (a) G225* or G225R, (b) L227*+L227R, + (c) V228* or V228R, or a combination thereof.

[0447] In another embodiment, the variant comprises (a) G225* or G225R, (b) L227*+L227R, + (c) P229* or P229R, or a combination thereof.

[0448] In another embodiment, the variant comprises (a) G225* or G225R, (b) L227*+L227R, + (c) V230* or V230R, or a combination thereof.

[0449] In another embodiment, the variant comprises (a) G225* or G225R, (b) V228* or V228R, + (c) P229* or P229R, or a combination thereof.

[0450] In another embodiment, the variant comprises (a) G225* or G225R, (b) V228* or V228R, + (c) V230* or V230R, or a combination thereof.

[0451] In another embodiment, the variant comprises (a) G225* or G225R, (b) P229* or P229R, + (c) V230* or V230R, or a combination thereof.

[0452] In another embodiment, the variant comprises (a) T226* or T226R, (b) L227* or L227R, and (c) V228* or V228R or composed thereof.

[0453] In another embodiment, the variant comprises (a) T226* or T226R, (b) L227* or L227R, + (c) P229* or P229R, or is composed of therewith.

[0454] In another embodiment, the variant comprises (a) T226* or T226R, (b) L227* or L227R, + (c) V230* or V230R, or composed thereof.

[0455] In another embodiment, the variant comprises (a) T226* or T226R, (b) V228* or V228R, + (c) P229* or P229R, or is composed of therewith.

[0456] In another embodiment, the variant comprises (a) T226* or T226R, (b) V228* or V228R, + (c) V230* or V230R, or composed thereof.

[0457] In another embodiment, the variant comprises (a) T226* or T226R, (b) P229* or P229R, + (c) V230* or V230R, or is composed of the latter.

[0458] In another embodiment, the variant includes (a) L227* or L227R, (b) V228* or V228R, or a combination thereof at positions corresponding to positions 227, 228, and 229.

[0459] In another embodiment, the variant comprises (a) L227* or L227R, (b) V228* or V228R, + (c) V230* or V230R, or composed thereof.

[0460] In another embodiment, the variant comprises (a) L227* or L227R, (b) P229* or P229R, + (c) V230* or V230R, or is composed of the latter.

[0461] In another embodiment, the variant comprises (a) V228* or V228R, (b) P229* or P229R, + (c) V230* or V230R, or a combination thereof.

[0462] In another embodiment, the variant comprises (a) T231TR or T231R, (b) G239* or G239E, + (c) Q249R or composed thereof.

[0463] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, + (d) L227* or L227R, or composed thereof.

[0464] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, + (d) V228* or V228R, or composed thereof.

[0465] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, + (d) P229* or P229R, or composed thereof.

[0466] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, + (d) V230* or V230R, or composed thereof.

[0467] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) L227* or L227R, + (d) V228* or V228R, or composed thereof.

[0468] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) L227* or L227R, and (d) P229* or P229R or composed thereof.

[0469] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) L227* or L227R, + (d) V230* or V230R, or composed thereof.

[0470] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) V228* or V228R, + (d) P229* or P229R, or composed thereof.

[0471] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) V228* or V228R, + (d) V230* or V230R, or composed thereof.

[0472] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) P229* or P229R, + (d) V230* or V230R, or composed thereof.

[0473] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) L227* or L227R, + (d) V228* or V228R, or composed thereof.

[0474] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) L227* or L227R, and (d) P229* or P229R or composed thereof.

[0475] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) L227* or L227R, + (d) V230* or V230R, or composed thereof.

[0476] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) V228* or V228R, + (d) P229* or P229R, or composed thereof.

[0477] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) V228* or V228R, + (d) V230* or V230R, or composed thereof.

[0478] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) P229* or P229R, and (d) V230* or V230R or thereof.

[0479] In another embodiment, the variant comprises (a) S224* or S224R, (b) L227* or L227R, (c) V228* or V228R, + (d) P229* or P229R, or consists of therewith.

[0480] In another embodiment, the variant comprises (a) S224* or S224R, (b) L227* or L227R, (c) V228* or V228R, + (d) V230* or V230R, or composed thereof.

[0481] In another embodiment, the variant comprises (a) S224* or S224R, (b) L227* or L227R, (c) P229* or P229R, + (d) V230* or V230R, or composed thereof.

[0482] In another embodiment, the variant comprises (a) S224* or S224R, (B) V228* or V228R, (c) P229* or P229R, + (d) V230* or V230R, or composed thereof.

[0483] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) L227* or L227R, + (d) V228* or V228R, or composed thereof.

[0484] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) L227* or L227R, + (d) P229* or P229R, or composed thereof.

[0485] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) L227* or L227R, and (d) V230* or V230R or composed thereof.

[0486] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) V228* or V228R, + (d) P229* or P229R, or composed thereof.

[0487] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) V228* or V228R, + (d) V230* or V230R, or composed thereof.

[0488] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) P229* or P229R, + (d) V230* or V230R, or composed thereof.

[0489] In another embodiment, the variant comprises (a) G225* or G225R, (b) L227* or L227R, (c) V228* or V228R, + (d) P229* or P229R, or composed thereof.

[0490] In another embodiment, the variant comprises (a) G225* or G225R, (b) L227* or L227R, (c) V228* or V228R, + (d) V230* or V230R, or composed thereof.

[0491] In another embodiment, the variant comprises (a) G225* or G225R, (b) V228* or V228R, (c) P229* or P229R, + (d) V230* or V230R, or composed thereof.

[0492] In another embodiment, the variant comprises (a) T226* or T226R, (b) L227* or L227R, (c) V228* or V228R, + (d) P229* or P229R, or consists of therewith.

[0493] In another embodiment, the variant comprises (a) T226* or T226R, (b) L227* or L227R, (c) V228* or V228R, + (d) V230* or V230R, or composed thereof.

[0494] In another embodiment, the variant comprises (a) T226* or T226R, (b) V228* or V228R, (c) P229* or P229R, + (d) V230* or V230R, or composed thereof.

[0495] In another embodiment, the variant comprises (a) L227* or L227R, (b) V228* or V228R, (c) P229* or P229R, + (d) V230* or V230R, or composed thereof.

[0496] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, (d) L227* or L227R, + (e) V228* or V228R, or composed thereof.

[0497] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, (d) L227* or L227R, and (e) P229* or P229R or composed thereof.

[0498] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, (d) L227* or L227R, + (e) V228* or V228R, or composed thereof.

[0499] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) L227* or L227R, (d) V228* or V228R, + (e) P229* or P229R, or composed thereof.

[0500] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) L227* or L227R, (d) V228* or V228R, + (e) V230* or V230R, or composed thereof.

[0501] In another embodiment, the variant comprises (a) S224* or S224R, (b) L227* or L227R, (c) V228* or V228R, (d) P229* or P229R, + (e) V230* or V230R, or composed thereof.

[0502] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) L227* or L227R, (d) V228* or V228R, + (e) P229* or P229R, or composed thereof.

[0503] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) L227* or L227R, (d) V228* or V228R, + (e) V230* or V230R, or composed thereof.

[0504] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) V228* or V228R, (d) P229* or P229R, + (e) V230* or V230R, or a combination thereof.

[0505] In another embodiment, the variant comprises (a) G225* or G225R, (b) L227* or L227R, (c) V228* or V228R, (d) P229* or P229R, + (e) V230* or V230R, or a combination thereof.

[0506] In another embodiment, the variant comprises (a) T226* or T226R, (b) L227* or L227R, (c) V228* or V228R, (d) P229* or P229R, + (e) V230* or V230R, or composed thereof.

[0507] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, (d) L227* or L227R, (e) V228* or V228R, + (f) P229* or P229R, or composed thereof.

[0508] In another embodiment, the variant comprises (a) S224* or S224R, (b) G225* or G225R, (c) T226* or T226R, (d) L227* or L227R, (e) V228* or V228R, + (f) V230* or V230R, or composed thereof.

[0509] In another embodiment, the variant comprises (a) S224* or S224R, (b) T226* or T226R, (c) L227* or L227R, (d) V228* or V228R, (e) P229* or P229R, + (f) V230* or V230R, or a combination thereof.

[0510] In another embodiment, the variant comprises (a) G225* or G225R, (b) T226* or T226R, (c) L227* or L227R, (d) V228* or V228R, (e) P229* or P229R, + (f) V230* or V230R, or a combination thereof.

[0511] In a third aspect, the present invention relates to lipase variants comprising Arg substitution at two or more positions corresponding to positions 2, 3, 4, 5, 8, 11, 37, 38, 39, 43, 101, 105, 106, 178, 199, 200, 224, 225, 227, 228, 229, 231, 236, 239, 244 and 249 of SEQ ID NO:1, wherein these lipase variants have lipase activity.

[0512] Therefore, in one embodiment, the variant comprises two substitutions corresponding to substitutions selected from the group consisting of: E1R+V2R; E1R+V3R; E1R+Q4R; E1R+D5R; E1R+L6R; E1R+F7R; E1R+N8R; E1R+Q9R; E1R+N11R; E1R+L12R; E1R+Q15R; E1R+T37R; E1R+Q38R; E1R+N39R; E1R+A40R; E1R+P42R; E1R+E43R; E1R+E45R; E1R+N73R; E1R+D167R; E1R+T192R; E1R+L193R; E1R+Y194R; E1R+T199 R;E1R+N200R;E1R+I202R;E1R+S217R;E1R+P218R;E1R+Y220R;E1R+W221R; E1R+S224R; E1R+S225R; E1R+T226R; E1R+L227R; E1R+V228R; E1R+P229R; E1 R+V230R; E1R+T231R; E1R+N233R; E1R+D234R; E1R+V236R; E1R+I238R; E1R+ E239R; E1R+A243R; E1R+T244R; E1R+G245R; E1R+N248R; E1R+Q249R; E1R+N25 1R; V2R+V3R; V2R+Q4R; V2R+D5R; V2R+L6R; V2R+F7R; V2R+N8R; V2R+Q9R; V2R +N11R; V2R+L12R; V2R+Q15R; V2R+T37R; V2R+Q38R; V2R+N39R; V2R+A40R; V2 R+P42R; V2R+E43R; V2R+E45R; V2R+N73R; V2R+D167R; V2R+T192R; V2R+L193 R;V2R+Y194R;V2R+T199R;V2R+N200R;V2R+I202R;V2R+S217R;V2R+P218R;V 2R+Y220R; V2R+W221R; V2R+S224R; V2R+S225R; V2R+T226R; V2R+L227R; V2R +V228R; V2R+P229R; V2R+V230R; V2R+T231R; V2R+N233R; V2R+D234R; V2R+V 236R; V2R+I238R; V2R+E239R; V2R+A243R; V2R+T244R; V2R+G245R; V2R+N24 8R; V2R+Q249R; V2R+N251R; V3R+Q4R; V3R+D5R; V3R+L6R; V3R+F7R; V3R+N8R;V3R+Q9R;V3R+N11R;V3R+L12R;V3R+Q15R;V3R+T37R;V3R+Q38R;V3R+N39R;V3R+A40R;V3R+P42R;V3R+E43R;V3R+E45R;V3R+N73R;V3R+D167R;V3R+T192R;V3R+L193R;V3R+Y194R;V3R+T199R;V3R+N200R;V3R+I202R;V3R+S217R;V3R+P218R;V3R+Y220R;V3R+W221R;V3R+S224R;V3R+S225R;V3R+T226R;V3R+L227R;V3R+V228R;V3R+P229R;V3R+V230R;V3R+T231R;V3R+N233R;V3R+D234R;V3R+V236R;V3R+I238R;V3R+E239R;V3R+A243R;V3R+T244R;V3R+G245R;V3R+N248R;V3R+Q249R;V3R+N251R;Q4R+D5R;Q4R+L6R;Q4R+F7R;Q4R+N8R;Q4R+Q9R;Q4R+N11R;Q4R+L12R;Q4R+Q15R;Q4R+T37R;Q4R+Q38R;Q4R+N39R;Q4R+A40R;Q4R+P42R;Q4R+E43R;Q4R+E45R;Q4R+N73R;Q4R+D167R;Q4R+T192R;Q4R+L193R;Q4R+Y194R;Q4R+T199R;Q4R+N200R;Q4R+I202R;Q4R+S217R;;Q4R+P218R;Q4R+Y220R;Q4R+W221R;Q4R+S224R;Q4R+S225R;Q4R+T226R;Q4R+L227R;Q4R+V228R;Q4R+P229R;Q4R+V230R;Q4R+T231R;Q4R+N233R;Q4R+D234R;Q4R+V236R;Q4R+I238R;Q4R+E239R;Q4R+A243R;Q4R+T244R;Q4R+G245R;Q4R+N248R;Q4R+Q249R;Q4R+N251R;D5R+L6R;D5R+F7R;D5R+N8R;D5R+Q9R;D5R+N11R;D5R+L12R;D5R+Q15R;D5R+T37R;D5R+Q38R;D5R+N39R;D5R+A40R;D5R+P42R;D5R+E43R;D5R+E45R;D5R+N73R;D5R+D167R;D5R+T192R;D5R+L193R;D5R+Y194R;D5R+T199R;D5R+N200R;D5R+I202R;D5R+S217R;D5R+P218R;D5R+Y220R;D5R+W221R;D5R+S224R;D5R+S225R;D5R+T226R;D5R+L227R;D5R+V228R;D5R+P229R;D5R+V230R;D5R+T231R;D5R+N233R;D5R+D234R;D5R+V236R;D5R+I238R;D5R+E239R;D5R+A243R;D5R+T244R;D5R+G245R;D5R+N248R;D5R+Q249R;D5R+N251R;L6R+F7R;L6R+N8R;L6R+Q9R;L6R+N11R;L6R+L12R;L6R+Q15R;L6R+T37R;L6R+Q38R;L6R+N39R;L6R+A40R;L6R+P42R;L6R+E43R;L6R+E45R;L6R+N73R;L6R+D167R;L6R+T192R;L6R+L193R;L6R+Y194R;L6R+T199R;L6R+N200R;L6R+I202R;L6R+S217R;L6R+P218R;L6R+Y220R;L6R+W221R;L6R+S224R;L6R+S225R;L6R+T226R;L6R+L227R;L6R+V228R;L6R+P229R;L6R+V230R;L6R+T231R;L6R+N233R;L6R+D234R;L6R+V236R;L6R+I238R;L6R+E239R;L6R+A243R L6R+T244R;L6R+G245R;L6R+N248R;L6R+Q249R;L6R+N251R;F7R+N8R;F7R+Q9R;F7R+N11R;F7R+L12R;F7R+Q15R;F7R+T37R;F7R+Q38R;F7R+N39R;F7R+A40R;F7R+P42R;F7R+E43R;F7R+E45R;F7R+N73R;F7R+D167R;F7R+T192R;F7R+L193R;F7R+Y194R;F7R+T199R;F7R+N200R;F7R+I202R;F7R+S217R;F7R+P218R;F7R+Y220R;F7R+W221R;F7R+S224R;F7R+S225R;F7R+T226R;F7R+L227R;F7R+V228R;F7R+P229R;F7R+V230R;F7R+T231R;F7R+N233R;F7R+D234R;F7R+V236R;F7R+I238R;F7R+E239R;F7R+A243R;F7R+T244R;F7R+G245R;F7R+N248R;F7R+Q249R;F7R+N251R;N8R+Q9R;N8R+N11R;N8R+L12R;N8R+Q15R;N8R+T37R;N8R+Q38R;N8R+N39R;N8R+A40R;N8R+P42R;N8R+E43R;N8R+E45R;N8R+N73R;N8R+D167R;N8R+T192R;N8R+L193R;N8R+Y194R;N8R+T199R;N8R+N200R;N8R+I202R;N8R+S217R;N8R+P218R;N8R+Y220R;N8R+W221R;N8R+S224R;N8R+S225R;N8R+T226R;N8R+L227R;N8R+V228R;N8R+P229R;N8R+V230R;N8R+T231R;N8R+N233R;N8R+D234R;N8R+V236R;N8R+I238R;N8R+E239R;N8R+A243R;N8R+T244R;N8R+G245R;N8R+N248R;N8R+Q249R;N8R+N251R;Q9R+N11R;Q9R+L12R;Q9R+Q15R;Q9R+T37R;Q9R+Q38R;Q9R+N39R;Q9R+A40R;Q9R+P42R;Q9R+E43R;Q9R+E45R;Q9R+N73R;Q9R+D167R;Q9R+T192R;Q9R+L193R;Q9R+Y194R;Q9R+T199R;Q9R+N200R;Q9R+I202R;Q9R+S217R;Q9R+P218R;Q9R+Y220R;Q9R+W221R;Q9R+S224R;Q9R+S225R;Q9R+T226R;Q9R+L227R;Q9R+V228R;Q9R+P229R;Q9R+V230R;Q9R+T231R;Q9R+N233R;Q9R+D234R;Q9R+V236R;Q9R+I238R;Q9R+E239R;Q9R+A243R;Q9R+T244R;Q9R+G245R;Q9R+N248R;Q9R+Q249R;Q9R+N251R;N11R+L12R;N11R+Q15R;N11R+T37R;N11R+Q38R;N11R+N39R;N11R+A40R;N11R+P42R;N11R+E43R;N11R+E45R;N11R+N73R;N11R+D167R;N11R+T192R;N11R+L193R;N11R+Y194R;N11R+T199R;N11R+N200R;N11R+I202R;N11R+S217R;N11R+P218R;N11R+Y220R;N11R+W221R;N11R+S224R;N11R+S225R;N11R+T226R;N11R+L227R;N11R+V228R;N11R+P229R;N11R+V230R;N11R+T231R;N11R+N233R;N11R+D234R;N11R+V236R;N11R+I238R;N11R+E239R;N11R+A243R;N11R+T244R;N11R+G245R;N11R+N248R;N11R+Q249R;N11R+N251R;L12R+Q15R;L12R+T37R;L12R+Q38R;L12R+N39R;L12R+A40R;L12R+P42R;L12R+E43R;L12R+E45R;L12R+N73R;L12R+D167R;L12R+T192R;L12R+L193R;L12R+Y194R;L12R+T199R;L12R+N200R;L12R+I202R;L12R+S217R;L12R+P218R;L12R+Y220R;L12R+W221R;L12R+S224R;L12R+S225R;L12R+T226R;L12R+L227R;L12R+V228R;L12R+P229R;L12R+V230R;L12R+T231R;L12R+N233R;L12R+D234R;L12R+V236R;L12R+I238R;L12R+E239R;L12R+A243R;L12R+T244R;L12R+G245R;L12R+N248R;L12R+Q249R;L12R+N251R;Q15R+T37R;Q15R+Q38R;Q15R+N39R;Q15R+A40R;Q15R+P42R;Q15R+E43R;Q15R+E45R;Q15R+N73R;Q15R+D167R;Q15R+T192R;Q15R+L193R;Q15R+Y194R;Q15R+T199R;Q15R+N200R;Q15R+I202R;Q15R+S217R;Q15R+P218R;Q15R+Y220R;Q15R+W221R;Q15R+S224R;Q15R+S225R;Q15R+T226R;Q15R+L227R;Q15R+V228R;Q15R+P229R;Q15R+V2 30R;Q15R+T231R;Q15R+N233R;Q15R+D234R;Q15R+V236R;Q15R+I238R;Q15 R+E239R;Q15R+A243R;Q15R+T244R;Q15R+G245R;Q15R+N248R;Q15R+Q249R ;Q15R+N251R;T37R+Q38R;T37R+N39R;T37R+A40R;T37R+P42R;T37R+E43R;T 37R+E45R; T37R+N73R; T37R+D167R; T37R+T192R; T37R+L193R; T37R+Y194R; T37R+T199R; T37R+N200R; T37R+I202R; T37R+S217R; T37R+P218R; T37R+Y2 20R; T37R+W221R; T37R+S224R; T37R+S225R; T37R+T226R; T37R+L227R; T37R+V228R; T37R+P229R; T37R+V230R; T37R+T231R; T37R+N233R; T37R+D234R; T37R+V236R; T37R+I238R; T37R+E239R; T37R+A243R; T37R+T244R; T37R+G245R; T37R+N248R; T37R+Q249R; T37R+N251R; Q38R+N39R; Q38R+A40R; Q38R+ P42R;Q38R+E43R;Q38R+E45R;Q38R+N73R;Q38R+D167R;Q38R+T192R;Q38R+ L193R;Q38R+Y194R;Q38R+T199R;Q38R+N200R;Q38R+I202R;Q38R+S217R;Q3 8R+P218R;Q38R+Y220R;Q38R+W221R;Q38R+S224R;Q38R+S225R;Q38R+T226 R;Q38R+L227R;Q38R+V228R;Q38R+P229R;Q38R+V230R;Q38R+T231R;Q38R+N 233R;Q38R+D234R;Q38R+V236R;Q38R+I238R;Q38R+E239R;Q38R+A243R;Q3 8R+T244R;Q38R+G245R;Q38R+N248R;Q38R+Q249R;Q38R+N251R;N39R+A40R;N39R+P42R;N39R+E43R;N39R+E45R;N39R+N73R;N39R+D167R;N39R+T192R;N39R+L193R;N39R+Y194R;N39R+T199R;N39R+N200R;N39R+I202R;N39R+S217R;N39R+P218R;N39R+Y220R;N39R+W221R;N39R+S224R;N39R+S225R;N39R+T226R;N39R+L227R;N39R+V228R;N39R+P229R;N39R+V230R;N39R+T231R;N39R+N233R;N39R+D234R;N39R+V236R;N39R+I238R;N39R+E239R;N39R+A243R;N39R+T244R;N39R+G245R;N39R+N248R;N39R+Q249R;N39R+N251R;A40R+P42R;A40R+E43R;A40R+E45R;A40R+N73R;A40R+D167R;A40R+T192R;A40R+L193R;A40R+Y194R;A40R+T199R;A40R+N200R;A40R+I202R;A40R+S217R;A40R+P218R;A40R+Y220R;A40R+W221R;A40R+S224R;A40R+S225R;A40R+T226R;A40R+L227R;A40R+V228R;A40R+P229R;A40R+V230R;A40R+T231R;A40R+N233R;A40R+D234R;A40R+V236R;A40R+I238R;A40R+E239R;A40R+A243R;A40R+T244R;A40R+G245R;A40R+N248R;A40R+Q249R;A40R+N251R;P42R+E43R;P42R+E45R;P42R+N73R;P42R+D167R;P42R+T192R;P42R+L193R;P42R+Y194R;P42R+T199R;P42R+N200R;P42R+I202R;P42R+S217R;P42R+P218R;P42R+Y220R;P42R+W221R;P42R+S224R;P42R+S225R;P42R+T226R;P42R+L227R;P42R+V228R;P42R+P229R;P42R+V230R;P42R+T231R;P42R+N233R;P42R+D234R;P42R+V236R;P42R+I238R;P42R+E239R;P42R+A243R;P42R+T244R;P42R+G2 45R;P42R+N248R;P42R+Q249R;P42R+N251R;E43R+E45R;E43R+N73R;E43R+ D167R;E43R+T192R;E43R+L193R;E43R+Y194R;E43R+T199R;E43R+N200R;E 43R+I202R;E43R+S217R;E43R+P218R;E43R+Y220R;E43R+W221R;E43R+S224 R;E43R+S225R;E43R+T226R;E43R+L227R;E43R+V228R;E43R+P229R;E43R+ V230R;E43R+T231R;E43R+N233R;E43R+D234R;E43R+V236R;E43R+I238R;E4 3R+E239R; E43R+A243R; E43R+T244R; E43R+G245R; E43R+N248R; E43R+Q249R; E43R+N251R; E45R+N73R; E45R+D167R; E45R+T192R; E45R+L193R; E45R+Y1 94R;E45R+T199R;E45R+N200R;E45R+I202R;E45R+S217R;E45R+P218R;E45 R+Y220R;E45R+W221R;E45R+S224R;E45R+S225R;E45R+T226R;E45R+L227R E45R+V228R; E45R+P229R; E45R+V230R; E45R+T231R; E45R+N233R; E45R+D234R; E45R+V236R; E45R+I238R; E45R+E239R; E45R+A243R; E45R+T244R; E45 R+G245R; E45R+N248R; E45R+Q249R; E45R+N251R; N73R+D167R; N73R+T192R; N73R+L193R; N73R+Y194R; N73R+T199R; N73R+N200R; N73R+I202R; N73R+S2 17R; N73R+P218R; N73R+Y220R; N73R+W221R; N73R+S224R; N73R+S225R; N73R+T226R; N73R+L227R; N73R+V228R; N73R+P229R; N73R+V230R; N73R+T231R;N73R+N233R;N73R+D234R;N73R+V236R;N73R+I238R;N73R+E239R;N73R+A243R;N73R+T244R;N73R+G245R;N73R+N248R;N73R+Q249R;N73R+N251R;D167R+T192R;D167R+L193R;D167R+Y194R;D167R+T199R;D167R+N200R;D167R+I202R;D167R+S217R;D167R+P218R;D167R+Y220R;D167R+W221R;D167R+S224R;D167R+S225R;D167R+T226R;D167R+L227R;D167R+V228R;D167R+P229R;D167R+V230R;D167R+T231R;D167R+N233R;D167R+D234R;D167R+V236R;D167R+I238R;D167R+E239R;D167R+A243R;D167R+T244R;D167R+G245R;D167R+N248R;D167R+Q249R;D167R+N251R;T192R+L193R;T192R+Y194R;T192R+T199R;T192R+N200R;T192R+I202R;T192R+S217R;T192R+P218R;T192R+Y220R;T192R+W221R;T192R+S224R;T192R+S225R;T192R+T226R;T192R+L227R;T192R+V228R;T192R+P229R;T192R+V230R;T192R+T231R;T192R+N233R;T192R+D234R;T192R+V236R;T192R+I238R;T192R+E239R;T192R+A243R;T192R+T244R;T192R+G245R;T192R+N248R;T192R+Q249R;T192R+N251R;L193R+Y194R;L193R+T199R;L193R+N200R;L193R+I202R;L193R+S217R;L193R+P218R;L193R+Y220R;L193R+W221R;L193R+S224R;L193R+S225R;L193R+T226R;L193R+L227R;L193R+V228R;L193R+P229R;L193R+V230R;L193R+T231R;L193R+N233R;L193R+D234R;L193R+V236R;L193R+I238R;L193R+E239R;L193R+A243R;L193R+T244R;L193R+G245R;L193R+N248R;L193R+Q249R;L193R+N251R;Y194R+T199R;Y194R+N200R;Y194R+I202R;Y194R+S217R;Y194R+P218R;Y194R+Y220R;Y194R+W221R;Y194R+S224R;Y194R+S225R;Y194R+T226R;Y194R+L227R;Y194R+V228R;Y194R+P229R;Y194R+V230R;Y194R+T231R;Y194R+N233R;Y194R+D234R;Y194R+V236R;Y194R+I238R;Y194R+E239R;Y194R+A243R;Y194R+T244R;Y194R+G245R;Y194R+N248R;Y194R+Q249R;Y194R+N251R;T199R+N200R;T199R+I202R;T199R+S217R;T199R+P218R;T199R+Y220R;T199R+W221R;T199R+S224R;T199R+S225R;T199R+T226R;T199R+L227R;T199R+V228R;T199R+P229R;T199R+V230R;T199R+T231R;T199R+N233R;T199R+D234R;T199R+V236R;T199R+I238R;T199R+E239R;T199R+A243R;T199R+T244R;T199R+G245R;T199R+N248R;T199R+Q249R;T199R+N251R;N200R+I202R;N200R+S217R;N200R+P218R;N200R+Y220R;N200R+W221R;N200R+S224R;N200R+S225R;N200R+T226R;N200R+L227R;N200R+V228R;N200R+P229R;N200R+V230R;N200R+T231R;N200R+N233R;N200R+D234R;N200R+V236R;N200R+I238R;N200R+E239R;N200R+A243R;N200R+T244R;N200R+G245R;N200R+N248R;N200R+Q249R;N200R+N251R;I202R+S217R;I202R+P218R;I202R+Y220R;I202R+W221R;I202R+S224R;I202R+S225R;I202R+T226R;I202R+L227R;I202R+V228R;I202R+P229R;I202R+V230R;I202R+T231R;I202R+N233R;I202R+D234R;I202R+V236R;I202R+I238R;I202R+E239R;I202R+A243R;I202R+T244R;I202R+G245R;I202R+N248R;I202R+Q249R;I202R+N251R;S217R+P218R;S217R+Y220R;S217R+W221R;S217R+S224R;S217R+S225R;S217R+T226R;S217R+L227R;S217R+V228R;S217R+P229R;S217R+V230R;S217R+T231R;S217R+N233R;S217R+D234R;S217R+V236R;S217R+I238R;S217R+E239R;S217R+A243R;S217R+T244R;S217R+G245R;S217R+N248R;S217R+Q249R;S217R+N251R;P218R+Y220R;P218R+W221R;P218R+S224R;P218R+S225R;P218R+T226R;P218R+L227R;P218R+V228R;P218R+P229R;P218R+V230R;P218R+T231R;P218R+N233R;P218R+D234R;P218R+V236R;P218R+I238R;P218R+E239R;P218R+A243R;P218R+T244R;P218R+G245R;P218R+N248R;P218R+Q249R;P218R+N251R;Y220R+W221R;Y220R+S224R;Y220R+S225R;Y220R+T226R;Y220R+L227R;Y220R+V228R;Y220R+P229R;Y220R+V230R;Y220R+T231R;Y220R+N233R;Y220R+D234R;Y220R+V236R;Y220R+I238R;Y220R+E239R;Y220R+A243R;Y220R+T244R;Y220R+G245R;Y220R+N248R;Y220R+Q249R;Y2 20R+N251R;W221R+S224R;W221R+S225R;W221R+T226R;W221R+L227R;W221 R+V228R;W221R+P229R;W221R+V230R;W221R+T231R;W221R+N233R;W221R+ D234R;W221R+V236R;W221R+I238R;W221R+E239R;W221R+A243R;W221R+T24 4R;W221R+G245R;W221R+N248R;W221R+Q249R;W221R+N251R;S224R+S225R ;S224R+T226R;S224R+L227R;S224R+V228R;S224R+P229R;S224R+V230R;S 224R+T231R; S224R+N233R; S224R+D234R; S224R+V236R; S224R+I238R; S224R+E239R; S224R+A243R; S224R+T244R; S224R+G245R; S224R+N248R; S224R+ Q249R; S224R+N251R; S225R+T226R; S225R+L227R; S225R+V228R; S225R+P229R; S225R+V230R; S225R+T231R; S225R+N233R; S225R+D234R; S225R+V236 R;S225R+I238R;S225R+E239R;S225R+A243R;S225R+T244R;S225R+G245R; S225R+N248R;S225R+Q249R;S225R+N251R;T226R+L227R;T226R+V228R;T22 6R+P229R; T226R+V230R; T226R+T231R; T226R+N233R; T226R+D234R; T226R+V236R; T226R+I238R; T226R+E239R; T226R+A243R; T226R+T244R; T226R+G 245R; T226R+N248R; T226R+Q249R; T226R+N251R; L227R+V228R; L227R+P229R; L227R+V230R; L227R+T231R; L227R+N233R; L227R+D234R; L227R+V236R;L227R+I238R; L227R+E239R; L227R+A243R; L227R+T244R; L227R+G245R; L227R+N248R; L227R+Q249R; L227R+N251R; V228R+P229R; V228R+V230R; V228 R+T231R;V228R+N233R;V228R+D234R;V228R+V236R;V228R+I238R;V228R+ E239R;V228R+A243R;V228R+T244R;V228R+G245R;V228R+N248R;V228R+Q24 9R;V228R+N251R;P229R+V230R;P229R+T231R;P229R+N233R;P229R+D234R ;P229R+V236R;P229R+I238R;P229R+E239R;P229R+A243R;P229R+T244R;P 229R+G245R;P229R+N248R;P229R+Q249R;P229R+N251R;V230R+T231R;V23 0R+N233R;V230R+D234R;V230R+V236R;V230R+I238R;V230R+E239R;V230R+ A243R;V230R+T244R;V230R+G245R;V230R+N248R;V230R+Q249R;V230R+N2 51R;T231R+N233R;T231R+D234R;T231R+V236R;T231R+I238R;T231R+E239 R; T231R+A243R; T231R+T244R; T231R+G245R; T231R+N248R; T231R+Q249R; T231R+N251R; N233R+D234R; N233R+V236R; N233R+I238R; N233R+E239R; N23 3R+A243R; N233R+T244R; N233R+G245R; N233R+N248R; N233R+Q249R; N233R+N251R; D234R+V236R; D234R+I238R; D234R+E239R; D234R+A243R; D234R+T 244R;D234R+G245R;D234R+N248R;D234R+Q249R;D234R+N251R;V236R+I23 8R;V236R+E239R;V236R+A243R;V236R+T244R;V236R+G245R;V236R+N248R;V236R+Q249R; V236R+N251R; I238R+E239R; I238R+A243R; I238R+T244R; I238R+G245R; I238R+N248R; I238R+ Q249R; I238R+N251R; E239R+A243R; E239R+T244R; E239R+G245R; E239R+N248R; E239R+Q249R; E239R+N251R; A 243R+T244R; A243R+G245R; A243R+N248R; A243R+Q249R; A243R+N251R; T244R+G245R; T244R+N248R; T244R+Q249R; T244R+N251R; G245R+N248R; G245R+Q249R; G245R+N251R; N248R+Q249R; N248R+N251R; and Q249R+N251R.

[0513] In one embodiment of all three aspects, the variant has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with SEQ ID NO:1. In another embodiment, the variant has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with SEQ ID NO:1. On the one hand, the number of modifiers is 1-20, such as 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifiers.

[0514] These variants may further include one or more additional modifications in one or more other locations.

[0515] These amino acid alterations can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function (such as polyhistidine fragments, antigenic epitopes, or binding domains).

[0516] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0517] Alternatively, these amino acid alterations result in changes to the physicochemical properties of the peptide. For example, amino acid changes can improve the peptide's thermal stability, alter its substrate specificity, and change its optimal pH.

[0518] For example, the lipase variant may further include one or more substitutions corresponding to positions 81-99 of SEQ ID NO:1 in the cap region, preferably R81Q; S83T; R84H; S85T; I86L, I86P, I86V, I86W; E87A, E87I, E87K, E87T, E87V; N88Q; I90L, I90M; G91A, G91L, G91N, G91Q, G91T; N92D, N92K; L93F; N94D, N94K, N94R; F95A, F95L, F95Y; D96E, D96I, D96L, D96T; L97F, L97M, L97P; K98D, K98E, K98I, K98Q; and E99K.

[0519] The lipase variant may further comprise one or more substitutions corresponding to any of the following positions: 27, 33, 38, 51, 56, 57, 58, 60, 69, 101, 106, 111, 150, 163, 198, 210, 211, 216, 220, 254, 255, 256, 263, 264, 265, 266, 267 and 269 of SEQ ID NO:1. In one aspect, the lipase variant further comprises one or more substitutions selected from the group consisting of: 27N, 27R; 33K, 33Q; 38A; 51I, 51L, 51V; 56K, 56Q, 56R, 56S; 57G, 57N; 58A; 60S; 69R; 101R; 106K, 101R; 111A; 131T; 149G; 150G; 163K, 163S; 198S; 210K, 210Q; 211L; 216P; 220F; 254S; 255A, 255I, 255T; 256K, 256T, 256V; 263Q; 264A, 264F, 264W; 265T; 266D; 267A; and 269N.

[0520] In a particular preferred embodiment, a variant of the invention has one of the following groups of modifications:

[0521]

[0522]

[0523] In another particular preferred embodiment, a variant of the invention has one of the following groups of modifications:

[0524]

[0525]

[0526]

[0527] The variants of the present invention have improved washing performance. In a preferred embodiment, these variants have a BRF (Benefit Risk Factor) higher than 1, such as higher than 2, such as higher than 3, such as higher than 4, such as higher than 5, such as higher than 6, such as higher than 7, such as higher than 8, such as higher than 9, such as higher than 10, such as higher than 11, such as higher than 12, such as higher than 13, such as higher than 14, such as higher than 15, compared to the lipase shown in SEQ ID NO:1.

[0528] In one embodiment, BRF is determined in a detergent composition containing anionic surfactants, particularly linear alkylbenzene sulfonates (LAS) and / or alcohol ethoxysulfates (AEOS).

[0529] In one embodiment, BRF is determined in a detergent composition containing a nonionic surfactant such as alcohol ethoxylate (AEO)0.

[0530] In one embodiment, BRF is determined in a detergent composition comprising one or more anionic and / or one or more nonionic surfactants.

[0531] In one embodiment, BRF was determined in a detergent composition comprising linear alkylbenzene sulfonate (LAS) and alcohol ethoxylate (AEO).

[0532] In one embodiment, BRF was determined using AMSA in Model Detergent J (6°dH, 0.8 g / L) and / or Model Detergent B (6°dH, 3.3 g / L) (see Example 3).

[0533] In one embodiment, a variant of the invention has one of the following groups of modifications:

[0534]

[0535]

[0536] In one embodiment, these variants exhibit improved washing performance, particularly a relative washing performance (RP) greater than 1, compared to the lipase shown in SEQ ID NO:1.

[0537] In one embodiment, relative washing performance (RP) is determined in a detergent composition containing anionic surfactants, particularly linear alkylbenzene sulfonates (LAS) and / or alcohol ethoxysulfates (AEOS).

[0538] In one embodiment, relative washing performance (RP) is determined in a detergent composition containing a nonionic surfactant such as an alcohol ethoxylate (AEO).

[0539] In one embodiment, relative washing performance (RP) is determined in a detergent composition containing a nonionic surfactant such as an alcohol ethoxylate (AEO).

[0540] In one embodiment, relative washing performance (RP) is determined in a detergent composition comprising one or more anionic and / or one or more nonionic surfactants.

[0541] In one embodiment, relative washing performance (RP) was determined in a detergent composition comprising linear alkylbenzene sulfonate (LAS) and alcohol ethoxylate (AEO).

[0542] In one embodiment, AMSA was used to determine the relative washing performance of a detergent composition selected from the group consisting of model detergent B, Detergent Diao, model detergent V, model detergent T, model detergent Y, and model detergent X (see Example 4).

[0543] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the lipase activity of the resulting mutant molecule is tested to identify the amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutations in the amino acids at the putative contact sites. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from comparison with related polypeptides. The amino acids of the catalytic triplet forming the lipase of SEQ ID NO:1 are S146, D201, and H258, and should not be altered.

[0544] In one embodiment, this variant exhibits improved washing performance compared to the parent enzyme.

[0545] In one embodiment, this variant exhibits improved catalytic efficiency compared to the parent enzyme.

[0546] In one embodiment, the variant exhibits an improved catalytic rate compared to the parent enzyme.

[0547] In one embodiment, this variant exhibits improved pH activity compared to the parent enzyme.

[0548] In one embodiment, this variant exhibits improved pH stability compared to the parent enzyme.

[0549] In one embodiment, the variant exhibits improved specific activity compared to the parent enzyme.

[0550] In one embodiment, this variant exhibits improved stability under storage conditions compared to the parent enzyme.

[0551] In one embodiment, this variant has improved substrate binding compared to the parent enzyme.

[0552] In one embodiment, this variant exhibits improved substrate cleavage compared to the parent enzyme.

[0553] In one embodiment, the variant has improved surface properties compared to the parent enzyme.

[0554] In one embodiment, the variant exhibits improved thermal stability compared to the parent enzyme.

[0555] Parental lipase

[0556] The parental lipase is a polypeptide having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1, and possessing lipase activity. In one aspect, the amino acid sequence of the parent differs from SEQ ID NO:1 by a maximum of 40 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0557] On the one hand, the parent contains or is composed of the amino acid sequence of SEQ ID NO:1.

[0558] On one hand, the parent is a fragment of SEQ ID NO:1 containing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids in SEQ ID NO:1.

[0559] On one hand, the parent is an allelic variant of the polypeptide of SEQ ID NO:1.

[0560] The polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0561] The parent peptide can be a fusion peptide or a cleavable fusion peptide, wherein another peptide is fused to the N-terminus or C-terminus of the peptide of the present invention. Fusion peptides are generated by fusing a polynucleotide encoding another peptide with the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they conform to reading frames, and that the expression of the fusion peptide is under the control of one or more identical promoters and terminators. Fusion peptides can also be constructed using intron technology, wherein the fusion peptide is generated post-translational (Cooper et al., 1993, EMBO J. [Journal of the European Society for Molecular Biology] 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0562] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3:568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63:3488-3493; Ward et al., 1995, B Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins; Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0563] The parent can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the parent is extracellularly secreted.

[0564] The parent can be a bacterial lipase. For example, the parent can be a Gram-positive bacterial polypeptide, such as Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. Lipases from Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma.

[0565] On one hand, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus scoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis lipase.

[0566] On the other hand, the parent is a lipase of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus lactis, or Streptococcus equi subsp. Zooepidemicus.

[0567] On the other hand, this parent does not produce Streptomyces chromopterus, Streptomyces viridisus, Streptomyces azureus, Streptomyces grayi, or Streptomyces purplish-blue.

[0568] The parent can be a fungal lipase. For example, the parent can be a yeast lipase, such as those from the genera *Candida*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*.Or filamentous fungal lipases, such as those from genera *Acremonium*, *Agaricus*, *Alternaria*, *Aspergillus*, *Aureobasidium*, *Botryospaeria*, *Ceriporiopsis*, *Chaetomidium*, *Chrysosporium*, *Claviceps*, *Cochliobolus*, *Coprinopsis*, and *Coptot*. ermes), Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaphthium The genera *Melanocarpus*, *Meripilus*, *Mucor*, *Myceliophthora*, *Neocallimastix*, *Neurospora*, *Paecilomyces*, *Penicillium*, *Phanerochaete*, *Piromyces*, *Poitrasia*, *Pseudoplectania*, and *Pseudotrichonymph* a) Lipases from the genera *Rhizomucor*, *Schizophyllum*, *Scytalidium*, *Talaromyces*, *Thermoascus*, *Thermomyces*, *Thielavia*, *Tolypocladium*, *Trichoderma*, *Trichophaea*, *Verticillium*, *Volvariella*, or *Xylaria*.

[0569] On the other hand, the parent is Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces sacchariformis, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces noviceum, or Saccharomyces ovalis lipase.

[0570] On the other hand, the parent species are *Acremonium cellulolyticus*, *Aspergillus aculeatus*, *Aspergillus awamori*, *Aspergillus foetidus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Chrysosporium inops*, *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium pannicola*, and *Chrysosporium quercetinum*. Queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium arcochroum Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolagrisea), Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thievora achromatica, Thievora albomyces, Thievora albopilosa, Thievora australeinsis, Thielaviafimeti, Thievora microspora, Thievora oospora The following are listed as lipases: ovispora, Thievora peruviana, Thievora setosa, Thievora spededonium, Thievora subthermophila, Thievora terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0571] On one hand, the parent is a lipase of *Thermomyces lanuginosus* (formerly known as *Humicola lanuginosa*), such as the lipase of SEQ ID NO:1.

[0572] It should be understood that, for the aforementioned species, this invention covers perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0573] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial and Cell Culture Collection (DSMZ), the Centralbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0574] The parent can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural habitat are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening another microorganism or a library of genomic DNA or cDNA from a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with one or more probes, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0575] Preparation of variants

[0576] The present invention also relates to a method for obtaining a variant having lipase activity, the method comprising: (a) at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 28, 29, 30, 31, 33, 37, 38, 39, 40, 42, 43, 45, 73, 91, 99, 101, 102, 105, 167, 178, 180, 192, 193, 194, 199, 200, 202, 210, 211, 217, 218, 220 corresponding to SEQ ID NO:1 (a) Introducing modifications into the parental lipase at one or more (e.g., several) positions of 221, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 236, 238, 239, 243, 244, 245, 248, 249, 250, 251, 252, 254 and 256, wherein the variant has lipase activity; and (b) recovering the variant.

[0577] Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0578] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0579] Site-directed mutagenesis can be achieved in vitro via PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent, followed by ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide, allowing the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acids Research] 18:7349-4966.

[0580] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0581] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0582] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.

[0583] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested. These relevant screening procedures are those disclosed in the following literature: Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other available methods include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0584] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0585] Semi-synthetic gene construction is achieved through a combination of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments combined with PCR technology. Therefore, defined regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can be amplified using error-prone or non-error-prone PCR. The polynucleotide subsequence can then be shuffled.

[0586] Polynucleotides

[0587] The present invention also relates to isolated polynucleotides encoding variants of the invention.

[0588] Nucleic acid constructs

[0589] The present invention also relates to a nucleic acid construct comprising a polynucleotide operably linked to one or more control sequences encoding a variant of the invention, the one or more control sequences guiding the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0590] Polynucleotides can be manipulated in a variety of ways to provide the expression of variants. Depending on the expression vector, manipulating the polynucleotide before insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are known in the art.

[0591] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell to express a polynucleotide encoding a variant of the present invention. The promoter contains a transcriptional control sequence that mediates the expression of the variant. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0592] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, and Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular). Microbiology [Molecular Microbiology] 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene [Gene] 69:301-315), Streptomyces agar hydrolase gene (dagA) and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 75:3727-3731), and tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 80:21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, ibid. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0593] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters obtained from the following genes: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium oxysporum amygdalinase (WO 00 / 56900), and Fusarium oxysporum Daria (WO 96 / 00787). 00 / 56900), Fusarium quinn (WO00 / 56900), Rhizopus miltiorrhiza lipase, Rhizopus miltiorrhiza aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylose Glycosidases, and *Trichoderma reesei* translation elongation factors, along with the NA2-tpi promoter (a modified promoter from an *Aspergillus* gene encoding neutral α-amylase, wherein the untranslated leader sequence has been replaced with an untranslated leader sequence from an *Aspergillus* gene encoding triose phosphate isomerase; non-limiting examples include a modified promoter from a *Aspergillus niger* neutral α-amylase gene, wherein the untranslated leader sequence has been replaced with an untranslated leader sequence from an *Aspergillus nidus* or *Aspergillus oryzae* triose phosphate isomerase gene); and mutant, truncated, and heterozygous promoters thereof. Other promoters are described in U.S. Patent No. 6,011,147.

[0594] In yeast hosts, useful promoters are derived from the following genes: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Other useful promoters in yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

[0595] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. This terminator is operatively linked to the 3' end of the polynucleotide encoding the variant. Any terminator that is functional in the host cell can be used in this invention.

[0596] The preferred terminator for bacterial host cells is derived from the genes of the following enzymes: Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0597] Preferred terminators for filamentous fungal host cells are derived from the following genes: Aspergillus nidulans acetamase, Aspergillus nidulans o-aminobenzoic acid synthase, Aspergillus niger glucosylamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor.

[0598] Preferred terminators for yeast host cells are derived from the following genes: *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described above by Romanos et al., 1992.

[0599] Control sequences can also be mRNA stabilizers downstream of the gene promoter and upstream of the coding sequence, which increase the expression of the gene.

[0600] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0601] The control sequence can also be a leader, which is the untranslated region of mRNA that is important for translation in the host cell. This leader is operatively linked to the 5' end of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell can be used.

[0602] Preferred leader sequences for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0603] Suitable leader sequences for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphodehydrogenase (ADH2 / GAP).

[0604] The control sequence can also be a polyadenylated sequence, i.e., a sequence operatively linked to the 3' end of a polynucleotide and recognized by the host cell during transcription as a signal to add polyadenylated residues to the transcribed mRNA. Any polyadenylated sequence that is functional in the host cell can be used.

[0605] Preferred polyadenylated sequences for use in filamentous fungal host cells were obtained from the genes of the following enzymes: Aspergillus nidulans o-aminobenzoic acid synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0606] Useful polyadenylated sequences in yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. [Molecular Cell Biology] 15: 5983-5990.

[0607] The control sequence can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and directs the variant into the cellular secretion pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked within the translation reading frame to a segment encoding the variant's coding sequence. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to that coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the variant's secretion. However, any signal peptide coding sequence that directs the expressed variant into the host cell's secretion pathway can be used.

[0608] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes of the following enzymes: Bacillus NCIB 11837 maltose amylase, Bacillus subtilis protease, Bacillus subtilis β-lactamase, Bacillus thermophilus α-amylase, Bacillus thermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0609] Effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of the following enzymes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Specific Humus cellulase, Specific Humus endoglucanase V, Specific Humus lipase, and Rhizomucormiehei aspartic protease.

[0610] Useful signal peptides for yeast host cells are derived from the genes for *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Sequences encoding other useful signal peptides are described by Romanos et al., 1992, ibid.

[0611] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active variant by catalytic or autocatalytic cleavage of the propeptide progenitor. The propeptide-coding sequence can be obtained from the genes of the following enzymes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae α-factor.

[0612] In the case where both the signal peptide and the propeptide sequence are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0613] Also desirable is the addition of regulatory sequences that modulate variant expression relative to the growth of the host cell. Examples of regulatory sequences are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 systems can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter and *Aspergillus oryzae* glucosylamylase promoter, the *Trichoderma reesei* cellobiose hydrolase I promoter, and the *Trichoderma reesei* cellobiose hydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0614] expression carrier

[0615] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more suitable restriction sites to allow insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0616] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0617] Vectors can be self-replicating vectors, i.e., vectors that exist as extrachromosomal entities and whose replication is independent of chromosome replication, such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. Vectors can contain any components necessary to ensure self-replication. Alternatively, a vector can be one that, when introduced into a host cell, integrates into the genome and replicates along with one or more chromosomes in which it has already been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0618] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, or protrophic traits such as auxotrophy.

[0619] Examples of selective markers for bacteria include the dal gene in Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, adeA (phosphogluconoylaminoimidazolium-succinate synthase), adeB (phosphogluconoylaminoimidazolium synthase), amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. Preferred genes for use in Aspergillus cells include the amdS and pyrG genes from Aspergillus nidulans or Aspergillus oryzae, and the bar gene from Streptomyces hygroscopicus. Preferred genes for use in Trichoderma cells include the adeA, adeB, amdS, hph, and pyrG genes.

[0620] Selective flags can be dual-selective flag systems as described in WO 2010 / 039889. In one aspect, a dual-selective flag is the hph-tk dual-selective flag system.

[0621] The vector preferably contains one or more elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0622] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the variant or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, the integrating element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have a high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. The integrating element can be any sequence homologous to the target sequence within the host cell genome. Furthermore, the integrating element can be a non-coding polynucleotide or a coding polynucleotide. On the other hand, the vector can be integrated into the host cell genome via non-homologous recombination.

[0623] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that functions in the cell to mediate autonomous replication. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0624] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and plasmids pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0625] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0626] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO00 / 24883). The AMA1 gene can be isolated and plasmids or vectors containing the gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0627] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the generation of variants. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selective marker gene along with the polynucleotide, wherein cells containing amplified copies of the selective marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selective reagent.

[0628] The methods for connecting the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0629] host cells

[0630] This invention also relates to recombinant host cells containing a polynucleotide operably linked to one or more control sequences encoding variants of the invention, said control sequences directing the generation of variants of the invention. A construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend largely on the gene encoding the variant and its origin.

[0631] The host cell can be any cell that is useful in the recombinant production of variants, such as prokaryotic or eukaryotic cells.

[0632] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0633] The bacterial host cell can be any Bacillus genus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0634] The bacterial host cell can also be any streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus.

[0635] The bacterial host cell can also be any Streptomyces cell, including but not limited to: non-chromogenic Streptomyces, insecticidal Streptomyces, sky blue Streptomyces, gray Streptomyces, and light blue Streptomyces cells.

[0636] DNA can be introduced into Bacillus cells through the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. [Molecular Genetics and Genomics] 168:111-115), competent cell transformation (see, for example, Young and Spizizen, 1961, J. Bacteriol. [Journal of Bacteriology] 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. [Journal of Molecular Biology] 56:209-221), electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques [Biotechnology] 6:742-751), or conjugation (see, for example, Koehler and Thorne, 1987, J. Bacteriol. [Journal of Bacteriology] 169:5271-5278). DNA can be introduced into E. coli cells by protoplast transformation (see, for example, Hanahan, 1983, J. Mol. Biol. [Journal of Molecular Biology] 166:557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. [Nucleic Acid Research] 16:6127-6145). DNA can be introduced into Streptomyces cells through protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).DNA can be introduced into Streptococcus cells by means of natural competence (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0637] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0638] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0639] Fungal host cells can be yeast cells. As used herein, “yeast” includes Ascomycota (Endosporales), Basidiomycota, and yeasts belonging to the Deuteromycetes (Bacillus). Since yeast classification may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (Skinner, Passmore, and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0640] Yeast host cells can be cells of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Saccharomyces carlsbergensis*, *Saccharomyces cerevisiae*, *Saccharomyces diastaticus*, *Saccharomyces douglasii*, *Saccharomyces kluyveri*, *Saccharomyces norbensis*, *Saccharomyces oviformis*, or *Yarrowia lipolytica*. lipolytica cells.

[0641] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms within the phylum Eumycota and subphyla Oomycota (as defined by Hawksworth et al., 1995, ibid.). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentative.

[0642] The host cells of filamentous fungi can be cells from genera such as *Cladosporium*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureosporium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Pleurotus*, *Mucor*, *Pyrophyllus*, *Pleurotus*, *Neurophyllus*, *Penicillium*, *Penicillium*, *Phlebia*, *Pleurotus*, *Schizophyllum*, *Ascomycetes*, *Clostridium*, *Cytotrichum*, *Phytotrichum*, *Pleurotus*, *Schizophyllum*, *Basilaria*, *Thermophilus*, *Clostridium*, *Trametes*, or *Trichoderma*.

[0643] For example, the host cells of filamentous fungi can be *Aspergillus bubomori*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis saneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inopsinatus*, *Chrysosporium lucknowense*, and *Chrysosporium scutellatus*. merdarium, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Fusarium graminum, Fusarium graminum, Fusarium spp., Fusarium albinosum, Fusarium oxysporum, Fusarium spp., Fusarium graminum, Fusarium spp., Fusarium oxysporum ... radiata), Pleurotus eryngii, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma corningensis, Trichoderma longibranchii, Trichoderma reesei, or green Trichoderma cells.

[0644] Fungal cells can be transformed through a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in the following references: EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 81:1470-1474 and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78:147-156 and WO 96 / 00787. Yeast transformation can be performed using the procedures described in the following literature: Becker and Guarente in Abelson, JN and Simon, MI, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0645] Generation method

[0646] The present invention also relates to a method for generating variants, the method comprising (a) culturing the recombinant host cells of the present invention under conditions conducive to generating the variant; and optionally (b) recovering the variant.

[0647] The host cells are cultured in a suitable nutrient medium for generating the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of the variant in a laboratory or industrial fermenter. Using procedures known in the art, the culture occurs in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0648] These variants can be detected using methods specific to them known in the art. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of the variants.

[0649] Variants can be recovered using methods known in the art. For example, variants can be recovered from nutrient media through a variety of conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the entire fermentation broth can be recovered.

[0650] Variants can be purified to obtain substantially pure variants by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0651] In terms of alternatives, instead of recycling variants, the host cells of the present invention expressing variants are used as the source of variants.

[0652] Fermentation broth preparations or cell compositions

[0653] The present invention also relates to fermentation broth formulations or cell compositions comprising variants of the invention. The fermentation broth product further includes additional components used in the fermentation process, such as cells (including host cells containing genes encoding variants of the invention, which are used to produce variants of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killing whole culture medium containing organic acids, killed cells and / or cell debris, and a culture medium.

[0654] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cell) and secretion of proteins into the cell culture medium. This fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, the fermentation broth is unfractionated and contains used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, the fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0655] In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component (containing at least one organic acid of 1-5 carbons and / or its salt) and a second organic acid component (containing at least one organic acid of 6 or more carbons and / or its salt). In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, its salt, or a mixture of two or more of the foregoing.

[0656] In one aspect, the composition contains one or more organic acids and optionally further contains killed cells and / or cell debris. In one embodiment, these killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0657] These fermentation broth formulations or cell compositions may further contain preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

[0658] The cell-killing whole culture or composition may contain ungraded contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killing whole culture or composition contains used culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have grown to saturation under carbon-limited conditions that allow protein synthesis. In some embodiments, the cell-killing whole culture or composition contains used cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole culture or composition.

[0659] The whole culture medium or cell composition described herein is typically a liquid, but may contain insoluble components such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

[0660] The whole culture medium formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0661] Composition

[0662] The present invention also relates to compositions comprising the lipase variants of the present invention.

[0663] The non-limiting list of compositional components set forth below is suitable for use in these compositions, and the methods described herein may be consensually incorporated into certain aspects of the invention, for example, to aid or enhance cleaning performance, to treat a substrate to be cleaned, or to enhance the aesthetics of the composition, as in the case of fragrances, colorants, dyes, or the like. The level of any such component incorporated into any composition is in addition to any materials previously referenced for incorporation. The precise properties of these additional components and their incorporation levels will depend on the physical form of the composition and the nature of the cleaning operation in which the composition will be used. Although the components mentioned below are classified under a general heading according to their specific functionality, this is not to be construed as limiting, as it will be understood by one of ordinary skill in the art that the components may contain additional functionalities.

[0664] Unless otherwise stated, percentages are by weight (wt.%) of the composition. Suitable component materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibitors, dispersants, enzymes and enzyme stabilizers, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymeric dispersants, clay removers / anti-redeposition agents, brighteners, foam inhibitors, dyes, tinting dyes, fragrances, fragrance delivery systems, structural elastic agents, fabric softeners, carriers, water-soluble auxiliaries, processing aids, solvents, and / or pigments. Suitable examples and levels of use of such other components, in addition to those disclosed below, are given in US 5,576,282, US 6,306,812, and US 6,326,348, which are hereby incorporated by reference.

[0665] Therefore, in some embodiments, the present invention does not contain or contains one or more of the following auxiliary materials: surfactants, soaps, detergent builders, chelating agents, dye transfer inhibitors, dispersants, additional enzymes, enzyme stabilizers, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymer dispersants, clay removers / anti-redeposition agents, brighteners, foam inhibitors, dyes, fragrances, fragrance delivery systems, structural elastic agents, fabric softeners, carriers, water-soluble additives, processing aids, solvents, and / or pigments. However, when one or more components are present, such one or more components may be present as detailed below:

[0666] surfactants - The compositions according to the invention may contain surfactants or surfactant systems, wherein the surfactants may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, facultative zwitterionic surfactants, semipolar nonionic surfactants, and mixtures thereof. When present, the surfactant is typically present at levels ranging from 0.1 wt.% to 60 wt.%, from 0.2 wt.% to 40 wt.%, from 0.5 wt.% to 30 wt.%, from 1 wt.% to 50 wt.%, from 1 wt.% to 40 wt.%, from 1 wt.% to 30 wt.%, from 1 wt.% to 20 wt.%, from 3 wt.% to 10 wt.%, from 3 wt.% to 5 wt.%, from 5 wt.% to 40 wt.%, from 5 wt.% to 30 wt.%, from 5 wt.% to 15 wt.%, from 3 wt.% to 20 wt.%, from 3 wt.% to 10 wt.%, from 8 wt.% to 12 wt.%, from 10 wt.% to 12 wt.%, from 20 wt.% to 25 wt.%, or from 25 wt.% to 60 wt.%.

[0667] Suitable anionic detergency surfactants include sulfate and sulfonate detergency surfactants.

[0668] Suitable sulfonate detergency surfactants include alkylbenzene sulfonates, which in one aspect are C 10-13 Alkylbenzene sulfonates. Suitable alkylbenzene sulfonates (LAS) can be obtained by sulfonating commercially available linear alkylbenzenes (LABs); suitable LABs include low-2-phenyl LABs, such as... or Other suitable LABs include high-2-phenyl LABs, such as Suitable anionic detergency surfactants are alkylbenzene sulfonates obtained via DETAL catalytic processes, but other synthetic routes (such as HF) may also be suitable. In one approach, magnesium salts of LAS are used.

[0669] Suitable sulfate detergency surfactants include alkyl sulfates, which in one aspect are C 8-18 Alkyl sulfates, or mainly C 12 Alkyl sulfates.

[0670] Another suitable sulfate detergency surfactant is alkylalkoxylated sulfate, which is alkylethoxylated sulfate on one hand and C on the other. 8-18 Alkyl alkoxylated sulfates, on the one hand, are C 8-18 Alkyl ethoxylated sulfates, typically alkyl alkoxylated sulfates have an average degree of alkoxylation from 0.5 to 20 or from 0.5 to 10, and are typically C10-2 ... 8-18 Alkyl ethoxylated sulfates having an average degree of ethoxylation of 0.5 to 10, 0.5 to 7, 0.5 to 5, or 0.5 to 3.

[0671] Alkyl sulfates, alkylalkoxylated sulfates, and alkylbenzene sulfonates can be linear or branched, substituted or unsubstituted.

[0672] The detergency surfactant can be a medium-chain branched detergency surfactant, in one aspect being a medium-chain branched anionic detergency surfactant, and in another aspect being a medium-chain branched alkyl sulfate and / or a medium-chain branched alkylbenzene sulfonate, such as a medium-chain branched alkyl sulfate. In one aspect, the medium chain branch is C 1-4 Alkyl groups, typically methyl and / or ethyl.

[0673] Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates, and disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES, AEOS, or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

[0674] Suitable nonionic detergency surfactants are selected from the following groups: C8-C 18 Alkyl ethoxylates, for example C6-C 12 Alkylphenol alkoxylates, wherein the alkoxyl unit may be an ethyleneoxy unit, an propyleneoxy unit, or a mixture thereof; C 12 -C 18 alcohols and C6-C 12 Condensations of alkylphenols with ethylene oxide / propylene oxide block polymers, for example C 14 -C 22 Medium-chain branched alcohols; C 14 -C 22 Medium-chain branched alkyl alkoxylates, typically having an average degree of alkoxylation from 1 to 30; alkyl polysaccharides, in one respect as alkyl polyglycosides; polyhydroxy fatty acid amides; ether-terminated poly(alkoxylated) alcohol surfactants; and mixtures thereof.

[0675] Suitable nonionic detergency surfactants include alkyl polysaccharides and / or alkyl alkoxylated alcohols.

[0676] On the one hand, nonionic detergency surfactants include alkylalkoxylated alcohols, and on the other hand, they are C 8-18 Alkyl alkoxylated alcohols, such as C 8-18 Alkyl ethoxylated alcohols, wherein the alkyl alkoxylated alcohols may have an average degree of alkoxylation ranging from 1 to 50, from 1 to 30, from 1 to 20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may be C 8-18Alkyl ethoxylated alcohols have an average degree of ethoxylation ranging from 1 to 10, from 1 to 7, more commonly from 1 to 5, or from 3 to 7. Alkyl alkoxylated alcohols can be straight-chain or branched, and substituted or unsubstituted. Suitable nonionic surfactants include...

[0677] Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA) or fatty acid glucosamide (FAGA)), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0678] Suitable cationic detergency surfactants include alkylpyridine compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl trisulfite compounds, and mixtures thereof.

[0679] Suitable cationic detergency surfactants are quaternary ammonium compounds with the following formula: (R)(R1)(R2)(R3)N + X - Where R is a straight or branched, substituted or unsubstituted C 6-18 The alkyl or alkenyl moiety, R1 and R2 are independently selected from the methyl or ethyl moiety, R3 is a hydroxy, hydroxymethyl, or hydroxyethyl moiety, and X is an anion that provides charge neutrality; suitable anions include halides, such as chlorides; sulfates; and sulfonates. Suitable cationic detergency surfactants are mono-C 6-18 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride. A highly suitable cationic detergency surfactant is mono-C... 8-10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, mono-C 10-12 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride and mono-C 10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride.

[0680] Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quaternary ammonium salts, and combinations thereof.

[0681] Suitable amphoteric / zombie surfactants include amine oxides and betaines (such as alkyl dimethyl betaine, sulfobetaine) or combinations thereof.

[0682] The amine-neutralized anionic surfactants of the present invention—including both anionic surfactants and auxiliary anionic co-surfactants—can be present in acidic form, and said acidic form can be neutralized to form a surfactant salt desired for use in the detergent compositions of the present invention. Typical neutralizing agents include metal counterionic bases, such as hydroxides, for example, NaOH or KOH. Further preferred agents for neutralizing the anionic surfactants of the present invention and auxiliary anionic surfactants or co-surfactants in their acidic form include ammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitable non-limiting examples include monoethanolamine, diethanolamine, triethanolamine, and other straight-chain or branched alkanolamines known in the art; for example, highly preferred alkanolamines include 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amine neutralization can be carried out to a complete or partial extent; for example, a portion of the anionic surfactant mixture can be neutralized by sodium or potassium, and a portion of the anionic surfactant mixture can be neutralized by an amine or alkanolamine.

[0683] Non-limiting examples of semi-polar surfactants include amine oxides (AO), such as alkyl dimethylamine oxides.

[0684] Surfactant systems comprising one or more anionic surfactants, one or more other nonionic surfactants, and optionally other surfactants such as cationic surfactants may be preferred. A preferred weight ratio of anionic to nonionic surfactants is at least 2:1, or at least 1:1 to 1:10.

[0685] On one hand, the surfactant system may comprise a mixture of isoprene-like surfactants represented by formulas A and B:

[0686]

[0687] Where Y is CH2 or absent, and Z may be selected such that the resulting surfactant is selected from the following surfactants: alkyl carboxyl ester surfactants, alkyl polyalkoxy surfactants, alkyl anionic polyalkoxy sulfate surfactants, alkyl glycerol ester sulfonate surfactants, alkyl dimethylamine oxide surfactants, alkyl polyhydroxy-based surfactants, alkyl phosphate ester surfactants, alkyl glycerol sulfonate surfactants, alkyl polyglucose ester surfactants, alkyl polyphosphate ester surfactants, alkyl phosphonate surfactants, alkyl polyglycoside surfactants, alkyl monosaccharide surfactants, alkyl disaccharide surfactants, alkyl sulfosuccinate surfactants, alkyl disulfate surfactants, and alkyl disulfonic acid. Ester surfactants, alkyl sulfonated succinate surfactants, alkyl glucamide surfactants, alkyl taurine ester surfactants, alkyl sarcosine ester surfactants, alkyl glycine ester surfactants, alkyl hydroxyethyl sulfonate surfactants, alkyl dialkanolamide surfactants, alkyl monoalkanolamide surfactants, alkyl monoalkanolamide sulfate surfactants, alkyl dihydroxyacetamide surfactants, alkyl dihydroxyacetamide sulfate surfactants, alkyl glycerol ester surfactants, alkyl glycerol ester sulfate surfactants, alkyl glycerol ether surfactants, alkyl glycerol ether sulfate surfactants, alkyl methyl ester sulfonate surfactants, alkyl polyglycerol ether surfactants, alkyl polyglycerol ether sulfate surfactants Surfactants, alkyl sorbitan ester surfactants, alkyl aminoalkane sulfonate surfactants, alkylamidopropyl betaine surfactants, surfactants based on alkylallylated quaternary ammonium salts, surfactants based on alkyl monohydroxyalkyl-dialkylated quaternary ammonium salts, surfactants based on alkyl dihydroxyalkyl monoalkyl quaternary ammonium salts, alkylated quaternary ammonium salt surfactants, alkyl trimethylammonium quaternary ammonium salt surfactants, surfactants based on alkyl polyhydroxyalkyloxypropyl quaternary ammonium salts, alkyl glycerol ester quaternary ammonium salt surfactants, alkyl ethylene glycolamine quaternary ammonium salt surfactants, alkyl monomethyl dihydroxyethyl quaternary ammonium surfactants, alkyl dimethyl monohydroxyethyl quaternary ammonium surfactants, alkyl trimethylammonium surfactants, and surfactants based on alkyl imidazolines. Surfactants, olefin-2-yl-succinate surfactants, alkyl α-sulfonated carboxylic acid surfactants, alkyl α-sulfonated carboxylic acid alkyl ester surfactants, α-olefin sulfonate surfactants, alkylphenol ethoxylate surfactants, alkylbenzene sulfonate surfactants, alkyl sulfobetaine surfactants, alkyl hydroxysulfobetaine surfactants, alkyl ammonium carboxylic acid betaine surfactants, alkyl sucrose ester surfactants, alkyl alkanolamide surfactants, alkyl di(polyoxyethylene) monoalkylammonium surfactants, alkyl mono(polyoxyethylene) dialkylammonium surfactants, alkyl benzyl dimethylammonium surfactants, alkyl aminopropionate surfactants, alkylamidopropyl dimethylamine surfactants, or mixtures thereof;Furthermore, if Z is a charged portion, it is charged by a suitable metal or organic counterion. Suitable counterions include metal counterions, amines, or alkanolamines, such as C1-C6 alkanolams. More specifically, suitable counterions include Na+, Ca+, Li+, K+, Mg+, such as monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine, dimethylethanolamine, monoisopropanolamine, triisopropanolamine, 1-amino-3-propanol, or mixtures thereof. In one aspect, these compositions contain 5% to 97% of one or more non-isoprene surfactants; and one or more auxiliary cleaning additives; wherein the weight ratio of the surfactant of formula A to the surfactant of formula B is 50:50 to 95:5.

[0688] soap - The compositions described herein may contain soap. Without being theoretically limited, it is desirable to include soap because it partially acts as a surfactant and partially as a detergent builder, and can be used to suppress foaming; furthermore, it can advantageously interact with a variety of cationic compounds in the composition to enhance the softness of textile fabrics treated with the compositions of the present invention. Any soap known in the art for use in laundry detergents can be utilized. In one aspect, these compositions contain from 0 wt.% to 20 wt.%, from 0.5 wt.% to 20 wt.%, from 4 wt.% to 10 wt.%, or from 4 wt.% to 7 wt.%.

[0689] Examples of soaps useful in this article include oleic acid soaps, palmitic acid soaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soaps are in the form of mixtures of fatty acid soaps with different chain lengths and degrees of substitution. One such mixture is top-coated palm kernel fatty acid.

[0690] On one hand, the soap is selected from free fatty acids. Suitable fatty acids are saturated and / or unsaturated and can be obtained from natural sources such as plant or animal esters (e.g., palm kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tallow oil, castor oil, tallow and fish oil, fats and oils and mixtures thereof), or synthesized (e.g., via the oxidation of petroleum or via the Fisher-Tropsch process of hydrogenating carbon monoxide).

[0691] Examples of suitable saturated fatty acids for use in the compositions of the present invention include decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and benzyl acid. Suitable unsaturated fatty acids include palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and ricinoleic acid. Preferred examples of fatty acids are saturated Cn fatty acids, saturated Ci2-Ci4 fatty acids, and saturated or unsaturated Cn to Ci8 fatty acids and mixtures thereof.

[0692] When present, the weight ratio of the fabric-softening cationic auxiliary surfactant to the fatty acid is preferably from about 1:3 to about 3:1, more preferably from about 1:1.5 to about 1.5:1, and most preferably about 1:1.

[0693] The levels of soap and non-soap anionic surfactants mentioned herein are percentages by weight of the detergent composition specified on an acidic basis. However, as is generally understood in the art, in practice, sodium, potassium, or alkanol ammonium bases such as sodium hydroxide or monoethanolamine are used to neutralize anionic surfactants and soaps.

[0694] Water-soluble additives – The compositions of the present invention may comprise one or more water-soluble additives. Water-soluble additives are compounds that solubilize hydrophobic compounds in aqueous solutions (or conversely, solubilize polar substances in nonpolar environments). Typically, water-soluble additives possess both hydrophilic and hydrophobic characteristics (so-called amphiphilic properties, as known from surfactants); however, the molecular structure of water-soluble additives is generally unfavorable for spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler 2007, Current Opinion in Colloid & Interface Science 12: 121-128. Water-soluble additives do not exhibit a critical concentration above which self-aggregation, as observed with surfactants and lipids, occurs, resulting in micelles, thin layers, or other well-defined intermediate phases. Instead, many water-soluble additives exhibit a continuous type of aggregation process in which the size of the aggregates increases with increasing concentration. However, many water-soluble additives alter the phase behavior, stability, and colloidal properties of systems containing substances with polar and non-polar characteristics (including mixtures of water, oil, surfactants, and polymers). Water-soluble additives are routinely used in various industries, from pharmaceuticals and personal care to food and technical applications. The use of water-soluble additives in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0695] Detergents may contain 0 wt.% to 10 wt.%, for example 0 wt.% to 5 wt.%, 0.5 wt.% to 5 wt.%, or from 3 wt.% to 5 wt.%, of a water-soluble additive. Any water-soluble additive known in the art for use in detergents may be used. Non-limiting examples of water-soluble additives include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthylcarbamate, sodium hydroxynaphthyl sulfonate, sodium ethylhexyl sulfonate, and combinations thereof.

[0696] builder- The compositions of the present invention may comprise one or more detergent builders, co-builders, detergent builder systems, or mixtures thereof. When detergent builders are used, the cleaning composition will typically comprise from 0 wt.% to 65 wt.%, at least 1 wt.%, from 2 wt.% to 60 wt.%, or from 5 wt.% to 10 wt.%. In dishwashing cleaning compositions, the level of detergent builders is typically 40 wt.% to 65 wt.% or 50 wt.% to 65 wt.%. The composition may be substantially free of detergent builders; substantially free of zeolite and / or phosphates, meaning "no intentionally added." Typical zeolite detergent builders include zeolite A, zeolite P, and zeolite MAP. Typical phosphate detergent builders are sodium tripolyphosphate.

[0697] Builders and / or co-builders may be, in particular, chelating agents that form water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in detergents may be used. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethanol (MEA), iminodiethanol (DEA), and 2,2',2'-triazine (TEA), and carboxymethyl inulin (CMI), and combinations thereof.

[0698] Cleaning compositions may include a co-agent alone or in combination with a builder (e.g., a zeolite builder). Non-limiting examples of co-agents include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers of (acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphonates, as well as alkyl succinic acids or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-dimethylbis(phosphonic acid) (HEDP), ethylenediaminetetra(methylene)tetra(phosphonic acid) (EDTMPA), diethylenetriaminepenta(methylene)penta(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), and N-(2-sulfoethyl)aspartic acid (SE). AS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), p-aminobenzenesulfonic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), diethanolamine glycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotri(methylenephosphonic acid) (ATMP), and combinations thereof and salts thereof. Other exemplary builders and / or co-builders are described in, for example, WO 2009 / 102854, US 5,977,053.

[0699] Chelating agents and crystal growth inhibitors- The compositions described herein may contain chelating agents and / or crystal growth inhibitors. Suitable molecules include copper, ionic, and / or manganese chelating agents and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), disodium hydroxide of 1,2-dihydroxybenzene-3,5-disulfonic acid, ethylenediamine, diethylenetriamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin, and 2-phosphinocarboxybutane-1,2,4-tricarboxylic acid ( ). AM) and its derivatives. Typically, the composition may contain chelating agents or crystal growth inhibitors ranging from 0.005 wt.% to 15 wt.% or from 3.0 wt.% to 10 wt%.

[0700] bleaching components - Suitable bleaching components for incorporation into the methods and compositions of the present invention comprise one bleaching component or a mixture of more than one bleaching component. Suitable bleaching components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, and mixtures thereof. Typically, when using bleaching components, the compositions of the present invention may contain from 0 wt.% to 30 wt.%, from 0.00001 wt.% to 90 wt.%, from 0.0001 wt.% to 50 wt.%, from 0.001 wt.% to 25 wt.%, or from 1 wt.% to 20 wt.%. Examples of suitable bleaching components include:

[0701] (1) Preformed peracid: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of: preformed peroxyacids or their salts, typically peroxycarboxylic acids or their salts, or peroxysulfuric acid or its salts.

[0702] The pre-formed peroxyacid or its salt is preferably a peroxycarboxylic acid or its salt, typically having a chemical structure corresponding to the following chemical formula:

[0703]

[0704] Where: R 14 Selected from alkyl, aralkyl, cycloalkyl, aryl, or heterocyclic groups; R 14 The functional group can be straight-chain or branched, substituted or unsubstituted; and Y is any suitable counterion that achieves charge neutrality, preferably selected from hydrogen, sodium, or potassium. Preferably, R... 14 Is it a linear or branched, substituted or unsubstituted C? 6-9Alkyl group. Preferably, the peroxy acid or its salt is selected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, and their salts, or any combination thereof. Particularly preferred peroxy acids are phthalimide-peroxy-alkanoic acids, especially ε-phthalimide-peroxyhexanoic acid (PAP). Preferably, the peroxy acid or its salt has a melting point in the range of 30°C to 60°C.

[0705] The preformed peroxyacid or its salt can also be persulfate or its salt, typically having a chemical structure corresponding to the following chemical formula:

[0706]

[0707] Where: R 15 Selected from alkyl, aralkyl, cycloalkyl, aryl, or heterocyclic groups; R 15 The group can be straight-chain or branched, substituted or unsubstituted; and Z is any suitable counterion that achieves charge neutrality, preferably selected from hydrogen, sodium, or potassium. Preferably, R... 15 Is it a linear or branched, substituted or unsubstituted C? 6-9 Alkyl groups. Preferably, such bleaching components may be present in the compositions of the present invention in an amount from 0.01 wt.% to 50 wt.% or from 0.1 wt.% to 20 wt.%.

[0708] (2) Hydrogen peroxide sources include, for example, inorganic hydrogen peroxide salts, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, perphosphates, sodium salts of persilicates, and mixtures thereof. In one aspect of the invention, inorganic hydrogen peroxide salts are, for example, those selected from the group consisting of perborates, sodium salts of percarbonates, and mixtures thereof. When used, inorganic hydrogen peroxide salts are typically present in amounts from 0.05 wt% to 40 wt% or 1 wt% to 30 wt% of the total composition and are typically incorporated into such compositions as crystalline solids that can be coated. Suitable coatings include: inorganic salts, such as alkali metal silicates, carbonates, or borates, or mixtures thereof, or organic materials, such as water-soluble or water-dispersible polymers, waxes, oils, or fatty soaps. Preferably, such bleaching components may be present in the compositions of the invention in amounts from 0.01 wt.% to 50 wt.% or 0.1 wt.% to 20 wt.%.

[0709] (3) The term bleaching activator, as used herein, refers to a compound that reacts with hydrogen peroxide via hydrolysis to form a peracid. The peracid formed in this manner constitutes an activated bleaching agent. Suitable bleaching activators for use herein include those belonging to the ester, imide, or anhydride class. Suitable bleaching activators are those having R-(C=O)-L, wherein R is an alkyl group (preferably branched), having 6 to 14 carbon atoms when the bleaching activator is hydrophobic, or 8 to 12 carbon atoms, and having less than 6 carbon atoms or less than 4 carbon atoms when the bleaching activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives—especially benzenesulfonates. Suitable bleaching activators include dodecyloxybenzenesulfonate, decyloxybenzenesulfonate, decyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecyloxy)benzene-1-sulfonate (LOBS), 4-(decyloxy)benzene-1-sulfonate, 4-(decyloxy)benzoate (DOBS or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. The family of bleaching activators disclosed in EP 624154, and particularly preferred in that family, is triethyl acetyl citrate (ATC). ATC, or short-chain triglycerides (like triacetin), has the advantage of being environmentally friendly. Furthermore, triethyl acetylacetonate (ATC) and triacetin exhibit good hydrolytic stability in the product during storage and are effective bleaching activators. Finally, ATC is versatile because the citrate released during the hydrolysis reaction can act as a detergent builder. Alternatively, the bleaching system may contain peracids of the type of amide, imide, or sulfone. The bleaching system may also contain peracids such as 6-(phthalimide)percapanoic acid (PAP). Suitable bleaching activators are also disclosed in WO 98 / 17767. Although any suitable bleaching activator may be used, in one aspect of the invention, the cleaning compositions of this subject may contain NOBS, TAED, or mixtures thereof. When present, peracids and / or bleaching activators are typically present in the composition in amounts of 0.1 wt.% to 60 wt.%, 0.5 wt.% to 40 wt.%, or 0.6 wt.% to 10 wt.%, depending on the fabric and home care composition. One or more hydrophobic peracids or their precursors may be used in combination with one or more hydrophilic peracids or their precursors. Preferably, such bleaching components may be present in the compositions of the present invention in an amount of 0.01 wt.% to 50 wt.% or 0.1 wt.% to 20 wt.%.

[0710] The amounts of hydrogen peroxide source and peracid or bleaching activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

[0711] (4) Diacyl peroxides – Preferred diacyl peroxide bleaching types include those selected from the following formula: R 1 -C(O)-OO-(O)CR 2 , where R 1 Indicates C6-C 18 Alkyl groups, preferably comprising a straight chain having at least 5 carbon atoms and optionally containing one or more substituents (e.g., –N). + (CH3)3, -COOH or -CN) and / or one or more interruptions (e.g. -CONH- or -CH=CH-) inserted between adjacent carbon atoms of an alkyl group at C6-C 12 Alkyl groups, and R 2 This indicates an aliphatic group compatible with the peroxide moiety, such that R 1 and R 2 Together they contain a total of 8 to 30 carbon atoms. In a preferred aspect, R 1 and R 2 It is a straight-chain, unsubstituted C6-C 12 Alkyl chain. Most preferably, R 1 and R 2 They are the same. Diacyl peroxide (where R) 1 and R 2 All are C6-C 12 Alkyl groups are particularly preferred. Preferably, R groups (R...) 1 Or R 2 At least one, most preferably only one, loop without branches or drooping at position α, or preferably no loop without branches or drooping at positions α or β, or most preferably no loop without branches or drooping at positions α, β, or γ. In another preferred embodiment, DAP may be asymmetric, such that R 1 The acyl group is preferably rapidly hydrolyzed to produce a superacid, but the hydrolysis of the R2 acyl group is slow.

[0712] Tetraacyl peroxides with bleaching properties are preferably selected from tetraacyl peroxides of the following formula: R 3 -C(O)-OO-C(O)-(CH2) n -C(O)-OO-C(O)-R 3 , where R 3 It represents C1-C9 alkyl or C3-C7 groups, and n represents an integer from 2 to 12 or 4 to 10 (inclusive).

[0713] Preferably, the diacyl and / or tetraacyl peroxide bleaching agents are present in an amount sufficient to provide at least 0.5 ppm, at least 10 ppm, or at least 50 ppm by weight of the detergent. In a preferred embodiment, these bleaching agents are present in an amount sufficient to provide from 0.5 to 300 ppm, or from 30 ppm to 150 ppm by weight of the detergent.

[0714] Preferably, the bleaching component includes bleaching catalysts (5 and 6).

[0715] (5) Preferably, organic (non-metallic) bleaching catalysts are included, comprising bleaching catalysts capable of accepting oxygen atoms from peroxy acids and / or their salts and transferring those oxygen atoms to an oxidizable substrate. Suitable bleaching catalysts include, but are not limited to: imine salt cations and polyions; imine zwitterions; modified amines; modified amine oxides; N-sulfonyl imides; N-phosphoryl imides; N-acyl imides; thiadiazole dioxides; perfluoroimides; cyclic glycoketones and mixtures thereof.

[0716] Suitable imine-onium cations and polyions include, but are not limited to: N-methyl-3,4-dihydroisoquinolineonium tetrafluoroborate, prepared as described in Tetrahedron [Tetrahedron] 49(2):423-438 (1992) (e.g., compound 4, page 433); N-methyl-3,4-dihydroisoquinolineonium p-toluenesulfonate, prepared as described in US 5,360,569 (e.g., column 11, example 1); and N-octyl-3,4-dihydroisoquinolineonium p-toluenesulfonate, prepared as described in US 5,360,568 (e.g., column 10, example 3).

[0717] Suitable imine-onium zwitterions include, but are not limited to, N-(3-sulfopropyl)-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in US 5,576,282 (e.g., column 31, Example II); N-[2-(sulfonoxy)dodecyl]-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in US 5,817,614 (e.g., column 32, Example V); 2-[3-[(2-ethylhexyl)oxy]-2-(sulfonoxy)propyl]-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in WO 2005 / 047264 (e.g., page 18, Example 8); and ethyl 2-[3-[(2-butyloctyl)oxy]-2-(sulfonoxy)propyl]-3,4-dihydroisoquinolineonium, an inner salt.

[0718] Suitable modified amine oxygen transfer catalysts include, but are not limited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinoline alcohol, which can be prepared according to the method described in Tetrahedron Letters 28(48):6061-6064 (1987). Suitable modified amine oxide oxygen transfer catalysts include, but are not limited to, sodium 1-hydroxy-N-oxo-N-[2-(sulfonoxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

[0719] Suitable N-sulfonylimide oxygen transfer catalysts include, but are not limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide prepared according to the procedure described in Journal of Organic Chemistry 55(4):1254-61 (1990).

[0720] Suitable N-phosphonoimide oxygen transfer catalysts include, but are not limited to, [R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)phosphonamide, which can be prepared according to the method described in Journal of the Chemical Society, Chemical Communications 22:2569-70 (1994).

[0721] Suitable N-acylimide oxygen transfer catalysts include, but are not limited to, [N(E)]-N-(phenylmethylene)acetamide, which can be manufactured according to the procedure described in the Polish Journal of Chemistry 77(5):577-590 (2003).

[0722] Suitable thiadiazole dioxide oxygen transfer catalysts include, but are not limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, which can be prepared according to the method described in US 5,753,599 (column 9, Example 2).

[0723] Suitable perfluoroimine oxygen transfer catalysts include, but are not limited to, (Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butyrylimide fluoride, which can be prepared according to the method described in Tetrahedron Letters 35(34):6329-30(1994).

[0724] Suitable cyclic glycoketone oxygen transfer catalysts include, but are not limited to, 1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexanedione-2,6-pyranose, as prepared in US 6,649,085 (column 12, Example 1).

[0725] Preferably, the bleaching catalyst comprises imine ions and / or carbonyl functional groups, and is generally capable of forming oxaziridinium and / or diethylene oxide functional groups upon accepting oxygen atoms, particularly from peroxy acids and / or their salts. Preferably, the bleaching catalyst comprises oxaziridinium functional groups and / or is capable of forming oxaziridinium functional groups upon accepting oxygen atoms, particularly from peroxy acids and / or their salts. Preferably, the bleaching catalyst comprises cyclic imine ion functional groups, preferably wherein the cyclic portion has a ring size of five to eight atoms (including nitrogen atoms), preferably six atoms. Preferably, the bleaching catalyst comprises aryl imine ion functional groups, preferably bicyclic aryl imine functional groups, and more preferably 3,4-dihydroisoquinolineonium functional groups. Typically, the imine functional group is a quaternary imine functional group and is typically capable of forming a quaternary peroxyimine cation functional group when accepting an oxygen atom, particularly when accepting an oxygen atom from a peroxy acid and / or its salt. In one aspect, the detergent composition comprises a logP having a value not greater than 0, not greater than -0.5, not greater than -1.0, not greater than -1.5, not greater than -2.0, not greater than -2.5, not greater than -3.0, or not greater than -3.5. o / w The bleaching component. The following describes in more detail the components used to determine logP. o / w The method.

[0726] Typically, bleaching agents can produce X values ​​ranging from 0.01 to 0.30, from 0.05 to 0.25, or from 0.10 to 0.20. SO The types of bleaching are described in more detail below for determining X. SO The method. For example, bleaching components with an isoquinolineonium structure can produce bleaching species with a peroxyimine cation structure. In this example, X SO It is X, a type of peroxide imine cation bleaching. SO .

[0727] Preferably, the bleaching catalyst has a chemical structure corresponding to the following chemical formula:

[0728]

[0729] Where: n and m are independently 0 to 4, preferably both n and m are 0; each R 1Independently selected from substituted or unsubstituted groups, the group being selected from the group consisting of: hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocyclic, fused heterocyclic, nitro, halogroup, cyano, sulfonate, alkoxy, ketone, carboxyl, and alkoxycarbonyl; and any two R groups in the connected positions 1 Substituents can merge to form fused aryl, fused carbocyclic, or fused heterocyclic groups; each R 2 Independently selected from substituted or unsubstituted groups, which are independently selected from the group consisting of: hydrogen, hydroxyl, alkyl, cycloalkyl, alkylaryl, aryl, aralkyl, alkylene, heterocyclic, alkoxy, arylcarbonyl, carboxyalkyl, and amide groups; any R 2 It can be used with any other R 2 They are combined to form part of a common ring; any harmonic R 2 They can merge to form a carbonyl group; and any two Rs can be combined to form a carbonyl group. 2 They can merge to form substituted or unsubstituted fused unsaturated portions; R 3 It is C1 to C 20 Substituted or unsubstituted alkyl groups; R 4 Is it hydrogen or Q? t -A portion, where: Q is a branched or unbranched alkene, t = 0 or 1, and A is an anionic group selected from the group consisting of: OSO3 - SO3 - CO2 - OCO2 - OPO3 2- OPO3H - and OPO2 - ;R 5 Is it hydrogen or -CR? 11 R 12 -YG b -Y c -[(CR 9 R 10 ) y -O] k -R 8 Part, wherein: each Y is independently selected from the following groups: O, S, NH, or NR. 8 And each R 8 Independently selected from the group consisting of alkyl, aryl, and heteroaryl groups, wherein the moiety is substituted or unsubstituted, and whether substituted or unsubstituted, the moiety has fewer than 21 carbons; each G is independently selected from the group consisting of CO, SO2, SO, PO, and PO2; R 9 and R 10 Independently selected from the group consisting of: H and C1-C4 alkyl groups; R 11 and R 12Independently choose from the following groups: H and alkyl, or when combined to form a carbonyl group; b = 0 or 1; c can = 0 or 1, but if b = 0, c must = 0; y is an integer from 1 to 6; k is an integer from 0 to 20; R 6 It is H, or an alkyl, aryl, or heteroaryl moiety; said moiety is substituted or unsubstituted; and if X is present, it is a suitable charge-balanced counterion when R 4 When the form is hydrogen, X is preferably present. Suitable X includes, but is not limited to: chlorides, bromides, sulfates, methyl sulfates, sulfonates, p-toluenesulfonates, and boron tetrafluoride phosphates.

[0730] In one aspect of the invention, the bleaching catalyst has a structure corresponding to the following formula:

[0731]

[0732] Where R 13 It is a branched alkyl group containing three to 24 carbon atoms (including branched carbon atoms) or a straight-chain alkyl group containing one to 24 carbon atoms; preferably, R 13 It is a branched alkyl group containing eight to 18 carbon atoms or a straight-chain alkyl group containing eight to eighteen carbon atoms; preferably, R 13 The following are selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isononyl, isodecyl, isothidecyl, isothidecyl, and isopentachidyl; preferably, R 13 Choose from the group consisting of: 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, isotridecyl and isopentadecanyl.

[0733] Preferably, in addition to the bleaching catalyst, particularly an organic bleaching catalyst, the bleaching component also contains a peracid source. The peracid source may be selected from (a) pre-formed peracid; (b) percarbonate, perborate, or persulfate (hydrogen peroxide source), preferably in combination with a bleaching activator; and (c) perhydrolase and esters, used to form the peracid in situ in the presence of water during textile or hard surface treatment steps.

[0734] When present, based on the composition, peracids and / or bleaching activators are typically present in the composition in amounts from 0.1 wt.% to 60 wt.%, from 0.5 wt.% to 40 wt.%, or from 0.6 wt.% to 10 wt.%. One or more hydrophobic peracids or their precursors may be used in combination with one or more hydrophilic peracids or their precursors.

[0735] The amounts of hydrogen peroxide source and peracid or bleaching activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

[0736] (6) Metal-containing bleaching catalysts – the bleaching component can be provided by a catalytic metal complex. One type of metal-containing bleaching catalyst is a catalytic system comprising a transition metal cation (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation) with defined bleaching catalytic activity, an auxiliary metal cation (e.g., zinc or aluminum cation) with little or no bleaching catalytic activity, and an insulator having defined stability constants for both the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts. Such catalysts are disclosed in US 4,...

Claims

1. A lipase variant of a parental lipase derived from the strain Thermomyces lanuginosus, wherein the variant is a variant modified at position 232 corresponding to SEQ ID NO: 1, wherein the modification is an insertion of R232RR and the amino acid sequence of the parental lipase is as shown in SEQ ID NO: 1, and wherein the lipase variant has lipase activity and higher relative washing performance than the lipase shown in SEQ ID NO:

1.

2. The variant of claim 1, having a higher benefit risk factor, higher relative washing performance and / or lower relative odor compared to the lipase shown in SEQ ID NO:

1.

3. A composition comprising a variant as described in any one of claims 1-2.

4. The composition of claim 3, further comprising a surfactant.

5. Use of the variants as described in any one of claims 1-2 for hydrolyzing lipase substrates.

6. A method for cleaning a surface, the method comprising bringing the surface into contact with a variant as described in any one of claims 1-2.

7. A method for hydrolyzing a lipase substrate, the method comprising treating the lipase substrate with a lipase variant as described in any one of claims 1-2.

8. A polynucleotide encoding a variant as described in any one of claims 1-2, wherein the polynucleotide is operatively linked to one or more control sequences that direct the generation of a lipase variant in a recombinant host cell.

9. A nucleic acid construct comprising the polynucleotide as described in claim 8.

10. An expression vector comprising the polynucleotide as described in claim 8.

11. A host cell comprising the nucleic acid construct of claim 9 or the expression vector of claim 10, wherein the host cell is not a plant variety.

12. A method for producing a lipase variant, the method comprising: a. Culture the host cells as described in claim 11 under conditions suitable for expressing the variant; as well as b. Recycle the variant.

13. A method for producing a variant as described in any one of claims 1-2, the method comprising: c. Cultivate transgenic plants or plant cells containing polynucleotides encoding the variant under conditions conducive to the production of the variant; as well as d. Recycle the variant.

14. A whole culture medium formulation or cell culture composition comprising a variant as described in any one of claims 1-2.

15. The variant of claim 1, wherein the variant has a relative washing performance greater than 1.00 when compared with the lipase shown in SEQ ID NO:

1.

16. The variant of claim 1, wherein the variant has a relative washing performance greater than 2.00 when compared with the lipase shown in SEQ ID NO:

1.

17. The variant of claim 1, wherein the relative washing performance is determined using AMSA model J RP: 6°dH, 0.8 g / L or AMSA model B RP: 6°dH, 3.3 g / L or AMSA model B RP: 15°dH, 3.3 g / L.

18. The variant of claim 15, wherein the relative washing performance is determined in a detergent containing an anionic surfactant.

19. The variant of claim 15, wherein the relative washing performance is determined in a detergent comprising linear alkylbenzene sulfonate (LAS) and / or alcohol ethoxysulfate (AEOS).

20. The variant of claim 15, wherein the relative washing performance is determined in a detergent composition comprising a nonionic surfactant.

21. The variant of claim 15, wherein the relative washing performance is determined in a detergent composition comprising an alcohol ethoxylate (AEO).

22. The variant of claim 15, wherein the relative washing performance is determined using AMSA in model detergent B, Diao detergent, model detergent V, model detergent T, model detergent Y or model detergent X.

23. A composition comprising a variant as claimed in any one of claims 1-2 or 15-22.

24. The composition of claim 23, wherein the composition is a detergent composition.

25. The composition of claim 23, wherein the composition is a liquid or powder laundry detergent composition.

26. The composition according to any one of claims 23-25, wherein the composition further comprises a surfactant.

27. Use of the variants as claimed in any of claims 1-2 or 15-22 for hydrolyzing lipase substrates.

28. A method for cleaning a surface, the method comprising contacting the surface with a variant as described in any one of claims 1-2 or 15-22.

29. A method for hydrolyzing a lipase substrate, the method comprising treating the lipase substrate with a lipase variant as described in any one of claims 1-2 or 15-22.

30. A polynucleotide encoding a variant as claimed in any one of claims 1-2 or 15-22, wherein the polynucleotide is operatively linked to one or more control sequences that direct the generation of a lipase variant in a recombinant host cell.

31. A nucleic acid construct comprising the polynucleotide as described in claim 30.

32. An expression vector comprising the polynucleotide as described in claim 30.

33. A host cell comprising a nucleic acid construct as described in claim 31 or an expression vector as described in claim 32, wherein the host cell is not a plant variety.

34. A method for producing a lipase variant, the method comprising: a. Culture the host cells as described in claim 33 under conditions suitable for expressing the variant; as well as b. Recycle the variant.

35. A method for producing a variant as described in any one of claims 1-2 or 15-22, the method comprising: a. Cultivating transgenic plants or plant cells containing polynucleotides encoding the variant under conditions conducive to the production of the variant; as well as b. Recycle the variant.

36. A whole culture medium formulation or cell culture composition comprising a variant as described in any one of claims 1-2 or 15-22.

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