Novel serine protease variants

Abstract

The present application relates to novel serine protease variants.

Description

Technical Field

[0001] This application relates to novel serine protease variants. Background Technology

[0002] Proteases participate in various functions, such as digestion, absorption, and defense in organisms, and are classified into serine proteases, cysteine ​​proteases, aspartic proteases, and metalloproteinases based on the structure of their active sites. Among these enzymes, serine proteases (or serine endopeptidases) are characterized by the presence of active serine residues at their active sites that cleave peptide bonds in proteins, where serine acts as a nucleophilic amino acid at the active site of the protease (Hedstrom, 2002. Chem Rev 102:4501-4524).

[0003] Serine proteases have been used in a variety of applications. In addition to therapeutic applications for treating human diseases (e.g., dissolving blood clots), serine proteases are used not only as components of laundry detergents and contact lens cleaners, but also for the modification of milk proteins, degumming of silk, soaking of leather, hair removal, synthesis of oligopeptides, recovery of silver from lung X-ray films, and the production and improvement of feed and food (Korean Patent Publication No. 10-2005-0068750). Summary of the Invention

[0004] Technical issues

[0005] There is a need in this field to develop serine proteases with improved thermal stability, increased activity, and other properties to achieve greater industrial cost-effectiveness and efficiency.

[0006] Technical solution

[0007] One objective of this application is to provide a serine protease variant.

[0008] Another objective of this application is to provide a polynucleotide encoding a serine protease variant and a vector containing said polynucleotide.

[0009] Another objective of this application is to provide a microorganism comprising at least one of a serine protease variant, a polynucleotide encoding a serine protease variant, and a vector comprising said polynucleotide.

[0010] Another objective of this application is to provide a feed composition comprising at least one of a serine protease variant and a microorganism expressing a serine protease variant.

[0011] Beneficial effects

[0012] Compared with existing serine proteases, the serine protease variant of this application has superior activity and can therefore be used industrially.

[0013] Brief description of the attached figures

[0014] Figure 1 The positions of mutated residues in the tertiary structure of a serine protease variant derived from Thermobifida fusca are shown.

[0015] Detailed description of the invention

[0016] The contents of this application are described in detail below. Furthermore, each description and embodiment disclosed in this application can be applied herein to different descriptions and embodiments. In other words, all combinations of the various components disclosed in this application are included within the scope of this application. Moreover, the scope of this application should not be limited by the description provided below.

[0017] Furthermore, those skilled in the art will recognize or be able to confirm various equivalent embodiments of the specific implementation of this application using no more than conventional experiments. Such equivalent embodiments are intended to be included within the scope of this application.

[0018] One aspect of this application provides a serine protease variant.

[0019] As used in this article, the term "serine protease" refers to an enzyme that belongs to the protease subgroup and has proteolytic activity. Specifically, a serine protease can be an enzyme that degrades proteins by hydrolyzing peptide bonds and has an active serine residue at its active site, and more specifically, an enzyme with a spatial arrangement of amino acid residues of histidine, aspartic acid, and serine (which may be referred to as a catalytic triplet), but is not limited thereto.

[0020] The serine protease according to this application may be derived from microorganisms of the genera *Thermobifida*, *Nocardiopsis*, *Actinorugispora*, or *Spinactinospora*, but is not limited thereto. Specifically, in this application, the wild-type serine protease may be derived from the following serine proteases: *Thermobifida fusca*, *Thermobifida celulosilytica*, *Thermobifida halotolerans*, *Actinorugispora endohytica*, *Spinactinospora alkalitolerans*, *Nocardiopsis composta*, or *Nocardiopsis potens*, but is not limited thereto.

[0021] In one embodiment, the serine protease of this application may be a polypeptide comprising the amino acid sequence shown in SEQ ID NO:31, substantially consisting of the amino acid sequence shown in SEQ ID NO:31, or consisting of the amino acid sequence shown in SEQ ID NO:31, but not limited thereto. In one embodiment, the amino acid sequence of SEQ ID NO:31 may be derived from the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:2, but is not limited thereto.

[0022] In one embodiment, the serine protease of this application may comprise any amino acid sequence from SEQ ID NO:49 to 54, substantially composed of any amino acid sequence from SEQ ID NO:49 to 54, or composed of any amino acid sequence from SEQ ID NO:49 to 54, but is not limited thereto. In one embodiment, the amino acid sequences of SEQ ID NO:49 to 54 may be derived from any amino acid sequence from SEQ ID NO:67 to 72, but is not limited thereto.

[0023] The serine protease of this application may include, but is not limited to, any sequence having the same activity as the amino acid sequence described above. Furthermore, the serine protease may comprise, or consist substantially of, any of the amino acid sequences in SEQ ID NO:31 and 49 to 54, or an amino acid sequence having at least 60% homology or identity with any of the amino acid sequences in SEQ ID NO:31 and 49 to 54, but is not limited thereto. Specifically, the amino acid sequence may comprise any one of the amino acid sequences shown in SEQ ID NO:31 and 49 to 54 or an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with any one of the amino acid sequences shown in SEQ ID NO:31 and 49 to 54. Furthermore, it is clearly understood that any protein having amino acid sequences that include deletions, modifications, substitutions, or additions within a portion of its sequence falls within the scope of this application, provided that the amino acid sequence has the aforementioned homology or identity and an effect equivalent to that of the protein.

[0024] In other words, although the expressions used in this application are "a protein or polypeptide having the amino acid sequence shown in SEQ ID NO:" and "a protein or polypeptide comprising the amino acid sequence shown in SEQ ID NO:", it is clear that any protein having amino acid sequences that include deletions, modifications, substitutions, or additions in a portion of its sequence can also be used in this application, as long as the protein has the same or equivalent activity as the polypeptide composed of the corresponding amino acid sequence. For example, "a polypeptide comprising the amino acid sequence of SEQ ID NO:31" clearly belongs to "a polypeptide composed of the amino acid sequence of SEQ ID NO:31", as long as the former has the same or equivalent activity as the latter.

[0025] As used in this article, the terms “homology” or “identity” refer to the degree of correlation between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity are used interchangeably.

[0026] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms and can be used together with a default gap penalty established by the program to be used. Essentially, homologous or identical sequences can typically hybridize with each other along the entire sequence or at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence under moderately or highly stringent conditions. Polynucleotides containing degenerate codon substitutions are also considered in hybridization.

[0027] Sequence homology, similarity, or identity between any two given polynucleotides or polypeptides can be determined using known computer algorithms, such as the “FASTA” program, by using the default parameters of Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443–453) can be used, which is performed in the Needleman program of the European Molecular Biology Open Software Suite (EMBOSS) package (Rice et al., 2000, Trends Genet. 16:276–277) (version 5.0.0 or later) (including the GCG package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF, et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994 and CARILLO et al. (1988) SIAM). (J Applied Math 48:1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information database can be used to determine homology, similarity, or identity.

[0028] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using computer programs such as GAP, for example Needleman et al., (1970), J Mol Biol. 48:443, and programs disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In short, the GAP program defines similarity as a value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP procedure may include: (1) a binary comparison matrix (containing the same value 1 and different values ​​0) and a weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, published in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979) (or EDNAFULL (EMBOSS version of NCBI NUC4.4) instead of the matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a penalty of 10 for gap opening and 0.5 for gap extension); (3) no penalty for end gaps.

[0029] Furthermore, sequence homology, similarity, or identity between any two given polynucleotides or polypeptides can be confirmed by comparing their sequences under defined, stringent conditions via Southern hybridization, and the appropriate hybridization conditions are within the scope of this application and can be determined by methods well known to those skilled in the art.

[0030] In one embodiment of the protein having a serine protease as described above, the serine protease variant provided in this application may refer to a variant in which an amino acid at a specific position is substituted, thereby having more than 100% of the enzymatic activity compared to the protein before the mutation.

[0031] In one specific embodiment, the variant provided in this application may have more than 100% of the enzyme activity compared to the wild-type enzyme including any of the amino acid sequences in SEQ ID NO:31 and 49 to 54, particularly about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% or higher of the increased enzyme activity, but is not limited thereto.

[0032] The term “about” includes, but is not limited to, all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and all values ​​in the range equal to or similar to the value following the term “about”.

[0033] As used herein, the term "variant" refers to a polypeptide obtained by conserving the substitution and / or modification of at least one different amino acid in the sequence while retaining the function or properties of the protein. Due to the substitution, deletion, or addition of several amino acids, the variant has an amino acid sequence different from the identified sequence. Such variants can typically be identified by modifying one of the aforementioned polypeptide sequences and evaluating the properties of the modified polypeptide. That is, the ability of the variant relative to the native protein can be enhanced, unchanged, or diminished.

[0034] In addition, some variants may include those in which at least a portion (such as the N-terminal leader sequence or transmembrane domain) has been removed. Other variants may include those in which a portion has been removed from the N-terminus and / or C-terminus of the mature protein, or a portion has been added to the N-terminus and / or C-terminus of the mature protein.

[0035] The term “variant” can also be used interchangeably with other terms, such as modified / mutated protein, modification, modified peptide, mutant, mutant protein, and divergent, and any term used to indicate variation can also be used without restriction.

[0036] Variants may have enhanced activity compared to natural wild-type or unmodified proteins, but are not limited to this.

[0037] As used herein, the term "conservative substitution" refers to the substitution of one amino acid by another amino acid having similar structure and / or chemical properties. For example, a variant may have one or more conserved substitutions while retaining one or more biological activities. Such amino acid substitutions can typically occur based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues. For example, among charged amino acids with side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include glutamic acid and aspartic acid; among uncharged amino acids with side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine; among amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine. Variants may also include the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, peptides can be conjugated to the N-terminal signal (or leader) sequence of proteins involved in protein transfer, either co-translationally or post-translationally. The peptide can also be conjugated to different sequences or linkers to identify, purify, or synthesize peptides.

[0038] As used in this article, "serine protease variant" refers to a polypeptide that includes a substitution of at least one amino acid in the amino acid sequence of a polypeptide having serine protease activity.

[0039] The serine protease variant according to this application may include the substitution of amino acids at positions 12 and / or 116 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO:31 with other amino acids. Specifically, the serine protease variant may include the substitution of amino acids at positions 12 and / or 116 of SEQ ID NO:31, and may include amino acid sequences having at least 60% and less than 100% homology or identity with any of the amino acid sequences in SEQ ID NO:31 and 49 to 54.

[0040] In one embodiment, the serine protease variant of this application may include substitutions of amino acids at positions corresponding to the 12th and / or 116th amino acids of SEQ ID NO:31, and may have at least 60% and less than 100% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) homology or identity with any of the amino acid sequences in SEQ ID NO:31 and 49 to 54, but is not limited thereto.

[0041] Meanwhile, since the 12th and 116th amino acids from the N-terminus of SEQ ID NO:31 correspond to the 12th and 116th amino acids from the N-terminus of SEQ ID NO:49 to 54, the description of the positions of amino acids based on SEQ ID NO:31 can also be applied to the 12th and 116th amino acids of any amino acid sequence in SEQ ID NO:49 to 54.

[0042] In one embodiment, the serine protease variant of this application may include substitutions of amino acids at positions corresponding to the 12th and / or 116th amino acids of SEQ ID NO: 54, and may include amino acid sequences having at least 60% and less than 100% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) homology or identity with any of the amino acid sequences in SEQ ID NO: 52 to 54. Specifically, the serine protease variant may include a substitution of an amino acid at the position corresponding to the 12th amino acid of SEQ ID NO:54, and may have at least 60% and less than 100% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) homology or identity with the amino acid sequence of SEQ ID NO:54, but is not limited thereto.

[0043] In one embodiment, the serine protease variant of this application can be a protein in which all amino acids corresponding to positions 12, 116, or 12 and 116 from the N-terminus in any amino acid sequence of SEQ ID NO:31 and 49 to 54 are replaced by other amino acids. "Other amino acids" refers to amino acids different from the original amino acids and is not limited thereto, as long as they are amino acids other than the original amino acids.

[0044] In one embodiment, the serine protease variant according to this application may be a variant in which the phenylalanine at position 12 of any of the amino acid sequences in SEQ ID NO: 31 and 49 to 51 is replaced by glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, tryptophan, valine, methionine, or threonine; and / or the asparagine at position 116 is replaced by glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine, or threonine, but is not limited thereto.

[0045] In one embodiment, the serine protease variant according to this application may be a variant in which the proline at position 12 of any of the amino acid sequences in SEQ ID NO: 52 to 54 is replaced by phenylalanine, glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, asparagine, glutamine, histidine, serine, tyrosine, isoleucine, leucine, lysine, tryptophan, valine, methionine, or threonine; and / or the asparagine at position 116 is replaced by glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine, or threonine, but is not limited thereto.

[0046] Specifically, the variant can be a protein in which the amino acid at position 12 of any of the amino acid sequences in SEQ ID NO:31 and 49 to 54 is replaced by tyrosine (Y), serine (S), alanine (A), or arginine (R); the amino acid at position 116 is replaced by aspartic acid (D), serine (S), threonine (T), or glycine (G); or the amino acids at positions 12 and 116 of the amino acid sequence in SEQ ID NO:31 are replaced by tyrosine (Y) and aspartic acid (D), tyrosine (Y) and serine (S), serine (S) and aspartic acid (D), serine (S) and threonine (T), or alanine (A) and glycine (G), respectively, but are not limited thereto. In one embodiment, the serine protease variant can be a variant in which the proline at position 12 of any of the amino acid sequences in SEQ ID NO:52 to 54 is replaced by tyrosine, alanine, serine, or arginine, but is not limited thereto.

[0047] It is clearly understood that variants in which the amino acid at position 12 and / or position 116 of any of the amino acid sequences in SEQ ID NO:31 and 49-54 is replaced by other amino acids include variants in which the amino acid corresponding to the said position is replaced by other amino acids.

[0048] Additionally, variants include those where the amino acid at position 12 (from the N-terminus) and / or position 116 (corresponding to any of the amino acid sequences in SEQ ID NO:31 and 49 to 54) is selected from any of the amino acid sequences in SEQ ID NO:31 and 49 to 54 or is combined with an amino acid sequence selected from SEQ ID NO:31 and 49 to 54. Any amino acid sequence of NO:31 and 49 to 54 has other amino acid substitutions in an amino acid sequence with at least 60% (60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) homology or identity.

[0049] In one embodiment of the variant, a variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO:31 is replaced by other amino acids may comprise any of the amino acid sequences of SEQ ID NO:32 to 39, substantially consisting of any of the amino acid sequences of SEQ ID NO:32 to 39, or consisting of any of the amino acid sequences of SEQ ID NO:32 to 39, but is not limited thereto.

[0050] In one embodiment of the variant, a variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO:49 is replaced by other amino acids may include the amino acid sequence of SEQ ID NO:55 or 56, consist essentially of the amino acid sequence of SEQ ID NO:55 or 56, or consist of the amino acid sequence of SEQ ID NO:55 or 56, but is not limited thereto.

[0051] In one embodiment of the variant, a variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO:50 is replaced by other amino acids may include the amino acid sequence of SEQ ID NO:57 or 58, consist essentially of the amino acid sequence of SEQ ID NO:57 or 58, or consist of the amino acid sequence of SEQ ID NO:57 or 58, but is not limited thereto.

[0052] In one embodiment of the variant, a variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO:51 is replaced by other amino acids may include the amino acid sequence of SEQ ID NO:59 or 60, consist essentially of the amino acid sequence of SEQ ID NO:59 or 60, or consist of the amino acid sequence of SEQ ID NO:59 or 60, but is not limited thereto.

[0053] In one embodiment of the variant, a variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO:52 is replaced by other amino acids may include the amino acid sequence of SEQ ID NO:61 or 62, consisting essentially of the amino acid sequence of SEQ ID NO:61 or 62, or consisting of the amino acid sequence of SEQ ID NO:61 or 62, but is not limited thereto.

[0054] In one embodiment of the variant, a variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO:53 is replaced by other amino acids may include the amino acid sequence of SEQ ID NO:63 or 64, consisting essentially of the amino acid sequence of SEQ ID NO:63 or 64, or consisting of the amino acid sequence of SEQ ID NO:63 or 64, but is not limited thereto.

[0055] In one embodiment of the variant, a variant in which the amino acid at the position corresponding to the 12th amino acid and / or the 116th amino acid in the amino acid sequence of SEQ ID NO:54 is replaced by other amino acids may include the amino acid sequence of SEQ ID NO:65 or 66, consisting essentially of the amino acid sequence of SEQ ID NO:65 or 66, or consisting of the amino acid sequence of SEQ ID NO:65 or 66, but is not limited thereto.

[0056] In one embodiment, the serine protease variant of this application may include positions corresponding to the 12th and / or 116th positions of any amino acid sequence in SEQ ID NO:31 and 49 to 54, where other amino acids are substituted; may have 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, but less than 100% sequence homology with any amino acid sequence in SEQ ID NO:31 and 49 to 54; and may have serine protease activity.

[0057] The serine protease variants of this application may have enhanced activity compared to unmodified peptides, native wild-type peptides, or unmodified peptides, but are not limited thereto. Furthermore, it is clearly understood that any protein with deletions, modifications, substitutions, or additions in a portion of its amino acid sequence is within the scope of this application, provided that the protein possesses the aforementioned homology and equivalent effects to the described protein.

[0058] Furthermore, it is clearly understood that, apart from mutations in the 12th and / or 116th amino acids or mutations at their respective positions, any variants of the amino acid sequence of the corresponding SEQ ID NO, whether with the addition of nonsense sequences in the forward or reverse direction, or with naturally occurring mutations, or with silent mutations, are not excluded from the scope of this application, provided that the variant has the same or equivalent activity as the variant according to this application.

[0059] Meanwhile, the mature region of the NCBI reference sequence WP_016188200.1 (SEQ ID NO:40) corresponds to the amino acid sequence of SEQ ID NO:31 of this application, and the sequence of SEQ ID NO:40 excluding the signal peptide corresponds to SEQ ID NO:2 of this application.

[0060] As can be clearly understood from the above, the serine protease variants of this application may include the deletion or addition of amino acids that have a minor impact on the properties and secondary structure of the serine protease, wherein the amino acids at positions corresponding to the 12th and / or 116th amino acids of SEQ ID NO:31 are substituted with different amino acids. Furthermore, through sequence alignment known in the art, those skilled in the art can clearly understand that positions 12th and 116th from the N-terminus of SEQ ID NO:31 of this application correspond to positions 193rd and 297th of SEQ ID NO:40 and positions 163rd and 267th of SEQ ID NO:2, and SEQ ID NO:31 is included in both SEQ ID NO:40 and SEQ ID NO:2.

[0061] Therefore, regarding the amino acid sequences of SEQ ID NO:2 and 40, each comprising the amino acid sequence of SEQ ID NO:31, the serine protease variants of this application include variants in which the amino acids corresponding to the 12th and 116th positions of SEQ ID NO:31 (the 163rd and / or 267th amino acids in SEQ ID NO:2 and the 193rd and / or 297th amino acids in SEQ ID NO:40) are respectively substituted. Furthermore, the description given above regarding the amino acids of SEQ ID NO:31 and the 12th and 116th positions also applies to SEQ ID NO:2 and its 163rd and 267th amino acids and SEQ ID NO:40 and its 193rd and 297th amino acids.

[0062] In one embodiment, the serine protease variant of this application may include an amino acid sequence in which the amino acids at positions 12 and 116 corresponding to SEQ ID NO:31 are substituted with other amino acids and may have sequence homology of at least 60% and less than 100% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) with SEQ ID NO:2. In another embodiment, the serine protease variant of this application may include the 163rd and / or 266th amino acids of SEQ ID NO:2 being replaced by other amino acids, and may have at least 60% and less than 100% sequence homology with SEQ ID NO:2, and may have at least 60% sequence homology with any of the amino acid sequences of SEQ ID NO:3 to 10, but is not limited thereto.

[0063] It is also clearly understood that variants of polypeptides comprising any of the amino acid sequences in SEQ ID NO:49 to 54, including the substitution of the 12th and / or 116th amino acids from the N-terminus corresponding to SEQ ID NO:49 to 54, are also included within the scope of the serine proteases of this application.

[0064] The sequence of a polypeptide containing any of the amino acid sequences in SEQ ID NO:49 to 54 can be, for example, the amino acid sequence described in GenBank accession number KUP96625.1 (SEQ ID NO:67), NCBI reference sequence WP_068687914.1 (SEQ ID NO:68), NCBI reference sequence WP_133739400.1 (SEQ ID NO:69), NCBI reference sequence WP_179641868.1 (SEQ ID NO:70), NCBI reference sequence WP_184391208.1 (SEQ ID NO:71), NCBI reference sequence WP_017594871.1 (SEQ ID NO:72), etc.

[0065] Those skilled in the art can identify the amino acids corresponding to the 12th and / or 116th positions from the N-terminus of SEQ ID NO:49-54 in SEQ ID NO:67-72 by sequence alignment known in the art, and apply the description of the 12th and / or 116th positions from the N-terminus of SEQ ID NO:49-54.

[0066] In one embodiment, the serine protease variant of this application may include replacing the 12th and / or 116th amino acids corresponding to any of the amino acid sequences in SEQ ID NO:49 to 54 with other amino acids, and may have at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) homology or identity with any of the amino acid sequences in SEQ ID NO:67 to 72.

[0067] In one embodiment, the serine protease variant of this application may include a substitution of the amino acid corresponding to position 12 in the amino acid sequence shown in SEQ ID NO:54, and may have at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher) homology or identity with the amino acid sequence shown in any one of SEQ ID NO:70 to 72. As an example, serine protease variants may further include a substitution of the amino acid corresponding to position 116 in the amino acid sequence shown in SEQ ID NO:54.

[0068] In one embodiment, the serine protease variant of this application may include a substitution of the amino acid corresponding to position 198 of the amino acid sequence shown in SEQ ID NO:67. The variant may have at least 60% homology or identity with SEQ ID NO:67, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. For example, a variant may include an amino acid sequence in which the amino acid at position 12 corresponding to SEQ ID NO:49 is replaced by another amino acid, and has at least 70% sequence identity with SEQ ID NO:49. As an example, a serine protease variant may further include a substitution of the amino acid at position 302 corresponding to the amino acid sequence shown in SEQ ID NO:67.

[0069] In one embodiment, the serine protease variant of this application may include a substitution of the amino acid corresponding to position 178 of the amino acid sequence shown in SEQ ID NO:68. The variant may have at least 60% homology or identity with SEQ ID NO:68, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. For example, a variant may include an amino acid sequence in which the amino acid at position 12 corresponding to SEQ ID NO:50 is replaced by another amino acid, and has at least 70% sequence identity with SEQ ID NO:50. As an example, a serine protease variant may further include a substitution of the amino acid at position 282 corresponding to the amino acid sequence shown in SEQ ID NO:68.

[0070] In one embodiment, the serine protease variant of this application may include a substitution of the amino acid corresponding to position 207 of the amino acid sequence shown in SEQ ID NO:69. The variant may have at least 60% homology or identity with SEQ ID NO:69, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. For example, a variant may include an amino acid sequence in which the amino acid at position 12 corresponding to SEQ ID NO:51 is replaced by another amino acid, and has at least 70% sequence identity with SEQ ID NO:51. As an example, a serine protease variant may further include a substitution of the amino acid at position 311 corresponding to the amino acid sequence shown in SEQ ID NO:69.

[0071] In one embodiment, the serine protease variant of this application may include a substitution of the amino acid at position 203 corresponding to the amino acid sequence shown in SEQ ID NO:70. The variant may have at least 60% homology or identity with SEQ ID NO:70, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. For example, a variant may include an amino acid sequence in which the amino acid at position 12 corresponding to SEQ ID NO:52 is replaced by another amino acid, and has at least 70% sequence identity with SEQ ID NO:52. As an example, a serine protease variant may further include a substitution of the amino acid at position 303 corresponding to the amino acid sequence shown in SEQ ID NO:70.

[0072] In one embodiment, the serine protease variant of this application may include a substitution of the amino acid at position 201 corresponding to the amino acid sequence shown in SEQ ID NO:71. The variant may have at least 60% homology or identity with SEQ ID NO:71, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. For example, a variant may include an amino acid sequence in which the amino acid at position 12 corresponding to SEQ ID NO:53 is replaced by another amino acid, and has at least 70% sequence identity with SEQ ID NO:53. As an example, a serine protease variant may further include a substitution of the amino acid at position 304 corresponding to the amino acid sequence shown in SEQ ID NO:71.

[0073] In one embodiment, the serine protease variant of this application may include a substitution of the amino acid at position 201 corresponding to the amino acid sequence shown in SEQ ID NO:72. The variant may have at least 60% homology or identity with SEQ ID NO:72, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. For example, a variant may include an amino acid sequence in which the amino acid at position 12 corresponding to SEQ ID NO:54 is replaced by another amino acid, and has at least 70% sequence identity with SEQ ID NO:54. As an example, a serine protease variant may further include a substitution of the amino acid at position 304 corresponding to the amino acid sequence shown in SEQ ID NO:72.

[0074] However, the serine protease variants of this application are not limited to those mentioned above.

[0075] As used herein, the term "corresponding to" refers to an amino acid residue at the position described in the protein or polypeptide, or an amino acid residue that is similar to, identical to, or homologous to the position described in the protein or polypeptide. Identifying the amino acid at the corresponding position can be a specific amino acid that identifies a sequence that references a particular sequence. As used herein, the term "corresponding region" generally refers to a similar or corresponding position in the related or reference protein. For example, any amino acid sequence can be aligned to SEQ ID NO:31, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO:31. For example, sequence alignment algorithms (such as those described in this application) can identify the position of an amino acid or the position where it has been modified (e.g., substituted, inserted, or deleted) compared to the query sequence (also referred to as the "reference sequence").

[0076] For this type of alignment, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443–453) and the Needle program in the EMBOSS software package (The European Molecular Biology OpenSoftware Suite, Rice et al., 2000, Trends Genet.16:276–277) can be used, but the alignment program is not limited to these. The corresponding amino acid residues can be identified through multiple sequence alignment. Examples of known multiple sequence alignment programs in this field include MUSCLE (multiple sequence alignment by logarithmic expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (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 537:39–64; Katoh and Toh, 2010, Bioinformatics 26:1899–1900), and EMBOSS. EMMA, using ClustalW (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22:4673–4680), and can use the basic parameters of each program, but the program is not limited to this.

[0077] Another aspect of this application provides a polynucleotide encoding a serine protease variant.

[0078] As used in this article, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are linked together by covalent bonds in a long chain shape, and generally refers to a DNA or RNA chain of a certain or longer length, and more specifically, to a polynucleotide fragment that encodes a variant.

[0079] The polynucleotide encoding the serine protease variant of this application may include, but is not limited to, any polynucleotide sequence encoding a serine protease variant with enhanced activity according to this application. In one embodiment, the gene encoding the wild-type serine protease of this application may be derived from microorganisms of the genera *Thermobifida*, *Nocardiopsis*, *Actinorugispora*, or *Spinactinospora*, specifically *Thermobifida fusca*, *Thermobifida celulosilytica*, *Thermobifida halotolerans*, *Actinorugispora endohytica*, *Spinactinospora alkalitolerans*, *Nocardiopsis composta*, or *Nocardiopsis potens*, but is not limited to these.

[0080] Within the scope of not altering the amino acid sequence, the polynucleotides of this application may include various modifications made in the coding region of the amino acid sequence of the polypeptide due to codon degeneracy or taking into account the preferred codons of the living organism in which the polypeptide will be expressed. Specifically, any polynucleotide sequence may include coding variants in which, but not limited to, the amino acids at the N-terminal positions of the 12th and / or 116th amino acids of any amino acid sequence corresponding to SEQ ID NO: 31 and 49 to 54 are substituted with other amino acids.

[0081] For example, the polynucleotide of this application may be a polynucleotide sequence encoding a variant of this application. Specifically, the variant is a polypeptide consisting of any amino acid sequence in SEQ ID NO:32 to 39 and 55 to 66, or a polypeptide having a certain homology with the polypeptide, but is not limited thereto.

[0082] In one embodiment, the polynucleotide sequence encoding a polypeptide consisting of any of the amino acid sequences in SEQ ID NO:32 to 39 may consist of any of the polynucleotide sequences in SEQ ID NO:41 to 48, but is not limited thereto.

[0083] As described above, the serine protease variants of this application include variants in which an amino acid corresponding to position 12 and / or position 116 of a sequence is substituted in a polypeptide containing any of the amino acid sequences in SEQ ID NO: 31 and 49 to 54. Therefore, it is clearly understood that polynucleotide sequences encoding such serine protease variants are also included within the scope of this application.

[0084] As an example, variants in SEQ ID NO:2 and SEQ ID NO:40 where the amino acids at positions 12 and 116 corresponding to SEQ ID NO:31 (amino acid 163 and / or amino acid 267 of SEQ ID NO:2 and amino acid 193 and / or amino acid 297 of SEQ ID NO:40) are substituted are also included within the scope of the serine proteases of this application. Therefore, polynucleotide sequences encoding these variants are also included within the scope of this application. For example, a polynucleotide sequence encoding a serine protease variant may be a sequence encoding any of the amino acid sequences in SEQ ID NO:3-10, specifically, a sequence composed of any of the polynucleotide sequences in SEQ ID NO:23-30, but is not limited thereto.

[0085] Additionally, it may include any sequence encoding a protein with variant activity, wherein, under stringent conditions, by hybridization with a probe that can be prepared from a known gene sequence (e.g., a sequence that is fully or partially complementary to a nucleotide sequence), the amino acid at the position corresponding to the 12th and / or 116th amino acid, starting from the N-terminus, of any of SEQ ID NO:31 and 49 to 54 in the variant is replaced by other amino acids, but is not limited thereto.

[0086] The term "strict conditions" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically disclosed in the literature. For example, strict conditions may include conditions under which genes with high homology (at least 40%, particularly at least 90%, more particularly at least 95%, even more particularly at least 97%, and even more particularly at least 99% homology) hybridize, but genes with lower homology do not hybridize; or typical washing conditions for Southern hybridization, i.e., washing is performed once, particularly twice or three times, at salt concentrations and temperatures corresponding to 60°C, 1×SSC, and 0.1% SDS, particularly 60°C, 0.1×SSC, and 0.1% SDS, more particularly 68°C, 0.1×SSC, and 0.1% SDS. However, strict conditions are not limited to these and can be appropriately adjusted by those skilled in the art according to their purpose.

[0087] Hybridization requires two nucleotides to have complementary sequences, but due to the strictness of hybridization, mismatches between bases are possible. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, this application may include not only substantially similar nucleotide sequences, but also isolated polynucleotide fragments complementary to the entire sequence.

[0088] Specifically, homologous polynucleotides can be detected using the hybridization conditions described above at a Tm value of 55°C. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately controlled by those skilled in the art for their purposes.

[0089] The appropriate stringency of polynucleotide hybridization can depend on the length and complementarity of the polynucleotides, parameters well known in the art.

[0090] Another aspect of this application provides a vector containing a multinucleotide encoding a serine protease variant of this application.

[0091] As used herein, the term "vector" refers to a DNA construct containing a nucleotide sequence encoding a target protein polynucleotide operatively linked to an appropriate control sequence to express the target polynucleotide in a suitable host cell. The control sequence may include a promoter capable of initiating transcription, any operon sequence for controlling transcription, a sequence encoding an appropriate mRNA ribosome binding site, and sequences for controlling transcription and translation termination. After transformation into a suitable host cell, the vector may replicate or function independently of the host genome, or it may integrate into the genome itself.

[0092] As used herein, the term "operably ligated" refers to the functional ligation of a polynucleotide sequence encoding the target protein of this application to a promoter sequence that initiates and mediates polynucleotide transcription. Operable ligations can be prepared using genetic recombination techniques known in the art, and site-specific DNA cutting and ligation can be prepared using restriction enzymes, ligases, etc., known in the art, but are not limited thereto.

[0093] There are no particular limitations on the vectors used in this application; any vector known in the art may be used. Examples of commonly used vectors may include plasmids, granules, viruses, and bacteriophages in their natural or recombinant forms. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and vectors based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, and pUB110 can be used as plasmid vectors. Specifically, vectors such as pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pSM704 may be used. There are no particular limitations on the vectors that can be used in this application; any known expression vector may be used.

[0094] In one embodiment, using a vector for chromosome insertion in cells, a polynucleotide encoding a variant target in the chromosome can be replaced with a mutated polynucleotide. Insertion of the polynucleotide into the chromosome can be performed by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may further include a selection marker to confirm chromosome insertion. The selection marker is used to select cells transformed by the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted. Examples of selection markers may include markers that provide a selective phenotype, such as drug resistance, auxotrophic phenotype, resistance to cytotoxic agents, or expression of a surface mutant polypeptide. In an environment treated with a selective agent, only cells expressing the selection marker can survive or exhibit a different phenotype, thus allowing selection of cells to be transformed.

[0095] Another aspect of this application provides a host cell comprising at least one of the serine protease variant of this application, a polynucleotide encoding the variant, and a vector comprising the polynucleotide.

[0096] In particular, the host cell can be a microorganism.

[0097] Microorganisms containing at least one of a serine protease variant, a polynucleotide encoding that variant, and a vector containing that polynucleotide can be prepared, but are not limited to, microorganisms prepared by transformation with a vector containing a polynucleotide encoding the variant.

[0098] Microorganisms can be microorganisms that express serine protease variants.

[0099] As used herein, the term "to be expressed / expressed / expressed protein" refers to the state in which the target protein is introduced into or expressed in a microorganism. For the purposes of this application, "target protein" may be the aforementioned serine protease variant.

[0100] Specifically, the term "introduced protein" can refer to the expression of activity of a specific protein in a microorganism that initially did not possess the protein, or the expression of enhanced activity of the protein compared to its endogenous activity or its activity before modification. For example, protein introduction can refer to the introduction of a polynucleotide encoding a specific protein into the chromosome of a microorganism, or the introduction of a vector containing a polynucleotide encoding a specific protein into a microorganism, thereby exhibiting protein activity.

[0101] Microorganisms can be recombinant microorganisms. Recombination can be achieved through genetic modification (such as transformation).

[0102] As used herein, the term "transformation" refers to the process of introducing a vector containing a polynucleotide encoding a target protein into a host cell to allow the protein encoded by the polynucleotide to be expressed in the host cell. Whether the transformed polynucleotide is inserted into the chromosome of the host cell or located outside the chromosome, both forms of transformed polynucleotides are within the scope of this application, provided that the transformed polynucleotide is expressed in the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide can be introduced into the host cell in any form, as long as the polynucleotide is introduced into the host cell and expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the necessary elements for self-replication. The expression cassette typically includes a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the polynucleotide. The expression cassette can be a self-replicating expression vector. Alternatively, the polynucleotide can be introduced into the host cell in its original form and operably linked to a sequence required for expression in the host cell, but is not limited thereto. Methods for transformation include any methods for introducing the polynucleotide into the cell and can be performed using suitable standard techniques known in the art. For example, conversion methods include, but are not limited to, electroporation, calcium phosphate (Ca(H2PO4)2, CaHPO4 or Ca3(PO4)2) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, natural sensing (e.g., see Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), and lithium acetate-DMSO method.

[0103] The recombinant microorganism can be a microorganism whose serine protease activity is enhanced according to this application.

[0104] "Enhanced activity" can refer to an increase in the activity of a specific protein in a microorganism compared to its endogenous activity or its activity before modification. The term "endogenous activity" can refer to the activity of a specific protein possessed by the parent strain of the microorganism before transformation, when the microorganism is transformed by genetic mutations caused by natural or artificial factors.

[0105] Specifically, the enhancement of the activity of the protein variant in this application can be achieved by at least one of the following methods: increasing the intracellular copy number of the gene encoding the protein variant, introducing a mutation into the expression control sequence of the gene encoding the protein variant, replacing the expression control sequence of the gene encoding the protein variant with a sequence having stronger activity, replacing the chromosomal gene encoding a wild-type protein with serine protease activity with the gene encoding the protein variant, and further introducing a mutation into the gene encoding the protein variant to enhance the activity of the protein variant, but not limited thereto.

[0106] Next, mutations can be introduced into the nucleic acid sequence through deletion, insertion, non-conservative substitution, conserved substitution, or a combination of the above methods to further enhance the activity of the expression control sequence. Alternatively, the expression control sequence can be modified to increase the expression of multiple nucleotides by replacing the nucleic acid sequence with a nucleic acid sequence that has stronger activity, but this is not limited to these methods. The expression control sequence may include promoters, operon sequences, ribosome binding site coding sequences, sequences used to regulate transcription and translation, etc., but is not limited to these.

[0107] Strong promoters used to replace endogenous promoters can be linked upstream of polynucleotide expression units, but the promoters are not limited to this. Examples of known strong promoters include the cj1 to cj7 promoters (US7662943B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the λ phage PR promoter, and the P... L Promoters include, but are not limited to, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, yccA promoter, etc.

[0108] In addition, modifications to polynucleotide sequences on chromosomes can be made by introducing mutations into the expression control sequence through deletion, insertion, non-conservative substitution, conservative substitution, or a combination of the above methods to further enhance the activity of the polynucleotide sequence, or by replacing the sequence with a modified polynucleotide sequence that has stronger activity, but are not limited to these methods.

[0109] Typically, the introduction and enhancement of protein activity can increase the activity or concentration of the corresponding protein from the wild-type or unmodified microbial strain by 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500% to a maximum of 1000% or 2000%, but is not limited thereto.

[0110] The host cell or microorganism according to this application can be any microorganism that expresses a serine protease variant via a polynucleotide containing this application or a vector containing this application. Specifically, examples of host cells or microorganisms may include strains of microorganisms belonging to the genera *Escherichia*, *Serratia*, *Erwinia*, *Enterobacteria*, *Providencia*, *Salmonela*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, *Corynebacterium*, or *Bacilus*, etc. Specifically, host cells or microorganisms may be *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus amyloliquefaciens*, *Bacillus velezensis*, *Escherichia coli*, or *Corynebacterium glutamicum*. The strains of *Aspergillus oryzae* (glutamicum) or *Aspergillus oryzae*, more specifically, *Bacillus subtilis*, but not limited to these.

[0111] Another aspect of this application provides a method for preparing the serine protease variant of this application.

[0112] Methods for preparing variants of this application may include the steps of culturing at least one of a serine protease variant of this application, a polynucleotide encoding the variant, and a vector containing the polynucleotide.

[0113] As used herein, the term "culture" refers to the growth of host cells under appropriately regulated environmental conditions. The culture methods described in this application can be performed using suitable culture media under appropriate culture conditions known in the art. Those skilled in the art can readily adjust and use such culture methods according to the selected strain. Specifically, the culture can be batch culture, continuous culture, and fed-batch culture, but is not limited to these.

[0114] As used herein, "culture medium" refers to a substance that is a mixture of nutrients required for the culture of host cells, providing essential nutrients and growth factors (including water) necessary for survival and growth. Specifically, any culture medium can be used without particular limitation as the culture medium and other culture conditions for culturing the host cells of this application, as long as it is a conventional culture medium for culturing host cells. However, the host cells of this application can be cultured under aerobic conditions in a conventional culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0115] In one embodiment, the method for preparing a variant of the present application may further include the step of recovering the variant of the present application expressed in the culture step.

[0116] In another embodiment, variants expressed during the culture step can be recovered using methods known in the art to which this application pertains. For example, variants can be recovered from nutrient media using conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0117] The recovery method may be to collect variants using suitable methods known in the art, depending on the culture method of the host cells used in this application, such as batch, continuous, or fed-batch culture methods. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out method), extraction, sonication, ultrafiltration, dialysis, various chromatographic methods (e.g., molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography), HPLC, and combinations of these methods may be used to recover variants from the culture medium or host cells using suitable methods known in the art.

[0118] In another embodiment, variants expressed by the host cells during the culture step may not be recycled. In this embodiment, the host cells expressing the variant can themselves be used as a source of the variant.

[0119] Another aspect of this application provides a feed composition comprising at least one of the serine protease variant of this application and a microorganism expressing the serine protease variant.

[0120] The serine protease variant contained in the feed composition of the variants of this application may be included in the feed composition in such a way that the microorganism expressing the serine protease variant is included in the feed composition, or may be in the form of being isolated and purified from the microorganism expressing the serine protease variant, but this application is not limited thereto.

[0121] As used herein, the term "feed composition" means any preparation of any natural or artificial food, diet, etc., or a dietary component intended for animal consumption, ingestion, digestion, or suitability, and feed can be prepared in a variety of forms known in the art.

[0122] Feed compositions can be feed additives.

[0123] There are no particular restrictions on the type of feed, and feeds commonly used in the art can be used. Non-limiting examples of feeds may include: vegetable feeds, such as grains, roots / fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, melons, and grain by-products; and animal feeds, such as proteins, inorganic materials, oils and fats, minerals, single-cell proteins, animal plankton, or food. These feeds may be used alone or in combination of at least two.

[0124] The feed composition of this application may further include at least one selected from the following: organic acids, such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates, such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphates (polyphosphates); and natural antioxidants, such as polyphenols, catechins, α-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid.

[0125] The feed composition of this application may further include at least one selected from the following: excipients, such as amino acids, minerals, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal agents, and microbial preparations in various forms of probiotics; grains (e.g., powdered or ground wheat, oats, barley, corn, and rice); plant protein feeds, including rapeseed, beans, and sunflower as the main components; animal protein feeds, such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products (e.g., dry components formed from various types of dry milk powder and whey powder); lipids (e.g., active ingredients, such as any animal fats and vegetable oils liquefied by heating); and additives, such as nutritional supplements, digestion and absorption promoters, growth promoters, and preventative agents.

[0126] The feed composition of this application may be in the form of a dry or liquid formulation, and may further include excipients for use in the feed. Excipients for use in the feed may be, for example, zeolite, corn flour, rice bran, etc., but are not limited thereto.

[0127] In addition to serine protease variants, the feed compositions of this application may also include enzyme preparations. For example, the feed compositions may further include at least one enzyme selected from: lipid-degrading enzymes, such as lipases; phytases that degrade phytic acid into phosphate and phosphatidylinositol; amylases that catalyze the hydrolysis of α-1,4-glycosidic bonds contained in starch, glycogen, etc.; phosphatases that catalyze the hydrolysis of organophosphates; maltases that catalyze the formation of two glucose molecules from maltose; and invertases that catalyze the hydrolysis of sucrose into a glucose-fructose mixture. However, the feed compositions of this application are not limited thereto.

[0128] The feed composition of this application can be administered to animals alone or in combination with other feed additives contained in an edible carrier. Furthermore, the feed composition can be readily administered as a feed additive or supplemental feed, applied directly to livestock feed or separately from feed, or administered in a single oral formulation or in combination with other ingredients. Additionally, the daily dosage can be used as a once-daily or multiple-daily dose as commonly known in the art.

[0129] Examples of animals to which the feed composition of this application is applied may include, but are not limited to, livestock such as beef cattle, dairy cattle, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs and cats; and poultry such as chicks, hens, chickens, roosters, ducks, geese, turkeys, quails and small birds.

[0130] The amount of serine protease variant included in the feed composition of this application is not particularly limited and can be appropriately adjusted according to the purpose. In one embodiment, a serine protease variant may be included in an appropriate amount for degrading protein source substances while remaining in the digestive tract of livestock for a long period of time, as is generally known in the art to which this application pertains, but the amount of serine protease variant is not limited thereto.

[0131] Another aspect of this application provides a food composition comprising at least one of the serine protease variant of this application and a microorganism expressing the serine protease variant. The serine protease variant can be used in liquid or solid food compositions. Furthermore, the food can be a powder, pill, beverage, tea, or additive to a general food.

[0132] In one implementation, the food may be a group of foods that require proteases, such as dairy products, health functional foods for improving bowel motility and weight loss, and health functional foods for preventing hypertension.

[0133] In another embodiment, serine protease variants can be included in various food compositions as food solubilizers, food softeners, and meat improvers. In various other embodiments, serine protease variants can be added to baking mixtures during the gluten network breakdown step. Alternatively, serine protease variants can be used to catalyze the hydrolysis of food proteins (e.g., milk proteins). Alternatively, serine protease variants can be included in various food compositions to provide or prepare flavorings, reduce bitterness, modify emulsifying properties, generate bioactive peptides, or reduce allergenic antigens in proteins. However, these are merely illustrative embodiments, and the uses of serine protease variants are not limited thereto.

[0134] The amount of serine protease variant contained in the food composition of this application can be appropriately adjusted by those skilled in the art for their purposes.

[0135] Another aspect of this application provides a descaling composition comprising at least one of the serine protease variant of this application and a microorganism expressing the serine protease variant.

[0136] The descaling composition of this application may be in the form of first and second aqueous descaling compositions, non-aqueous liquid descaling compositions, cast solids, coarse particles, granules, compressed tablets, gels, pastes, or slurries. The descaling composition can be used to remove stubborn food stains, food residue films, and other small amounts of food.

[0137] The descaling compositions according to this application may be provided in the form of a descaling composition for cleaning hard surfaces, a descaling composition for cleaning fabrics, a descaling composition for washing dishes, a descaling composition for oral hygiene, a descaling composition for cleaning dentures, or a contact lens cleaning solution. However, the descaling compositions of this application are not limited thereto.

[0138] Another aspect of this application provides a pharmaceutical composition comprising at least one of the serine protease variant of this application and a microorganism expressing the serine protease variant.

[0139] The pharmaceutical compositions of this application can be used as pharmaceutical compositions of digestive enzymes to improve digestive diseases, digestive disorders and postoperative abnormalities of the digestive tract, thrombolytic or antithrombotic compositions that can be directly applied to blood clots to dissolve fibrin, anti-inflammatory drugs used as part of the body's defense system to remove inflammatory substances or necrotic tissue, or anti-inflammatory drugs to reduce edema after surgery or wounds.

[0140] Depending on the method of use or purpose, the pharmaceutical composition may further include a pharmaceutically acceptable or nutritionally acceptable carrier, excipient, diluent, or auxiliary ingredient. The carrier, excipient, or diluent may include, but is not limited to, at least one selected from, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and saline solution.

[0141] In addition to the uses described above, the serine protease variants of this application, or microorganisms expressing serine protease variants, can also be used for the production of cosmetics, leather processing, pharmaceutical preparation, diagnostic reagent preparation, waste treatment, and the preparation of chemicals for academic research. However, this is merely an exemplary description of the purposes, and the serine protease variants can also be used for any other purpose known in the art of denaturing, degrading, or removing proteinaceous substances. Example

[0142] The present application will be described in more detail below with reference to the following embodiments and experimental examples. However, these embodiments and experimental examples are for illustrative purposes only and are not intended to limit the scope of the present application.

[0143] Example 1. Screening of serine protease variants derived from Thermobifida fusca

[0144] Example 1-1: Preparation of a serine protease library derived from Thermobifida fusca

[0145] Random mutations were introduced into the gene encoding an amino acid (SEQ ID NO:31) corresponding to the maturation region of a serine protease derived from *Thermobifida fusca* using error-prone PCR. Diversify was then used. TM Error-prone PCR was performed using the PCR RandomMutagenesis Kit (Clontech, catalog number 630703), and the PCR conditions used are described in Table 1 below. The mutations were confirmed to be introduced at a frequency of 6.2 mutations / kb.

[0146] Table 1

[0147]

[0148]

[0149] Using the primers shown in Table 2 below, the PCR fragments obtained in the above process were ligated into a vector using the In-FusionR HD Cloning Kit (Clontech) and transformed into DH5α cells to obtain colonies. The plasmids in the resulting colonies were purified to obtain a size of approximately 5 × 10⁻⁶. 4 The library.

[0150] Table 2

[0151] Template DNA (pBE-S-TAP) SEQ ID NO:1 forward primer SEQ ID NO: 13 reverse primer SEQ ID NO: 14

[0152] Examples 1-2: Screening of serine protease libraries derived from Thermobifida fusca

[0153] The protease library prepared in Example 1-1 was used to transform and screen Bacillus subtilis strain LB700, which readily releases proteins. Screening was performed using a two-stage method. In the first stage, the Bacillus subtilis strain transformed with the library was inoculated onto 2% skim milk plates, and desired colonies were selected based on halo size. Transformation of Bacillus subtilis was performed according to the Groningen method, and the composition of the skim milk used in the screening is shown in Table 3 below.

[0154] Table 3

[0155]

[0156]

[0157] (per 1L)

[0158] The second stage involved reselecting colonies selected in the first stage using azocasein colorimetric assay. Brain-heart perfusion (BHI, bd, catalog number 53286) liquid medium containing kanamycin antibiotic was added to 96-well plates, and the colonies selected in the first stage were inoculated into them, followed by incubation at 37°C for 20 to 24 hours. After incubation, a supernatant containing the enzyme was obtained by centrifugation, and the supernatant was mixed with an equal volume of 2% (w / v) azocasein as substrate, and then reacted at 37°C for 1 hour. The reaction was terminated by adding 3 volumes of 10% trichloroacetic acid (TCA) to the enzyme reaction solution, and the coagulated protein was removed by centrifugation. A colorimetric reaction was performed by mixing the product with an equal volume of NaOH, and the absorbance was measured at 440 nm to compare the degree of color development. Using this method, colonies with an absorbance increase of 150% or more compared to wild-type serine proteases were selected.

[0159] Example 2. Preparation and activity evaluation of selected variants

[0160] Example 2-1: Variant Preparation

[0161] As a result of analysis of the screened variant sequences, it was confirmed that amino acids 12 (phenylalanine, Phe) and 116 (asparagine, Asn) of SEQ ID NO:31 were substituted with tyrosine (Tyr) and aspartic acid (Asp), respectively. Figure 1 The location of the mutation based on the amino acid sequence of SEQ ID NO: 2 is shown in the figure. Two selected mutations (F12 and N116) were reintroduced into the pBE-S-TAP plasmid as single mutations via site-directed mutagenesis. The activity of strains with double mutations and single mutations was compared with that of the wild-type strain. Primers used to prepare the variants are shown in Table 4 below.

[0162] Table 4

[0163]

[0164]

[0165] Example 2-2: Activity Evaluation

[0166] After transforming Bacillus subtilis strain LB700 with the prepared plasmid, the activity of the transformed strain was evaluated using N-succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Sigma, catalog number S7388, hereinafter referred to as SUC-AAPF-pNA) peptide as a substrate. The transformed Bacillus subtilis strain was inoculated into brain-heart perfusion (BHI, bd, catalog number 53286) liquid medium containing kanamycin antibiotic and cultured at 37°C for 20 to 24 hours. A portion of the culture medium, excluding cells, was mixed with 25 mM Tris-HCl (pH 7.5) buffer and 1 mM Suc-AAPF-pNA, and then reacted at 37°C for 30 minutes. The absorbance of the reaction solution was measured at 410 nm. The extinction coefficient of p-nitroaniline produced by the enzyme known in the literature was 8800 M at 410 nm. -1 cm -1 The enzyme units were calculated based on this (Barrett, AJ, Cathepsin G. Methods Enzymol., 80, Pt.C, 561-565, (1981)). The measured activities are shown in Table 5 below.

[0167] Table 5

[0168] Enzyme activity (units / mL) wild type 16.3 F12Y 34.0 F12YN116D 63.8

[0169] The measurements confirmed that the activities of the F12Y and F12YN116D variants were approximately 2.1-fold and 3.9-fold higher than the wild type at pH 7.5 and 37°C, respectively.

[0170] Examples 2-3: Evaluation of thermal stability

[0171] The experiments described below were conducted to demonstrate the effect of introducing mutations on thermal stability.

[0172] Specifically, after placing the samples used in Examples 2-2 at room temperature, 70°C, 80°C, and 90°C for 5 minutes, respectively, the enzyme activity of the samples was measured for activity evaluation. The measured activities are shown in Table 6 below.

[0173] Table 6

[0174]

[0175] The measurements confirmed that even at 80°C, the F12Y and F12YN116D variants exhibited approximately 2-fold and 4-fold higher enzyme activities than the wild-type strain, respectively. This demonstrates that the serine protease variants of this application maintain high activity even at high temperatures, and therefore can be effectively used in industrial applications.

[0176] Example 3. Preparation and screening of saturated mutagenesis libraries

[0177] Example 3-1. Preparation of saturated mutagenesis library of F12 and N116 residues

[0178] To confirm the effect of replacing F12 and N116 residues (i.e., the previously selected variants) with residues other than tyrosine and aspartic acid on activity, a saturated mutagenesis library of these two residues was prepared.

[0179] Two PCR fragments were obtained using pBE-S-TAP plasmid as template and primer pairs SEQ ID NO:11 and 12, and SEQ ID NO:13 and 14, respectively. The fragments were ligated using the In-Fusion HD cloning kit and then transformed into DH5α cells to obtain colonies. The plasmids in the obtained colonies were purified to obtain cells approximately 4 × 10⁻⁶ in size. 3 The library.

[0180] Table 7

[0181] Template DNA (pBE-S-TAP) SEQ ID NO:1 Saturation mutagenesis_F_1 SEQ ID NO: 19 Saturated mutagenesis_R_1 SEQ ID NO: 20 Saturation mutagenesis_F_2 SEQ ID NO: 21 Saturated mutagenesis_R_2 SEQ ID NO: 22

[0182] Example 3-2: Screening and activity evaluation of saturated mutagenesis libraries

[0183] The saturated mutagenesis libraries prepared in Example 3-1 were screened in the same manner as in Examples 1-2. The activities of these variants were evaluated by screening, sequence analysis, and using Suc-AAPF-pNA as a substrate. Variants exhibiting the same or increased activity compared to the F12YN116D variant were selected.

[0184] Table 8

[0185] Enzyme activity (units / mL) wild type 23.52 F12YN116D 65 F12YN116S 77.35 F12SN116D 61.75 F12SN116T 117.65 F12AN116G 94.9 F12A 94.25 F12R 58.5

[0186] The results showed that the F12 and N116 variants had increased activity, even though their residues were each replaced by different amino acids other than tyrosine and aspartic acid as demonstrated in Example 2 (e.g., F12S, F12A, F12R, N116S, N116T, N116G).

[0187] Example 3-3: Preparation and Activity Evaluation of F12S Variant

[0188] After the mutant (F12S) was reintroduced into the pBE-S-TAP plasmid as a single mutant through site-directed mutagenesis, the activity of the variant was compared with that of the wild type.

[0189] The activity of the variant (F12S) was evaluated using Suc-AAPF-pNA as a substrate pair.

[0190] [Table 9]

[0191]

[0192]

[0193] The measurements confirmed that the activity of the F12S variant increased by approximately 1.5 times.

[0194] Example 4. Confirmation of the effect of residues 12 and 116 of a serine protease-like protein derived from Thermobifida fusca.

[0195] Example 4-1: Preparation of wild type and variant

[0196] To investigate whether the amino acid residues corresponding to positions 12 and 116 of SEQ ID NO:31 affect the increased activity of other serine proteases with sequence homology to SEQ ID NO:31, the 12th and 116th residues of serine proteases with sequence homology of 87.2%, 81.8%, 81.3%, 73.8%, 69.9%, and 66.7%, respectively, were replaced with tyrosine (Y) and aspartic acid (D). The activities of the serine proteases were then compared with those of the wild-type proteases. The source and sequence information of each serine protease are shown in Table 10.

[0197] [Table 10]

[0198]

[0199] The variants shown in SEQ ID NO:55 to 66 were prepared by replacing residues 12 and 116 of various serine proteases with tyrosine (Y) and aspartic acid (D).

[0200] Example 4-2: Activity Evaluation

[0201] The prepared plasmid was transformed and expressed in Bacillus subtilis LB700 strain, and its activity was evaluated using the same method as described in Examples 2-2. The measured activities are shown in Table 11.

[0202] [Table 11]

[0203]

[0204]

[0205] The measurements confirmed that when a mutation was introduced at residue 12, the activity of six proteins with sequence homology to serine proteases derived from *Thermobifidafusca* also increased, as did the serine proteases derived from *Thermobifidafusca*. Based on these facts, residues 12 and 116 are confirmed to be important residues exhibiting serine protease activity, and that enzyme activity can be enhanced by substituting these residues with different amino acids, as demonstrated by SEQ ID NO: 31. Therefore, the serine protease variant with increased enzyme activity of this application can be effectively used in industry.

[0206] In summary, those skilled in the art will understand that this application can be implemented in other specific forms without altering the technical concept or essential characteristics of this application. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of this application. Rather, this application is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the substance and scope of this application as defined in the appended claims. sequence list <110> CJ CheilJedang Corporation <120> New serine protease variants <130> OPA22007 <150> KR 10-2021-0032885 <151> 2021-03-12 <160> 72 <170> KoPatentIn 3.0 <210> 1 <211> 6922 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> pBE‐S‐TFP <400> 1 actagtgttc ttttctgtat gaaaatagtt atttcgagtc tctacggaaa tagcgagaga tgatatacct aatagagat aaaatcatct caaaaaaatg ggtctactaa aatattattc catctattac aataaattca cagaatagtc ttttaagtaa gtctactctg aacttaagca aaaggagagg gacgcgtgtg agaagcaaaa aattgtggat cagcttgttg tttgcgttaa cgttaatctt tacgatggcg ttcagcaaca tgtctgcgca ggctgcggcc ggtgcacata 360. tgcaagagct ggcgttgaaa cgggacctcg gcctctctga cgcagaagta gccgaactcc gggctgctga ggcggaagcg gtcgagctcg aggaggagct ccgcgattca ttagggtcag 420 acttcggcgg tgtatatctg gatgctgaca ccaccgaat tacggtcgcg gtaaccgacc cggcagcggt aagtcgtgtc gacgcggatg atgtcacagt tgatgttgtc gatttcgggg 540 aaacagcttt gaatgatttt gtggcttcat taaatgccat tgccgacacg gcagacccta 600 aagtcactgg atggtatacc gatctcgaaa gtgatgccgt agtcattacg accttgcgtg 660 gcgggactcc tgctgccgag gaacttgctg agagagcggg tctcgacgaa agagccgttc 720 ggattgtgga agaagatgaa gaaccacaga gcttggctgc aattattggt ggaaacccgt 780 actatttcgg aaattacaga tgcagtatcg ggtttagtgt ccgtcagggc tctcaaacgg 840 gattcgcgac cgcaggccac tgcggatcca cggggacgcg tgtgtcttct cttcaggaa 900 cagttgcagg aagttatttc ccgggtcgcg atatgggctg ggtgcggatt acatcagcag 960 atactgtaac accactcgta aatcggtata atgggggaac tgttacggtc actgggtcac 1020 aagaagctgc caccggatcc tccgtttgtc gctctggagc aacaacgggc tggcgctgcg 1080 gaactatcca atcaaaaaac caaacggttc gctatgcaga agggactgtt actggtttaa 1140 caagaactac agcctgtgct gaaggtgggg attctggagg gccatggctc acaggtagcc 1200 aggcgcaagg ggttacaagc ggcggaacag gcgattgcag aagtggaggg attacctttt 1260 tccaaccaat caatccattg cttagctatt tcggccttca attagtgacc ggctgaaagc 1320 ttgtcgacct gcagtctaga catcaccatc atcaccacta atgcggtagt ttatcacagt 1380 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc 1440 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc 1500 tatttcactt tttgcattct acaaactgca taactattat gtaaatcgct cctttttagg 1560 tggcacaaat gtgaggcatt ttcgctcttt ccggcaacca cttccaagta aagtataaca 1620 cactatactt tatattcata aagtgtgtgc tctgcgaggc tgtcggcagt gccgaccaaa 1680 accataaaac ctttaagacc tttctttttt ttacgagaaa aaagaaacaa aaaaacctgc 1740 cctctgccac ctcagcaaag gggggttttg ctctcgtgct cgtttaaaaa tcagcaaggg 1800 acaggtagta ttttttgaga agatcactca aaaaatctcc acctttaaac ccttgccaat 1860 ttttattttg tccgttttgt ctagcttacc gaaagccaga ctcagcaaga ataaaatttt 1920 tattgtcttt cggtttcta gtgtaacgga caaaaccact caaaataaa aagatacaag 1980 agaggtctct cgtatctttt attcagcaat cgcgcccgat tgctgaacag attaataata 2040 gattttagct ttttatttgt tgaaaaaagc taatcaaatt gttgtcggga tcaattactg 2100 caaagtctcg ttcatcccac cactgatctt ttaatgatgt attggggtgc aaaatgccca 2160 aaggcttaat atgttgatat aattcatcaa ttccctctac ttcaatgcgg caactagcag 2220 taccagcaat aaacgactcc gcacctgtac aaaccggtga atcattacta cgagagcgcc 2280 agccttcatc acttgcctcc catagatgaa tccgaacctc attacacatt agaactgcga 2340 atccatcttc atggtgaacc aaagtgaaac ctagtttatc gcaataaaaa cctatactct 2400 ttttaatatc cccgactggc aatgccggga tagactgtaa cattctcacg cataaaatcc 2460 cctttcattt tctaatgtaa atctattacc ttattattaa ttcaattcgc tcataattaa 2520 tcctttttct tattacgcaa aatggcccga tttaagcaca ccctttattc cgttaatgcg 2580 ccatgacagc catgataatt actaatacta ggagaagtta ataaatacga gcaaaaggcc 2640 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 2700 ccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 2760 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 2820 tgccgcttac cggatacctg tccgccttc tcccttcggg aagcgtggcg cttctcata 2880 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 2940 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 3000 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 3060 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 3120 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 3180 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggtttttt gtttgcaagc 3240 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 3300 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tttcaaaaa 3360 ggatcttcac ctagatcctt ttaaattaaa aatgaagtttt taaatcaatc taaagtatat 3420 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3480 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3540 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3600 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3660 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 3720 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 3780 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 3840 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 3900 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 3960 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 4020 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 4080 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 4140 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 4200 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 4260 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 4320 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 4380 agaaaaataa acaataggg gttccgcgca cattccccg aaaagtgcca cctgacgtct 4440 aagaaaccat tattatcatg acattaacct ataaaatag gcgtatcacg aggccctttc 4500 gtctcgcgcg ttcggtgat gacggtgaaa acctctgaca gtaaccaaca tgattaacaa 4560 ttattagagg tcatcgttca aaatggtatg cgttttgaca catccactat atatccgtgt 4620 cgttctgtcc actcctgaat cccattccag aaattctcta gcgattccag aagtttctca 4680 gagtcggaaa gttgaccaga cattacgaac tggcacagat ggtcataacc tgaaggaaga 4740 tctgattgct taactgcttc agttaagacc gaagcgctcg tcgtataaca gatgcgatga 4800 tgcagaccaa tcaacatggc acctgccatt gctacctgta cagtcaagga tggtagaaat 4860 gttgtcggtc cttgcacacg aatattacgc catttgcctg catattcaaa cagctcttct acgataaggg cacaaatcgc atcgtggaac gtttgggctt ctaccgattt agcagtttga tacactttct ctaagtatcc acctgaatca taaatcggca aatagagaa aaattgacca tgtgtaagcg gccaatctga ttccacctga gatgcataat ctagtaga ctcttcgcta tcaaattca cttccacctt ccactcaccg gttgtccatt catggctgaa ctctgcttcc 5220. tctgttgaca tgacacacat catctcaata tccgaatagg gcccatcagt ctgacgacca agagagccat aaacaccat agccttaaca tcatccccat atttatccaa tattcgttcc ttaatttcat gaacaatctt cattctttct tctctagtca ttattattgg tccattcact attctcattc ccttttcaga tattttaga tttgcttttc tattaga tattttggaga gcaccgttct tattcagcta ttataactc gtcttccta gcatccttca atccttttaa 5520. 5520. 5520. 5520. 5520. 5520. 5520. 5520. 5520. 5520. 5520 aaatatttt tccgttccca attccacatt gcaatatag aaaatccatc ttcatcggct ttttcgtcat catctgtatg aatcaaatcg ccttcttctg tgtcatcaag gtttaatttt 5640 ttatgtattt cttttaacaa accaccatag gagattaacc ttttacggtg taaaccttcc 5700 tccaaatcag acaaacgttt caaattcttt tcttcatcat cggtcataaa atccgtatcc 5760 tttacaggat attttgcagt ttcgtcaatt gccgattgta tatccgattt atatttattt 5820 ttcggtcgaa tcatttgaac ttttacattt ggatcatagt ctaatttcat tgcctttttc 5880 caaaattgaa tccattgttt ttgattcacg tagttttctg tattcttaaa ataagttggt 5940 tccacacata ccaatacatg catgtgctga ttataagaat tatctttatt atttattgtc 6000 acttccgttg cacgcataaa accaacaaga tttttattaa tttttttata ttgcatcatt 6060 cggcgaaatc cttgagccat atctgacaaa ctcttattta attcttcgcc atcataaaca 6120 tttttaactg ttaatgtgag aaacaaccaa cgaactgttg gcttttgttt aataacttca 6180 gcaacaacct tttgtgactg aatgccatgt ttcattgctc tcctccagtt gcacattgga 6240 caaagcctgg atttacaaaa ccacactcga tacaactttc tttcgcctgt ttcacgattt 6300 tgtttatact ctaatatttc agcacaatct tttactcttt cagccttttt aaattcaaga 6360 atatgcagaa gttcaaagta atcaacatta gcgattttct tttctctcca tggtctcact 6420 tttccacttt ttgtcttgtc cactaaaacc cttgattttt catctgaata aatgctacta 6480 ttaggacaca taatattaaa agaaaccccc atctatttag ttatttgttt agtcacttat 6540<000\0604> aactttaaca gatggggttt ttctgtgcaa ccaattttaa gggttttcaa tactttaaaa 6600 cacatacata ccaacacttc aacgcacctt tcagcaacta aaataaaaat gacgttattt 6660 ctatatgtat caagataaga aagaacaagt tcaaaaccat caaaaaaaga caccttttca 6720<\ ggtgcttttt ttattttata aactcattcc ctgatctcga cttcgttctt tttttacctc 6780 tcggttatga gttagttcaa attcgttctt tttaggttct aaatcgtgtt tttcttggaa 6840 ttgtgctgtt ttatccttta ccttgtctac aaacccctta aaaacgtttt taaaggcttt 6900 taagccgtct gtacgttcct aa 6922 <210> 2 <211> 338 <212> PRT <213> Unknown <220> <221> <222> <223> Thermobifida fusca <400> 2 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Phe Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Asn Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 3 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163Y) <400> 3 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15<\ Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Tyr Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Asn Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 4 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163Y_N267D) <400> 4 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Tyr Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Asp Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 5 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163Y_N267S) <400> 5 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Tyr Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Ser Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 6 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163S_N267D) <400> 6 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Ser Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Asp Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 7 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163S_N267T) <400> 7 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Ser Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Thr Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 8 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163A_N267G) <400> 8 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Ala Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Gly Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 9 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163A) <400> 9 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Ala Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Asn Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335 Thr Gly <210> 10 <211> 338 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163R) <400> 10 Met Gln Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu 1 5 10 15 Val Ala Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu 20 25 30 Glu Leu Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp 35 40 45 Ala Asp Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val 50 55 60 Ser Arg Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly 65 70 75 80 Glu Thr Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp 85 90 95 Thr Ala Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp 100 105 110 Ala Val Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu 115 120 125 Leu Ala Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu 130 135 140 Glu Asp Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro 145 150 155 160 Tyr Tyr Arg Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln 165 170 175 Gly Ser Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly 180 185 190 Thr Arg Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro 195 200 205 Gly Arg Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr 210 215 220 Pro Leu Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser 225 230 235 240 Gln Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr 245 250 255 Gly Trp Arg Cys Gly Thr Ile Gln Ser Lys Asn Gln Thr Val Arg Tyr 260 265 270 Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu 275 280 285 Gly Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly 290 295 300 Val Thr Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe 305 310 315 320 Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val 325 330 335​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <211> twenty three <212> DNA <213> Artificial Sequence <220> <221> <222> <223> pBE-S-vector-F <400> 13 aagcttgtcg acctgcagtc tag 23 <210> 14 <211> twenty four <212> DNA <213> Artificial Sequence <220> <221> <222> <223> pBE-S-vector-R <400> 14 ctgtggttct tcatcttctt ccac 24 <210> 15 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> TAP_F12Y_F <400> 15 gtggaaaccc gtactattac ggaaattaca gatgcag 37 <210> 16 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> TAP_F12Y_R <400> 16 ctgcatctgt aatttccgta atagtacggg tttccac 37 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> TAP_N116D_F <400> 17 ggaactatcc aatcaaaaga ccaaacggtt cgctatgc 38 <210> 18 <211> 38 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> TAP_N116D_R <400> 18 gcatagcgaa ccgtttggtc ttttgattgg atagttcc 38 <210> 19 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Saturation mutagenesis_F_1 <400> 19 gtggaaaccc gtactatnnn ggaaattaca gatgcag 37 <210> 20 <211> 38 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Saturated mutagenesis_R_1 <400> 20 gcatagcgaa ccgtttgnnn ttttgattgg atagttcc 38 <210> twenty one <211> 38 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Saturation mutagenesis_F_2 <400> twenty one ggaactatcc aatcaaaann ncaaacggtt cgctatgc 38 <210> twenty two <211> 37 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Saturated mutagenesis_R_2 <400> twenty two ctgcatctgt aatttccnnn atagtacggg tttccac 37 <210> twenty three <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F12Y) <400> twenty three atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gactcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactattacg gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaaaa ccaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 24 <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163Y_N267D) <400> 24 atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gacttcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactattacg gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaaga ccaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 25 <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163Y_N267S) <400> 25 atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gacttcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactattacg gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaaag tcaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 26 <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163S_N267D) <400> 26 atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gactcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactatagtg gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaaga tcaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 27 <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163S_N267T) <400> 27 atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gacttcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactatagtg gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaaac tcaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 28 <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163A_N267G) <400> 28 atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gacttcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactatgctg gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaagg tcaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 29 <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163A) <400> 29 atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gactcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactatgctg gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaaaa tcaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 30 <211> 1017 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca (F163R) <400> 30 atgcaagagc tggcgttgaa acgggacctc ggcctctctg acgcagaagt agccgaactc 60 [[ID=K28]]cgggctgctg aggcggaagc ggtcgagctc gaggaggagc tccgcgattc attagggtca 120 gacttcggcg gtgtatatct ggatgctgac accaccgaaa ttacggtcgc ggtaaccgac 180 ccggcagcgg taagtcgtgt cgacgcggat gatgtcacag ttgatgttgt cgatttcggg 240 gaaacagctt tgaatgattt tgtggcttca ttaaatgcca ttgccgacac ggcagaccct 300 aaagtcactg gatggtatac cgatctcgaa agtgatgccg tagtcattac gaccttgcgt 360 ggcgggactc ctgctgccga ggaacttgct gagagagcgg gtctcgacga aagagccgtt 420 cggattgtgg aagaagatga agaaccacag agcttggctg caattattgg tggaaacccg 480 tactatagag gaaattacag atgcagtatc gggtttagtg tccgtcaggg ctctcaaacg 540 ggattcgcga ccgcaggcca ctgcggatcc acggggacgc gtgtgtcttc tccttcagga 600 acagttgcag gaagttattt cccgggtcgc gatatgggct gggtgcggat tacatcagca 660 gatactgtaa caccactcgt aaatcggtat aatgggggaa ctgttacggt cactgggtca 720 caagaagctg ccaccggatc ctccgtttgt cgctctggag caacaacggg ctggcgctgc 780 ggaactatcc aatcaaaaaa tcaaacggtt cgctatgcag aagggactgt tactggttta 840 acaagaacta cagcctgtgc tgaaggtggg gattctggag ggccatggct cacaggtagc 900 caggcgcaag gggttacaag cggcggaaca ggcgattgca gaagtggagg gattaccttt 960 ttccaaccaa tcaatccatt gcttagctat ttcggccttc aattagtgac cggctga 1017 <210> 31 <211> 187 <212> PRT <213> Unknown <220> <221> <222> <223> Thermobifida fusca_Mature Region <400> 31 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Phe Gly Asn Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser Gln Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 32<00C1441><211> 187 <212> PRT <213> Artificial Sequence (Note: There might be a misprint in the original Chinese, should be "Artificial Sequence") <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12Y) <400> 32 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Tyr Gly Asn Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser Gln Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125[[ID=了15]] Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 33 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12Y_N116D) It should be noted that there seems to be a misspelling in "了15" in the translation of line 15, which should be " ". This might be an error in the original text or during the copying process. If this is an important text, it is recommended to double-check the original source to ensure accuracy.<400> 33 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Tyr Gly Asn Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser Gln Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asp Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 34 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12Y_N116S) <400> 34 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Tyr Gly Asn Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser Gln Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Ser Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 35 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12S_N116D) <400> 35 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Ser Gly Asn Tyr Arg 1 5 10 15 [[ID=4​20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asp Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 36 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 37 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12A_N116G) <400> 37 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Ala Gly Asn Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser Gln Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Gly Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185<� <210> 38 <211> 187 <&12> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12A) It should be noted that there seems to be an incorrect tag "<� " in the original text which is likely a misrepresentation. I've translated it as best as possible while keeping the incorrect tag as is for the purpose of following the instructions. If this is a critical error in the original, it might need to be corrected before accurate translation work. Also, the tag "<&12>" is likely incorrect as well and is translated as is. If you can provide the correct tags, the translation will be more accurate. <400> 38 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Ala Gly Asn Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser Gln Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 39 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12R) <400> 39 Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr Arg Gly Asn Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser Gln Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg Val Ser Ser Pro Ser 35 40 45 Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Thr Gly 145 150 155 160 Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 180 185 <210> 40 <211> 368 <212> PRT <213> Unknown <220> <221> <222> <223> Thermobifida fusca <400> 40 Met Asn His Ser Ser Arg Arg Thr Thr Ser Leu Leu Phe Thr Ala Ala 1 5 10 15 Leu Ala Ala Thr Ala Leu Val Ala Ala Thr Thr Pro Ala Ser Ala Gln 20 25 30 Glu Leu Ala Leu Lys Arg Asp Leu Gly Leu Ser Asp Ala Glu Val Ala 35 40 45 Glu Leu Arg Ala Ala Glu Ala Glu Ala Val Glu Leu Glu Glu Glu Leu 50 55 60 Arg Asp Ser Leu Gly Ser Asp Phe Gly Gly Val Tyr Leu Asp Ala Asp 65 70 75 80 Thr Thr Glu Ile Thr Val Ala Val Thr Asp Pro Ala Ala Val Ser Arg 85 90 95 Val Asp Ala Asp Asp Val Thr Val Asp Val Val Asp Phe Gly Glu Thr 100 105 110 Ala Leu Asn Asp Phe Val Ala Ser Leu Asn Ala Ile Ala Asp Thr Ala 115 120 125 Asp Pro Lys Val Thr Gly Trp Tyr Thr Asp Leu Glu Ser Asp Ala Val 130 135 140 Val Ile Thr Thr Leu Arg Gly Gly Thr Pro Ala Ala Glu Glu Leu Ala 145 150 155 160 Glu Arg Ala Gly Leu Asp Glu Arg Ala Val Arg Ile Val Glu Glu Asp 165 170 175 Glu Glu Pro Gln Ser Leu Ala Ala Ile Ile Gly Gly Asn Pro Tyr Tyr 180 185 190 Phe Gly Asn Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Gln Gly Ser 195 200 205 Gln Thr Gly Phe Ala Thr Ala Gly His Cys Gly Ser Thr Gly Thr Arg 210 215 220 Val Ser Ser Pro Ser Gly Thr Val Ala Gly Ser Tyr Phe Pro Gly Arg 225 230 235 240 Asp Met Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu 245 250 255 Val Asn Arg Tyr Asn Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu 260 265 270 Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ala Thr Thr Gly Trp 275 280 285 Arg Cys Gly Thr Ile Gln Ser Lys Asn Gln Thr Val Arg Tyr Ala Glu 290 295 300 Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly 305 310 315 320 Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr 325 330 335 Ser Gly Gly Thr Gly Asp Cys Arg Ser Gly Gly Ile Thr Phe Phe Gln 340 345 350 Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu Gln Leu Val Thr Gly 355 360 365 <210> 41 <211> 564 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12Y) <400> 41 ttggctgcaa ttattggtgg aaacccgtac tattacggaa attacagatg cagtatcggg 60 tttagtgtcc gtcagggctc tcaaacggga ttcgcgaccg caggccactg cggatccacg 120 gggacgcgtg tgtcttctcc ttcaggaaca gttgcaggaa gttatttccc gggtcgcgat 180 atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaaaacca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480 gattgcagaa gtggagggat tacctttttc caaccaatca atccattgct tagctatttc 540 ggccttcaat tagtgaccgg ctga 564 <210> 42 <211> 564 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12Y_N116D) <400> 42 ttggctgcaa ttattggtgg aaacccgtac tattacggaa attacagatg cagtatcggg 60 tttagtgtcc gtcagggctc tcaaacggga ttcgcgaccg caggccactg cggatccacg 120 gggacgcgtg tgtcttctcc ttcaggaaca gttgcaggaa gttatttccc gggtcgcgat 180 s atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaagacca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480 gattgcagaa gtggagggat tacctttttc caaccaatca atccattgct tagctatttc 540 ggccttcaat tagtgaccgg ctga 564 <210> 43 <211> 564 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12Y_N116S) <400> 43 ttggctgcaa ttattggtgg aaacccgtac tattacggaa attacagatg cagtatcggg 60 tttagtgtcc gtcagggctc tcaaacggga ttcgcgaccg caggccactg cggatccacg 120 gggacgcgtg tgtcttctcc ttcaggaaca gttgcaggaa gttatttccc gggtcgcgat 180 atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaaagtca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480 gattgcagaa gtggagggat tacctttttc caaccaatca atccattgct tagctatttc 540 ggccttcaat tagtgaccgg ctga 564 <210> 44 <211> 564 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12S_N116D) <400> 44<tmp ttggctgcaa ttattggtgg aaacccgtac tatagtggaa attacagatg cagtatcggg 60 tttagtgtcc gtcagggctc tcaaacggga ttcgcgaccg caggccactg cggatccacg 120 gggacgcgtg tgtcttctcc ttcaggaaca gttgcaggaa gttatttccc gggtcgcgat 180 atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaagatca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480 gattgcagaa gtggagggat tacctttttc caaccaatca atccattgct tagctatttc 540 ggccttcaat tagtgaccgg ctga 564 <210> 45 <211> 564 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12S_N116T)<( <400> 45 ttggctgcaa ttattggtgg aaacccgtac tatagtggaa attacagatg cagtatcggg 60 tttagtgtcc gtcagggctc tcaaacggga ttcgcgaccg caggccactg cggatccacg 120 gggacgcgtg tgtcttctcc ttcaggaaca gttgcaggaa gttatttccc gggtcgcgat 180 atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 It should be noted that there seems to be a formatting issue with the tag <( in the original, which is likely a typo. I've translated it as <( as it's not clear what the correct form should be. If this is an error in the original, it may need to be corrected for a more accurate translation.gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaaactca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480​​​​​​​​​​​​​​​​​​​​​​​​​​​​​atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaaggtca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480 gattgcagaa gtggagggat tacctttttc caaccaatca atccattgct tagctatttc 540 ggccttcaat tagtgaccgg ctga 564 <210> 47 [[ID= 16]]<211> 564 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12A) <400> 47 ttggctgcaa ttattggtgg aaacccgtac tatgctggaa attacagatg cagtatcggg 60 tttagtgtcc gtcagggctc tcaaacggga ttcgcgaccg caggccactg cggatccacg 120 gggacgcgtg tgtcttctcc ttcaggaaca gttgcaggaa gttatttccc gggtcgcgat 180 atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaaaatca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480 gattgcagaa gtggagggat tacctttttc caaccaatca atccattgct tagctatttc 540 ggccttcaat tagtgaccgg ctga 564 <210> 48 <211> 564 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Thermobifida fusca_Mature Region (F12R) <400> 48 ttggctgcaa ttattggtgg aaacccgtac tatagaggaa attacagatg cagtatcggg 60 tttagtgtcc gtcagggctc tcaaacggga ttcgcgaccg caggccactg cggatccacg 120 gggacgcgtg tgtcttctcc ttcaggaaca gttgcaggaa gttatttccc gggtcgcgat 180 atgggctggg tgcggattac atcagcagat actgtaacac cactcgtaaa tcggtataat 240 gggggaactg ttacggtcac tgggtcacaa gaagctgcca ccggatcctc cgtttgtcgc 300 tctggagcaa caacgggctg gcgctgcgga actatccaat caaaaaatca aacggttcgc 360 tatgcagaag ggactgttac tggtttaaca agaactacag cctgtgctga aggtggggat 420 tctggagggc catggctcac aggtagccag gcgcaagggg ttacaagcgg cggaacaggc 480 gattgcagaa gtggagggat tacctttttc caaccaatca atccattgct tagctatttc 540 ggccttcaat tagtgaccgg ctga 564 <210> 49 <211> 187 <212> PRT <213> Unknown <220> <221> <222> <223> Thermobifida cellulosilytica <400> 49 Phe Ala Asp Val Ile Gly Gly Asn Pro Tyr Tyr Phe Gly Gly Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Lys Gly Ser Asp Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Glu Thr Gly Thr Leu Thr Arg Ser Pro Glu 35 40 45 Gly Val Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Leu Thr Gly Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asp 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Val Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Ile Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Ala Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Arg Thr Gly Gly Ile Thr Tyr Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Glu Leu Val Thr Gly 180 185 <210> 50 <211> 187 <212> PRT <213> Unknown <220> <221> <222> <223> Thermobifida halotolerans <400> 50 Phe Thr Asp Ile Ile Gly Gly Asn Pro Tyr Tyr Phe Asp Gly Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Arg Gly Ser Glu Ser Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Glu Glu Gly Thr Glu Thr Ser Asp Pro Glu 35 40 45 Gly Thr Val Ala Gly Ala Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 (55) 60 Arg Ile Thr Asp Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Glu Asn Val Thr Val Ala Gly Ser Arg Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Arg Ser Lys Asn Gln Thr Val Arg Tyr Ile Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Gly Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Thr Leu Gly Gly Val Thr Tyr Phe Gln Pro Leu Asn Pro Leu 165 170 175 Leu Ser His Phe Asp Leu Asp Leu Val Thr Gly 180 185 <210> 51 <211> 187 <212> PRT <213> Unknown <220> <221> <222> <223> Actinorugispora endophytica <400> 51 Leu Ala Asn Val Ile Gly Gly Asn Ala Tyr Tyr Phe Gly Gly Tyr Arg 1 5 10 15 Cys Ser Val Gly Phe Ser Val Arg His Ser Ser Gly Pro Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Asp Val Gly Thr Arg Thr Thr Ser Pro Thr 35 40 45 Gly Thr Ile Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Ser Tyr Ile Thr Val Thr Gly Ser Ser Glu Ala Ala Asn Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Asn Tyr Ala Glu Gly Ser Val Ala Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Ser 130 135 140 Trp Leu Thr Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​Phe Ala Asp Ile Ile Gly Gly Asn Ala Tyr Tyr Pro Gly Ser Ser Arg 1 5 10 15 Cys Ser Ile Gly Phe Ala Val Gln Gly Gly Phe Val Thr Ala Gly His 20 25 30 Cys Gly Ser Thr Gly Thr Arg Thr Ser Ser Pro Ser Gly Thr Val Ala 35 40 45 Gly Ser Trp Phe Pro Gly Arg Asp Met Gly Trp Val Arg Thr Gly Ser 50 55 60 Gly Asp Thr Pro Arg Pro Trp Val Asn Asn Tyr Arg Gly Gly Tyr Val 65 70 75 80 Thr Val Ala Gly Ser Gln Glu Ala Gly Ile Gly Ser Ser Val Cys Arg 85 90 95 Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln Ser Lys Asn 100 105 110 Gln Thr Val Arg Tyr Ser Gln Gly Ser Val Tyr Gly Leu Thr Arg Thr 115 120 125 Ser Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp Val Thr Gly 130 135 140 Asn Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Thr Trp 145 150 155 160 Gly Gly Thr Thr Tyr Phe Gln Pro Val Asn Pro Ile Leu Ser Gln Tyr 165 170 175 Gly Leu Arg Leu Val Thr Gly 180 <210> 53 <211> 186 <212> PRT <213> Unknown <220> <221> <222> <223> Nocardiopsis composta <400> 53 Phe Gly Asp Ile Val Gly Gly Asn Ala Tyr Tyr Pro Gly Gly Ser Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Gln Gly Gly Phe Ala Thr Ala Gly His 20 25 30 Cys Gly Ser Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly 35 40 45 Thr Val Ala Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg 50 55 60 Val Asn Ser Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly 65 70 75 80 Gly Arg Val Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser 85 90 95 Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Thr Ile Gln 100 105 110 Ala Lys Asn Gln Thr Val Arg Tyr Pro Glu Gly Thr Val Asn Gly Leu 115 120 125 Thr Arg Thr Thr Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp 130 135 140 Leu Ser Gly Asn Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Cys Gly Ser Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly 35 40 45 Thr Val Ala Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg 50 55 60 Val Asn Ser Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly 65 70 75 80 Gly Arg Val Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser 85 90 95 Ile Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln 100 105 110 Ala Lys Asn Gln Thr Val Arg Tyr Pro Gln Gly Thr Val Asn Gly Leu 115 120 125 Thr Arg Thr Asn Val Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp 130 135 140 Ile Ser Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn 145 150 155 160 Cys Ser Thr Gly Gly Thr Thr Phe Tyr Gln Pro Ile Asn Pro Ile Leu 165 170 175 Ser Gln Trp Gly Leu Thr Leu Thr Thr Gly 180 185 <210> 55 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida cellulosilytica_F12Y <400> 55 Phe Ala Asp Val Ile Gly Gly Asn Pro Tyr Tyr Tyr Gly Gly Tyr Arg 1 5 40 45[[ID=1"]] Cys Ser Ile Gly Phe Ser Val Arg Lys Gly Ser Asp Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Glu Thr Gly Thr Leu Thr Arg Ser Pro Glu 35 40 45 Gly Val Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Leu Thr Gly Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asp 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Val Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Ile Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Ala Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Arg Thr Gly Gly Ile Thr Tyr Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Glu Leu Val Thr Gly 180 185 <210> 56 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida cellulosilytica_F12YN116D <400> 56 Phe Ala Asp Val Ile Gly Gly Asn Pro Tyr Tyr Tyr Gly Gly Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Lys Gly Ser Asp Thr Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Glu Thr Gly Thr Leu Thr Arg Ser Pro Glu 35 40 45 Gly Val Val Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Leu Thr Gly Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asp 65 70 75 80 Gly Gly Thr Val Thr Val Thr Gly Ser Gln Glu Ala Val Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Ile Ile 100 105 110 Gln Ser Lys Asp Gln Thr Val Arg Tyr Ala Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Ala Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Arg Thr Gly Gly Ile Thr Tyr Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Gly Leu Glu Leu Val Thr Gly 180 185 <210> 57 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida halotolerans_F12Y <400> 57 Phe Thr Asp Ile Ile Gly Gly Asn Pro Tyr Tyr Tyr Asp Gly Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Arg Gly Ser Glu Ser Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Glu Glu Gly Thr Glu Thr Ser Asp Pro Glu 35 40 45 Gly Thr Val Ala Gly Ala Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Asp Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Glu Asn Val Thr Val Ala Gly Ser Arg Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Arg Ser Lys Asn Gln Thr Val Arg Tyr Ile Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Gly Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Thr Leu Gly Gly Val Thr Tyr Phe Gln Pro Leu Asn Pro Leu 165 170 175 Leu Ser His Phe Asp Leu Asp Leu Val Thr Gly 180 185 <210> 58 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Thermobifida halotolerans_F12YN116D <400> 58 Phe Thr Asp Ile Ile Gly Gly Asn Pro Tyr Tyr Tyr Asp Gly Tyr Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Arg Arg Gly Ser Glu Ser Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Glu Glu Gly Thr Glu Thr Ser Asp Pro Glu 35 40 45 Gly Thr Val Ala Gly Ala Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Asp Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn<00Q2226>65 70 75 80 Gly Glu Asn Val Thr Val Ala Gly Ser Arg Glu Ala Ala Thr Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Thr Ile 100 105 110 Arg Ser Lys Asp Gln Thr Val Arg Tyr Ile Glu Gly Thr Val Thr Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Pro 130 135 140 Trp Leu Thr Gly Ser Gln Gly Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Thr Leu Gly Gly Val Thr Tyr Phe Gln Pro Leu Asn Pro Leu 165 170 175 Leu Ser His Phe Asp Leu Asp Leu Val Thr Gly 180 185 <210> 59 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Actinorugispora endophytica_F12Y <400> 59 Leu Ala Asn Val Ile Gly Gly Asn Ala Tyr Tyr Tyr Gly Gly Tyr Arg 1 5 10 15 Cys Ser Val Gly Phe Ser Val Arg His Ser Ser Gly Pro Gly Phe Ala 20 25 30 Thr Ala Gly His Cys Gly Asp Val Gly Thr Arg Thr Thr Ser Pro Thr 35 40 45 Gly Thr Ile Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val 50 55 60 Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn 65 70 75 80 Gly Ser Tyr Ile Thr Val Thr Gly Ser Ser Glu Ala Ala Asn Gly Ser 85 90 95 Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile 100 105 110 Gln Ser Lys Asn Gln Thr Val Asn Tyr Ala Glu Gly Ser Val Ala Gly 115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Ser 130 135 140 Trp Leu Thr Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Thr Trp Gly Gly Thr Thr Tyr Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Asn Leu Thr Leu Val Thr Gly 180 185 <210> 60 <211> 187 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Actinorugispora endophytica_F12YN116D <400> 60[[ID=十六]] [[ID=十七]]Leu Ala Asn Val Ile Gly Gly Asn Ala Tyr Tyr Tyr Gly Gly Tyr Arg[[ID=十八]] [[ID=十九]]1 5 10 15[[ID=二十]] [[ID=二十一]]Cys Ser Val Gly Phe Ser Val Arg His Ser Ser Gly Pro Gly Phe Ala[[ID=二十二]] [[ID=二十三]]20 25 30[[ID=二十四]] ]>[[ID=二十五]]Thr Ala Gly His Cys Gly Asp Val Gly Thr Arg Thr Thr Ser Pro Thr[[ID=二十六]] [[ID=二十七]]35 40 45[[ID=二十八]] [[ID=二十九]]Gly Thr Ile Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met Gly Trp Val[[ID=三十]] [[ID=三十一]]50 55 60[[ID=三十二]] [[ID=三十三]]Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn Arg Tyr Asn[[ID=三十四]] [[ID=三十五]]65 70 75 80[[ID=三十六]] [[ID=三十七]]Gly Ser Tyr Ile Thr Val Thr Gly Ser Ser Glu Ala Ala Asn Gly Ser[[ID=三十八]] [[ID=三十九]]85 90 95[[ID=四十]] [[ID=四十一]]Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile[[ID=四十二]] [[ID=四十三]]100 105 110[[ID=四十四]] [[ID=四十五]]Gln Ser Lys Asp Gln Thr Val Asn Tyr Ala Glu Gly Ser Val Ala Gly [[ID=四十六]] It should be noted that there seems to be some incorrect numbering in the "[[ID=]]" tags in the original text (such as "[[ID=十六]]", "[[ID=十七]]", etc.). This may cause issues in the correct interpretation and processing of the content. It is recommended to check and correct these numbering errors if possible.115 120 125 Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser Gly Gly Ser 130 135 140 Trp Leu Thr Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly 145 150 155 160 Asn Cys Thr Trp Gly Gly Thr Thr Tyr Phe Gln Pro Ile Asn Pro Leu 165 170 175 Leu Ser Tyr Phe Asn Leu Thr Leu Val Thr Gly 180 185 <210> 61 <211> 183 <212> PRT<00​​​​​​​​​​​​​​​​​​​​​​​​​ 50 55 60 Gly Asp Thr Pro Arg Pro Trp Val Asn Asn Tyr Arg Gly Gly Tyr Val 65 70 75 80 Thr Val Ala Gly Ser Gln Glu Ala Gly Ile Gly Ser Ser Val Cys Arg 85 90 95 Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln Ser Lys Asn 100 105 110 Gln Thr Val Arg Tyr Ser Gln Gly Ser Val Tyr Gly Leu Thr Arg Thr 115 120 125 Ser Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp Val Thr Gly 130 135 140 Asn Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Thr Trp 145 150 155 160 Gly Gly Thr Thr Tyr Phe Gln Pro Val Asn Pro Ile Leu Ser Gln Tyr 165 170 175 Gly Leu Arg Leu Val Thr Gly 180 <210> 62 <211> 183 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Spinactinospora alkalitolerans_F12YN116D <400> 62 Phe Ala Asp Ile Ile Gly Gly Asn Ala Tyr Tyr Tyr Gly Ser Ser Arg 1 5 10 15 Cys Ser Ile Gly Phe Ala Val Gln Gly Gly Phe Val Thr Ala Gly His 20 25 30 Cys Gly Ser Thr Gly Thr Arg Thr Ser Ser Pro Ser Gly Thr Val Ala 35 40 45 Gly Ser Trp Phe Pro Gly Arg Asp Met Gly Trp Val Arg Thr Gly Ser 50 55 60 Gly Asp Thr Pro Arg Pro Trp Val Asn Asn Tyr Arg Gly Gly Tyr Val 65 70 75 80 Thr Val Ala Gly Ser Gln Glu Ala Gly Ile Gly Ser Ser Val Cys Arg 85 90 95 Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln Ser Lys Asp 100 105 110 Gln Thr Val Arg Tyr Ser Gln Gly Ser Val Tyr Gly Leu Thr Arg Thr 115 120 125 Ser Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp Val Thr Gly 130 135 140 Asn Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Thr Trp 145 150 155 160 Gly Gly Thr Thr Tyr Phe Gln Pro Val Asn Pro Ile Leu Ser Gln Tyr 165 170 175 Gly Leu Arg Leu Val Thr Gly 180 <210> 63 <211> 186 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Nocardiopsis composta_F12Y <400> 63 Phe Gly Asp Ile Val Gly Gly Asn Ala Tyr Tyr Tyr Gly Gly Ser Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Gln Gly Gly Phe Ala Thr Ala Gly His 20 25 30 Cys Gly Ser Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly 35 40 45 Thr Val Ala Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg 50 55 60 Val Asn Ser Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly 65 70 75 80 Gly Arg Val Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser 85 90 95 Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Thr Ile Gln 100 105 110 Ala Lys Asn Gln Thr Val Arg Tyr Pro Glu Gly Thr Val Asn Gly Leu 115 120 125 Thr Arg Thr Thr Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp 130 135 140 Leu Ser Gly Asn Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn 145 150 155 160 Cys Ser Ser Gly Gly Thr Thr Phe Phe Gln Pro Leu Asn Pro Ile Leu 165 170 175 Ser Gln Trp Gly Leu Thr Leu Thr Thr Gly 180 185 <210> 64 <211> 186 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Nocardiopsis composta_F12YN116D <400> 64 Phe Gly Asp Ile Val Gly Gly Asn Ala Tyr Tyr Tyr Gly Gly Ser Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Gln Gly Gly Phe Ala Thr Ala Gly His 20 25 30 Cys Gly Ser Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly 35 40 45 Thr Val Ala Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg 50 55 60 Val Asn Ser Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly 65 70 75 80 Gly Arg Val Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser 85 90 95 Val Cys Arg Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Thr Ile Gln 100 105 110 Ala Lys Asp Gln Thr Val Arg Tyr Pro Glu Gly Thr Val Asn Gly Leu 115 120 125 Thr Arg Thr Thr Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp 130 135 140 Leu Ser Gly Asn Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn 145 150 155 160 Cys Ser Ser Gly Gly Thr Thr Phe Phe Gln Pro Leu Asn Pro Ile Leu 165 170 175 Ser Gln Trp Gly Leu Thr Leu Thr Thr Gly 180 185 <210> 65 <211> 186 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Nocardiopsis potens_F12Y <400> 65 Phe Gly Asp Ile Val Gly Gly Asn Ala Tyr Tyr Tyr Gly Gly Ser Arg 1 5 10 15 Cys Ser Ile Gly Phe Ser Val Gln Gly Gly Phe Ala Thr Ala Gly His 20 25 30 Cys Gly Ser Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly 35 40 45 Thr Val Ala Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg 50 55 60 Val Asn Ser Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly 65 70 75 80 Gly Arg Val Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser 85 90 95 Ile Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln 100 105 110 Ala Lys Asn Gln Thr Val Arg Tyr Pro Gln Gly Thr Val Asn Gly Leu 115 120 125 Thr Arg Thr Asn Val Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp 130 135 140 Ile Ser Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn 145 150 155 160 Cys Ser Thr Gly Gly Thr Thr Phe Tyr Gln Pro Ile Asn Pro Ile Leu 165 170 175 Ser Gln Trp Gly Leu Thr Leu Thr Thr Gly 180 185 <210> 66 <211> 186 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Nocardiopsis potens_F12YN116D <400> 66 Phe Gly Asp Ile Val Gly Gly Asn Ala Tyr Tyr Tyr Gly Gly Ser Arg 1 5 10 15 [[ID=,38]]Cys Ser Ile Gly Phe Ser Val Gln Gly Gly Phe Ala Thr Ala Gly His 20 25 30 Cys Gly Ser Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly 35 40 45 Thr Val Ala Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg 50 55 60 Val Asn Ser Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly 65 70 75 80 Gly Arg Val Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser 85 90 95 Ile Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln 100 105 110 Ala Lys Asp Gln Thr Val Arg Tyr Pro Gln Gly Thr Val Asn Gly Leu 115 120 125 Thr Arg Thr Asn Val Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp 130 135 140 Ile Ser Gly Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn 145 150 155 160 Cys Ser Thr Gly Gly Thr Thr Phe Tyr Gln Pro Ile Asn Pro Ile Leu 165 170 175 Ser Gln Trp Gly Leu Thr Leu Thr Thr Gly 180 185 <210> 67 <211> 374 <212> PRT <213> Unknown <220> <221> <222> <223> Thermobifida cellulosilytica <400> 67 Met Asn Arg Pro Ser Thr Arg Arg Thr Val Arg Ala Leu Leu Thr Ala 1 5 10 15 Ala Leu Ala Ala Thr Ala Leu Thr Ala Pro Ala Ala Pro Ser Leu Ala 20 25 30 Gln Glu Ala Ser Gln Glu Ala Ala Leu Thr Arg Asp Leu Asp Leu Thr 35 40 45 Gly Thr Glu Val Ala Leu Leu Arg Ala Ala Glu Ser Glu Ala Met Asp 50 55 60 Arg Glu Glu Glu Leu Ser Ala Val Leu Gly Ser Asp Phe Gly Gly Val 65 70 75 80 Tyr Leu Ala Pro Glu Thr Gly Glu Val Thr Val Ala Val Thr Asp Pro 85 90 95 Ala Ala Val Pro Val Val Glu Gln Ser Gly Ala Thr Ala Gln Val Val 100 105 110 Thr Phe Gly Glu Thr Ala Leu Asn Asp Phe Val Asp Ser Leu Asn Ala 115 120 125 Val Ala Asp Arg Ala Asp Glu Gln Ile Thr Gly Trp Tyr Thr Asp Leu 130 135 140 Ala Ala Asp Thr Val Val Ile Thr Ala Phe Pro Gly Gly Ser Ala Ala 145 150 155 160 Ala Glu Glu Leu Ala Ala Leu Ala Gly Val Asp Glu Arg Ala Val Arg 165 170 175 Val Thr Glu Ser Ala Ala Arg Pro Gln Leu Phe Ala Asp Val Ile Gly 180 185 190 Gly Asn Pro Tyr Tyr Phe Gly Gly Tyr Arg Cys Ser Ile Gly Phe Ser 195 200 205 Val Arg Lys Gly Ser Asp Thr Gly Phe Ala Thr Ala Gly His Cys Gly 210 215 220 Glu Thr Gly Thr Leu Thr Arg Ser Pro Glu Gly Val Val Ala Gly Ser 225 230 235 240 Tyr Phe Pro Gly Arg Asp Met Gly Trp Val Arg Leu Thr Gly Ala Asp 245 250 255 Thr Val Thr Pro Leu Val Asn Arg Tyr Asp Gly Gly Thr Val Thr Val 260 265 270 Thr Gly Ser Gln Glu Ala Val Thr Gly Ser Ser Val Cys Arg Ser Gly 275 280 285 Ser Thr Thr Gly Trp Arg Cys Gly Ile Ile Gln Ser Lys Asn Gln Thr 290 295 300 Val Arg Tyr Ala Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala 305 310 315 320 Cys Ala Glu Ala Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln 325 330 335 Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Arg Thr Gly Gly 340 345 350 Ile Thr Tyr Phe Gln Pro Ile Asn Pro Leu Leu Ser Tyr Phe Gly Leu 355 360 365 Glu Leu Val Thr Gly Ala 370 <210> 68 <211> 354 <212> PRT[[ID=2)]] <213> Unknown <220> <221> <222> <223> Thermobifida halotolerans <400> 68 Met Val Ala Thr Ala Leu Thr Ala Ser Ala Thr Pro Ala Ser Ala Asp 1 5 10 15 Arg His Asp Ala Leu Lys Arg Asp Leu Gly Leu Thr Asp Ser Glu Val 20 25 30 Ala Arg Leu Arg Thr Ala Glu Thr Glu Ala Met Asp Arg Glu Ala Glu 35 40 45 Leu Arg Asp Thr Leu Gly Ser Asp Phe Gly Gly Val His Leu Asp Ala 50 55 60 Ala Ser Gly Glu Leu Thr Ile Ala Val Thr Asp Pro Glu Ala Val Pro 65 70 75 80 Thr Val Glu Arg Ala Gly Ala Asn Ala Glu Val Val Thr Phe Gly Glu 85 90 95 Ser Ala Leu Asn Gly Phe Val Asp Ser Leu Asn Ser Val Ala Asp Gln 100 105 110 Ala Asp Glu Gln Val Thr Gly Trp Tyr Ala Asp Ile Ala Asp Asp Ser 115 120 125 Val Val Ile Thr Val Arg Glu Gly Gly Thr Ala Ala Ala Glu Ala Leu 130 135 140 Val Ala Arg Ala Gly Val Asp Glu Arg Ala Val Arg Val Thr Lys Ser 145 150 155 160 Asp Glu Arg Pro Gln Leu Phe Thr Asp Ile Ile Gly Gly Asn Pro Tyr 165 170 175 Tyr Phe Asp Gly Tyr Arg Cys Ser Ile Gly Phe Ser Val Arg Arg Gly 180 185 190 Ser Glu Ser Gly Phe Ala Thr Ala Gly His Cys Gly Glu Glu Gly Thr 195 200 205 Glu Thr Ser Asp Pro Glu Gly Thr Val Ala Gly Ala Tyr Phe Pro Gly 210 215 220 Arg Asp Met Gly Trp Val Arg Ile Thr Asp Ala Asp Thr Val Thr Pro 225 230 235 240 Leu Val Asn Arg Tyr Asn Gly Glu Asn Val Thr Val Ala Gly Ser Arg 245 250 255 Glu Ala Ala Thr Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly 260 265 270 Trp Arg Cys Gly Thr Ile Arg Ser Lys Asn Gln Thr Val Arg Tyr Ile 275 280 285 Glu Gly Thr Val Thr Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly 290 295 300 Gly Asp Ser Gly Gly Pro Trp Leu Thr Gly Ser Gln Gly Gln Gly Val 305 310 315 320 Thr Ser Gly Gly Ser Gly Asn Cys Thr Leu Gly Gly Val Thr Tyr Phe 325 330 335 Gln Pro Leu Asn Pro Leu Leu Ser His Phe Asp Leu Asp Leu Val Thr 340 345 350 Gly Ala <210> 69 <211> 383 <212> PRT <213> Unknown <220> <221> <222> <223> Actinorugispora endophytica <400> 69 Met Lys Arg Ser Ser Val Phe Arg Ala Leu Gly Gly Thr Ile Leu Thr 1 5 10 15 Ala Gly Leu Val Ile Thr Ala Ala Pro Phe Ala Ser Ala Ala Pro Val 20 25 30 His Thr Glu Thr Thr Pro Thr Ala Ala Glu Ala Gly Asp Gln Leu Ser 35 40 45 Ala Leu Lys Arg Asp Leu Gly Leu Ser Thr Ala Glu Val Glu Glu Leu 50 55 60 Gln Ala Ala Glu Ala Glu Ala Met Asp Val Glu Glu Gly Leu Arg Glu 65 70 75 80 Thr Leu Gly Ser Asp Phe Gly Gly Ala His Phe Asp Ile Asp Ser Gly 85 90 95 Glu Leu Thr Val Ser Val Thr Asp Ala Ala Ala Val Ser Thr Val Glu 100 105 110 Ala Ala Gly Ala Asn Ala Glu Val Val Asp Phe Gly Glu Pro Ala Leu 115 120 125 Asp Ala Ile Val Glu Asp Leu Asn Thr Val Ala Glu Glu Ala Asp Asp 130 135 140 Ser Val Thr Gly Trp Tyr Val Asp Thr Ala Asp Asp Ser Val Val Ile 145 150 155 160 Thr Val Leu Glu Gly Asp Thr Glu Ala Ala Glu Ala Leu Val Ala Glu 165 170 175 Ala Asp Val Asp Gly Lys Ala Val Arg Val Glu Glu Thr Thr Glu Gln 180 185 190 Pro Lys Leu Leu Ala Asn Val Ile Gly Gly Asn Ala Tyr Tyr Phe Gly 195 200 205 Gly Tyr Arg Cys Ser Val Gly Phe Ser Val Arg His Ser Ser Gly Pro 210 215 220 Gly Phe Ala Thr Ala Gly His Cys Gly Asp Val Gly Thr Arg Thr Thr 225 230 235 240 Ser Pro Thr Gly Thr Ile Ala Gly Ser Tyr Phe Pro Gly Arg Asp Met 245 250 255 Gly Trp Val Arg Ile Thr Ser Ala Asp Thr Val Thr Pro Leu Val Asn 260 265 270 Arg Tyr Asn Gly Ser Tyr Ile Thr Val Thr Gly Ser Ser Glu Ala Ala 275 280 285 Asn Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys 290 295 300 Gly Thr Ile Gln Ser Lys Asn Gln Thr Val Asn Tyr Ala Glu Gly Ser 305 310 315 320 Val Ala Gly Leu Thr Arg Thr Thr Ala Cys Ala Glu Gly Gly Asp Ser 325 330 335 Gly Gly Ser Trp Leu Thr Gly Thr Gln Ala Gln Gly Val Thr Ser Gly 340 345 350 Gly Ser Gly Asn Cys Thr Trp Gly Gly Thr Thr Tyr Phe Gln Pro Ile 355 360 365 Asn Pro Leu Leu Ser Tyr Phe Asn Leu Thr Leu Val Thr Gly Ala 370 375 380 <210> 70 <211> 375 <212> PRT <213> Unknown <220> <221> <222> <223> Spinactinospora alkalitolerans <400> 70 Met Arg Lys Ser Pro Ile Ile Arg Ala Val Gly Gly Ala Ala Ile Thr 1 5 10 15 Phe Gly Leu Val Ile Ala Ala Ala Pro Phe Ala Ser Ala Asp Ser Gly 20 25 30 Ser Glu Thr Thr Ala Gly Ser Val Gly Gln Leu Gly Ala Met Gln Arg 35 40 45 Asp Leu Gly Leu Ser Ala Thr Glu Ala Thr Ala Leu Leu Asp Gln Glu 50 55 60 Glu Gln Ala Arg Thr Leu Glu Gly Glu Leu Arg Glu Thr Leu Gly Gly 65 70 75 80 Asp Phe Gly Gly Ala Val Phe Asp Ile Glu Ser Gly Glu Leu Thr Val 85 90 95 Ser Val Thr Asp Glu Asp Ala Val Asp Glu Val Arg Glu Ala Gly Ala 100 105 110 Glu Ala Glu Val Val Thr Tyr Gly Glu Gln Arg Leu Asp Ala Ile Val 115 120 125 Asp Asp Leu Asn Ala Thr Glu Asp Thr Ala Asp Glu Ser Val Thr Gly 130 135 140 Trp Tyr Val Asp Thr Ala Asp Asp Ser Val Val Val Thr Val Met Glu 145 150 155 160 Gly Glu Glu Ala Ala Ala Glu Lys Leu Ile Ala Thr Ala Asp Val Glu 165 170 175 Gly Thr Ala Val Arg Val Glu Glu Thr Thr Glu Gln Pro Glu Thr Phe 180 185 190 Ala Asp Ile Ile Gly Gly Asn Ala Tyr Tyr Pro Gly Ser Ser Arg Cys 195 200 205 Ser Ile Gly Phe Ala Val Gln Gly Gly Phe Val Thr Ala Gly His Cys 210 215 220 Gly Ser Thr Gly Thr Arg Thr Ser Ser Pro Ser Gly Thr Val Ala Gly 225 230 235 240 Ser Trp Phe Pro Gly Arg Asp Met Gly Trp Val Arg Thr Gly Ser Gly 245 250 255 Asp Thr Pro Arg Pro Trp Val Asn Asn Tyr Arg Gly Gly Tyr Val Thr 260 265 270 Val Ala Gly Ser Gln Glu Ala Gly Ile Gly Ser Ser Val Cys Arg Ser 275 280 285 Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln Ser Lys Asn Gln 290 295 300 Thr Val Arg Tyr Ser Gln Gly Ser Val Tyr Gly Leu Thr Arg Thr Ser 305 310 315 320 Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp Val Thr Gly Asn 325 330 335 Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Thr Trp Gly 340 345 350 Gly Thr Thr Tyr Phe Gln Pro Val Asn Pro Ile Leu Ser Gln Tyr Gly 355 360 365 Leu Arg Leu Val Thr Gly Ala 370 375 <210> 71 <211> 376 <212> PRT <213> unknown <220> <221> <222> <223> Nocardiopsis composta <400> 71 Met Arg Lys Ser Pro Tyr Ile Pro Leu Leu Gly Ala Ser Val Leu Ala 1 5 10 15 Leu Gly Met Ile Ala Ala Ser Pro Thr Ala Ala Ser Ala Asp Glu Ala 20 25 30 Thr Asp Ser Ser Pro Ala Arg Ala Leu Ala Ser Gly Leu Asp Met Ser 35 40 45 Thr Ala Gln Ala Ala Glu Leu Leu Asp Ala Glu Ala Gln Ala Arg Thr 50 55 60 Ala Glu Gln Glu Ala Arg Glu Leu Ala Gly Ala Ser Phe Ala Gly Ala 65 70 75 80 Val Phe Asp Ala Asp Thr Arg Lys Leu Thr Val Ser Val Thr Asp Ala 85 90 95 Ala Ala Ala Glu Ala Val Gln Ala Thr Gly Ala Glu Thr Arg Val Val 100 105 110 Glu Ala Ser Ala Asp Glu Leu Asp Ala Ala Val Ala Asp Leu Asn Ala 115 120 125 Glu Glu Arg Gly Leu Gly Ser Glu Ile Asp Gly Val Thr Gly Trp Tyr 130 135 140 Val Asp Gln Ala Ala Asn Glu Leu Val Val Thr Val Leu Asp Gly Glu 145 150 155 160 Thr Glu Ala Ala Glu Thr Leu Leu Asp Glu Ala Gly Val Asp Ser Val 165 170 175 Pro Val Arg Val Asp Gln Gly Ala Glu Gln Pro Glu Thr Phe Gly Asp 180 185 190 Ile Val Gly Gly Asn Ala Tyr Tyr Pro Gly Gly Ser Arg Cys Ser Ile 195 200 205 Gly Phe Ser Val Gln Gly Gly Phe Ala Thr Ala Gly His Cys Gly Ser 210 215 220 Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly Thr Val Ala 225 230 235 240 Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg Val Asn Ser 245 250 255 Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly Gly Arg Val 260 265 270 Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser Val Cys Arg 275 280 285 Ser Gly Ser Thr Thr Gly Trp Arg Cys Gly Thr Ile Gln Ala Lys Asn 290 295 300 Gln Thr Val Arg Tyr Pro Glu Gly Thr Val Asn Gly Leu Thr Arg Thr 305 310 315 320 Thr Ala Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp Leu Ser Gly 325 330 335 Asn Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Ser Ser 340 345 350 Gly Gly Thr Thr Phe Phe Gln Pro Leu Asn Pro Ile Leu Ser Gln Trp 355 360 365​​​​​​​​​​​​​​​​​​​​​​​​​​​Leu Gly Met Ile Ala Ala Ser Pro Ala Ala Ala Ser Ala Asp Glu Ala 20 25 30 Ala Asp Thr Ser Pro Ala Glu Ala Leu Ala Ser Gly Leu Asp Met Ser 35 40 45 Ala Ser Gln Ala Ala Asp Leu Leu Asp Ala Glu Ala Glu Ala Arg Gly 50 55 60 Thr Glu Ala Glu Ala Arg Glu Ala Ala Gly Gly Ser Phe Ala Gly Ala 65 70 75 80 Val Phe Asp Ala Glu Ser Gln Val Leu Thr Val Ser Val Thr Asp Ala 85 90 95 Ala Ala Ala Glu Ala Val Glu Ala Thr Gly Ala Glu Thr Arg Val Val 100 105 110 Glu Ala Ser Glu Asp Glu Leu Asp Ser Ala Val Ser Asp Leu Asn Ala 115 120 125 Glu Glu Ser Ser Leu Gly Ser Ala Ile Glu Gly Val Thr Gly Trp Tyr 130 135 140 Val Asp Pro Ala Ala Asn Glu Val Val Val Thr Val Leu Asp Gly Glu 145 150 155 160 Thr Ala Ala Ala Glu Thr Leu Leu Asp Glu Ala Gly Val Asp Gly Val 165 170 175 Pro Val Arg Ile Asp Glu Gly Ala Glu Gln Pro Glu Thr Phe Gly Asp 180 185 190 Ile Val Gly Gly Asn Ala Tyr Tyr Pro Gly Gly Ser Arg Cys Ser Ile 195 200 205 Gly Phe Ser Val Gln Gly Gly Phe Ala Thr Ala Gly His Cys Gly Ser 210 215 220 Gln Gly Thr Arg Val Thr Gly Gly Ala Gly Glu Ser Gly Thr Val Ala 225 230 235 240 Gly Ser Ile Phe Pro Gly Arg Asp Met Gly Trp Val Arg Val Asn Ser 245 250 255 Gly Trp Asn Pro Ser Pro Tyr Val Asn Asn Tyr Ser Gly Gly Arg Val 260 265 270 Leu Val Thr Gly Ser Gln Glu Ala Ser Val Gly Ala Ser Ile Cys Arg 275 280 285 Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln Ala Lys Asn 290 295 300 Gln Thr Val Arg Tyr Pro Gln Gly Thr Val Asn Gly Leu Thr Arg Thr 305 310 315 320 Asn Val Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Trp Ile Ser Gly 325 330 335 Ser Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Ser Thr 340 345 350 Gly Gly Thr Thr Phe Tyr Gln Pro Ile Asn Pro Ile Leu Ser Gln Trp 355 360 365 Gly Leu Thr Leu Thr Thr Gly Ala 370 375

Claims

1. Serine protease variants, which consist of the following: (a) The amino acid sequence shown in SEQ ID NO: 52, wherein the amino acid corresponding to position 12 of the amino acid sequence shown in SEQ ID NO: 52 is replaced by tyrosine (Y). (b) The amino acid sequence shown in SEQ ID NO: 53, wherein the amino acid corresponding to position 12 of the amino acid sequence shown in SEQ ID NO: 53 is replaced by tyrosine (Y), or (c) The amino acid sequence shown in SEQ ID NO: 54, wherein the amino acid corresponding to position 12 of the amino acid sequence shown in SEQ ID NO: 54 is replaced by tyrosine (Y).

2. Serine protease variants, which consist of the following: (a) The amino acid sequence shown in SEQ ID NO: 70, wherein the amino acid corresponding to position 203 of the amino acid sequence shown in SEQ ID NO: 70 is replaced by tyrosine (Y). (b) The amino acid sequence shown in SEQ ID NO: 71, wherein the amino acid corresponding to position 201 of the amino acid sequence shown in SEQ ID NO: 71 is replaced by tyrosine (Y), or (c) The amino acid sequence shown in SEQ ID NO: 72, wherein the amino acid at position 201 corresponding to the amino acid sequence shown in SEQ ID NO: 72 is replaced by tyrosine (Y).

3. A composition comprising any one of the serine protease variants according to claims 1 to 2.

4. A polynucleotide encoding a serine protease variant as described in any one of claims 1 to 2.

5. A vector comprising the polynucleotide of claim 4.

6. A host cell comprising at least one of the following: a serine protease variant according to any one of claims 1 to 2, a polynucleotide encoding the serine protease variant, and a vector comprising the polynucleotide.

7. A composition comprising at least one of the serine protease variant of any one of claims 1 to 2 and a microorganism expressing said serine protease variant.

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