A proteinase K mutant with prolonged tolerance to guanidine isothiocyanate in nucleic acid extraction lysis solution and its application

Abstract

The present invention discloses a proteinase K mutant with prolonged tolerance time to guanidine isothiocyanate in nucleic acid extraction lysis solution and its application. The present invention performs combined directional transformation on wild-type proteinase K, and on the basis of the amino acid sequence shown in SEQ ID NO.1, mutates the tyrosine at position 151 to alanine, the lysine at position 208 to histidine, the serine at position 273 to threonine, and the glycine at position 293 to alanine. The proteinase K mutant provided by the present invention is more tolerant to high concentrations of guanidine isothiocyanate than the wild-type proteinase K. At 25°C, the residual activity is not affected after 30 minutes of treatment with nucleic acid extraction lysis solution; after 60 minutes of treatment, it can still maintain more than 80% of the residual activity, while the residual activity of the wild-type proteinase K is only 28%. Thus, the tolerance of proteinase K to the nucleic acid extraction lysis solution of the nucleic acid extraction kit is improved.

Description

Technical Field

[0001] The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a proteinase K mutant capable of prolonging the tolerance time to guanidine isothiocyanate in nucleic acid extraction lysis solution and application thereof. Background Art

[0002] Proteinase K is a serine protease derived from Tritirachium album. It has high efficiency in protein cleavage and a wide spectrum of substrates. It can preferentially decompose ester bonds and peptide bonds adjacent to the C-termini of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids, and is often used to degrade proteins to produce short peptides. Proteinase K is widely used in the fields of in vitro diagnostics, detergents, feed industry, and sewage treatment. In the field of molecular diagnosis, proteinase K is an important component of virus sampling fluid, which can cleave viruses to release nucleic acids and eliminate nuclease (RNase) to prevent RNA degradation of viruses. It can also inactivate viruses, denature viral proteins, and make them "dead" and no longer infectious, thus improving the safety of transportation and detection. In the process of nucleic acid extraction, proteinase K can degrade proteins bound to nucleic acids and promote the separation of nucleic acids, which has great advantages in extracting high molecular weight nucleic acids. Proteinase K is also often used to degrade DNA enzymes and RNA enzymes in nucleic acids.

[0003] Under normal circumstances, the pH range of proteinase K is between 7.5-9.0, and proteinase K is active at 20℃-60℃. Proteinase K can still exist and have certain activity in an environment containing TritX-100, urea, SDS, and guanidine salts. This feature is also one of the reasons for its wide application. Nucleic acid extraction lysis buffer usually contains components such as isopropanol and guanidine isothiocyanate (3mol / L). Although wild-type proteinase K has certain activity in this environment, it can only be maintained for a short time, generally not exceeding 5min. This shortcoming brings unstable factors to the nucleic acid extraction process and affects the efficiency of nucleic acid extraction. After the outbreak of the new coronavirus in 2020, countries around the world have an increasing demand for proteinase K for molecular diagnosis. Wild-type proteinase K cannot maintain long-term activity in nucleic acid extraction lysis buffer, which affects the stability of the nucleic acid extraction process. Therefore, it is urgent to screen out proteinase K with greatly improved resistance to nucleic acid extraction lysis buffer. Summary of the invention

[0004] The purpose of the present invention is to provide a proteinase K mutant and its application that can prolong the tolerance time of guanidine isothiocyanate in nucleic acid extraction lysate in view of the current status and existing deficiencies of the prior art. The present invention performs a directed transformation of wild-type proteinase K by a rational design method, and performs combined directed mutation on four amino acid sites of proteinase K. The tolerance of proteinase K to the nucleic acid extraction lysate of the nucleic acid extraction kit is improved by directed mutation.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a proteinase K mutant having a prolonged tolerance to guanidine isothiocyanate in a nucleic acid extraction lysis solution, wherein the proteinase K mutant is selected from any one of the following:

[0007] (a) The amino acid sequence is derived from the sequence shown in SEQ ID NO.1 by mutation, wherein at least two amino acid residues selected from the following group are mutated: tyrosine at position 151 is mutated to alanine, lysine at position 208 is mutated to histidine, serine at position 273 is mutated to threonine, and glycine at position 293 is mutated to alanine;

[0008] or,

[0009] (b) the proteinase K mutant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the proteinase K of (a), wherein the amino acid residues Y151, K208, S273, and G293 corresponding to the amino acid sequence of SEQ ID NO: 1 are the same as those in the amino acid sequence of (a);

[0010] or,

[0011] (c) The proteinase K mutant is formed by adding or deleting 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the proteinase K mutant described in (a), wherein the Y151, K208, S273, and G293 positions corresponding to the amino acid sequence shown in SEQ ID NO:1 are the same as those in the amino acid sequence described in (a).

[0012] According to an embodiment of the present invention, the amino acid sequence of the mutant comprises mutations at the following sites: mutations of amino acid residues at any two sites including positions 151, 208, 2730 or 293 corresponding to SEQ ID NO.1.

[0013] In one embodiment, the amino acid sequence of the mutant is any one of the following 1)-5):

[0014] 1) The tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the lysine at position 208 is mutated to histidine;

[0015] 2) the tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the serine at position 273 is mutated to threonine;

[0016] 3) corresponding to the mutation of tyrosine at position 151 to alanine and glycine at position 293 to alanine in SEQ ID NO.1;

[0017] 4) corresponding to the mutation of lysine at position 208 to histidine and serine at position 273 to threonine in SEQ ID NO.1;

[0018] 5) The serine at position 273 corresponding to SEQ ID NO.1 is mutated to threonine and the glycine at position 293 is mutated to alanine;

[0019] The amino acid sequences of the above five mutants are shown in any one of SEQ ID NOs. 3-7.

[0020] According to an embodiment of the present invention, the amino acid sequence of the mutant comprises mutations of amino acid residues at any three positions of position 151, position 208, position 2730 or position 293.

[0021] In one embodiment, the amino acid sequence of the mutant is any one of the following 1)-4):

[0022] 1) The tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine, the lysine at position 208 is mutated to histidine, and the serine at position 273 is mutated to threonine;

[0023] 2) corresponding to SEQ ID NO.1, the tyrosine at position 151 is mutated to alanine, the lysine at position 208 is mutated to histidine, and the glycine at position 293 is mutated to alanine;

[0024] 3) corresponding to SEQ ID NO.1, the tyrosine at position 151 is mutated to alanine, the serine at position 273 is mutated to threonine, and the glycine at position 293 is mutated to alanine;

[0025] 4) corresponding to SEQ ID NO.1, the lysine at position 208 is mutated to histidine, the serine at position 273 is mutated to threonine, and the glycine at position 293 is mutated to alanine;

[0026] The amino acid sequences of the above four mutants are shown in any one of SEQ ID NOs. 8-11.

[0027] In a preferred embodiment, the proteinase K mutant is based on the amino acid sequence shown in SEQ ID NO.1, with the tyrosine at position 151 mutated to alanine, the lysine at position 208 mutated to histidine, the serine at position 273 mutated to threonine, and the glycine at position 293 mutated to alanine. The amino acid sequence of the proteinase K mutant is shown in SEQ ID NO:12.

[0028] In a second aspect, the present invention provides a polynucleotide encoding the proteinase K mutant described in the first aspect, and the nucleotide sequence of the polynucleotide can be optimized according to the codon preference of the expression system.

[0029] In a preferred embodiment, the polynucleotide of the proteinase K mutant having an amino acid sequence as shown in SEQ ID NO:12 has a nucleotide sequence as shown in SEQ ID NO.27.

[0030] In a third aspect, the present invention provides an expression vector, which contains the polynucleotide described in the second aspect; preferably, the expression vector can be selected from: Pichia pastoris expression vector pPinkα-HC, pPIC9K, pPICZα-C; further preferably, the expression vector is pPinkα-HC.

[0031] In a fourth aspect, the present invention provides a genetically engineered host cell, wherein the host cell contains the vector described in the third aspect, or the polynucleotide described in the second aspect is integrated into the genome; preferably, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the eukaryotic cell includes a yeast cell, a mold cell, an insect cell, a plant cell, a fungal cell or a mammalian cell; preferably, the prokaryotic cell includes an Escherichia coli cell or a Bacillus subtilis cell; further preferably, the yeast cell is Pichia pastoris pPINK.

[0032] In a fifth aspect, the present invention provides a method for producing the proteinase K mutant described in the first aspect, wherein the genetically engineered host cell described in the fourth aspect is propagated under suitable conditions and expressed by the polynucleotide encoding the proteinase K mutant described in the second aspect, and then the proteinase K mutant is isolated from the host cell culture.

[0033] In a sixth aspect, the present invention provides a method for increasing the retention time of the enzyme activity of proteinase K in a nucleic acid extraction lysis solution, the method comprising mutating the proteinase K of the amino acid sequence derived from Candida albicans such as shown in SEQ ID NO.1 at at least two amino acid residue sites selected from the following group: the tyrosine at position 151 is mutated to alanine, the lysine at position 208 is mutated to histidine, the serine at position 273 is mutated to threonine, and the glycine at position 293 is mutated to alanine.

[0034] In the seventh aspect, the present invention provides a composition comprising the proteinase K mutant described in the first aspect, wherein the composition contains an enzyme protectant or a substance for delaying enzyme decomposition; preferably, the ingredients of the composition, in addition to water, also include the following components: Tris 30mmol / L, Ca2+10mmol / L-50mmol / L, glycerol 5v%, and HCl is used to adjust the pH to 7.5-8.4.

[0035] In the eighth aspect, the present invention provides the use of the proteinase K mutant described in the first aspect, the genetically engineered host cell described in the fourth aspect, or the composition described in the seventh aspect for enzymatic hydrolysis of proteins; preferably, it is used to decompose ester bonds or peptide bonds adjacent to the C-termini of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids, and enzymatically hydrolyze proteins.

[0036] In a ninth aspect, the present invention provides a method for enzymatic hydrolysis of proteins, comprising: performing an enzymatic hydrolysis reaction using the proteinase K mutant described in the first aspect, the genetically engineered host cell expressing the mutant described in the fourth aspect or its cleavage product, or the composition described in the seventh aspect.

[0037] In a tenth aspect, the present invention provides a detection system or a detection kit for enzymatic protein hydrolysis, which comprises: the proteinase K mutant described in the first aspect, and the genetically engineered host cell expressing the mutant described in the fourth aspect or its cleavage product.

[0038] Beneficial effects of the present invention:

[0039] The proteinase K mutant provided by the present invention is more tolerant to high concentrations of guanidine isothiocyanate (such as 3 mol / L) than the wild-type proteinase K. At 25° C., the residual activity is not affected 30 minutes after treatment with nucleic acid extraction and lysis solution; after treatment for 60 minutes, more than 80% of the residual activity can still be maintained, while the residual activity of the wild-type proteinase K is only 28% at this time. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 .Plasmid map of Pichia pastoris expression vector pPinkα-HC; DETAILED DESCRIPTION

[0041] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0042] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0043] Definition and explanation:

[0044] As used herein, unless otherwise specified, the "mutant of proteinase K" and "mutant proteinase K" are used interchangeably and refer to a protein corresponding to the wild-type proteinase K (such as SEQ ID NO: 1) after one or more mutations selected from the following positions occur: tyrosine at position 151 mutates to alanine, lysine at position 208 mutates to histidine, serine at position 273 mutates to threonine, and glycine at position 293 mutates to alanine.

[0045] As used herein, if it is necessary to indicate the wild-type proteinase K, it will be indicated as "wild-type proteinase K", the protein with the amino acid sequence shown in SEQ ID NO: 1, or WT.

[0046] As used herein, "isolated" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and proteins in their natural state in living cells are not isolated and purified, but the same polynucleotides or proteins are isolated and purified if they are separated from other substances that exist with them in their natural state.

[0047] As used herein, "isolated proteinase K mutant" means that the proteinase K mutant is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the proteinase K mutant using standard protein purification techniques. Substantially pure protein can produce a single major band on a non-reducing polyacrylamide gel.

[0048] As used herein, "recombinant (R)" refers to proteins, genetic engineering vectors or cells obtained (or prepared in large quantities) by means of genetic engineering.

[0049] As used herein, "extended tolerance time to guanidine isothiocyanate in nucleic acid extraction lysate" means that compared with the wild-type proteinase K before modification, the tolerance time of guanidine isothiocyanate in nucleic acid extraction lysate of the mutant proteinase K is statistically extended, or is called a significant extension. For example, under the same reaction conditions / environment, the residual activity of the mutant proteinase K is not affected 30 minutes after treatment with nucleic acid extraction lysate; it can still maintain more than 80% residual activity after treatment for 60 minutes, while the residual activity of the wild-type proteinase K is only 28% at this time.

[0050] As used herein, the terms "comprising" or "including" include "including", "consisting essentially of", and "consisting of". The term "consisting essentially of" means that in addition to the essential ingredients or components, the composition / reaction system / kit may also contain minor ingredients and / or impurities that do not affect the active ingredients in small amounts.

[0051] Proteinase K mutants, nucleic acids encoding them and constructs

[0052] The proteinase K mutants of the present invention can be the products of chemical synthesis, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects and mammalian cells) using recombinant technology.

[0053] The present invention also includes fragments, derivatives and analogs of the proteinase K mutant. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity as the natural proteinase K mutant of the present invention. The protein fragments, derivatives or analogs of the present invention may be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having a substitution group in one or more amino acid residues, or (iii) proteins formed by the fusion of an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify this protein, or a fusion protein). According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art. However, the condition that needs to be met is that the amino acid sequence of the proteinase K mutant and its fragments, derivatives and analogs must contain the mutations specifically pointed out above in the present invention. Preferably, the mutation corresponds to the wild-type proteinase K (such as SEQ ID NO: 1), and a mutation selected from the following positions occurs to form a protein: tyrosine at position 151 mutates to alanine, lysine at position 208 mutates to histidine, serine at position 273 mutates to threonine, and glycine at position 293 mutates to alanine.

[0054] In the present invention, the term "proteinase K mutant" also includes (but is not limited to): the deletion, insertion and / or substitution of several (usually 1 to 20, preferably 1 to 10, and more preferably 1 to 8, 1 to 5, 1 to 3, or 1 to 2) amino acids, and the addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, the use of ` will not change the function of the protein. The term also includes active fragments and active derivatives of proteinase K mutants. However, among these variant forms, the mutations described above in the present invention must exist. Preferably, the mutation corresponds to the wild-type proteinase K (such as SEQ ID NO: 1), and the protein is constituted after the mutation selected from the following positions: the tyrosine at position 151 is mutated to alanine, the lysine at position 208 is mutated to histidine, the serine at position 273 is mutated to threonine, and the glycine at position 293 is mutated to alanine.

[0055] In the present invention, the term "proteinase K mutant" also includes (but is not limited to): a derived protein that retains its protein activity and has a sequence identity of more than 80%, preferably more than 85%, more preferably more than 90%, further more preferably more than 95%, such as more than 98%, more than 99% with the amino acid sequence of the proteinase K mutant. Similarly, in these derived proteins, the mutations described above in the present invention must exist. Preferably, the mutations correspond to the wild-type proteinase K (such as SEQ ID NO: 1), and one or more mutations selected from the following positions occur in the protein: tyrosine at position 151 is mutated to alanine, lysine at position 208 is mutated to histidine, serine at position 273 is mutated to threonine, and glycine at position 293 is mutated to alanine.

[0056] The present invention also provides analogs of the proteinase K mutant. The difference between these analogs and the proteinase K mutant may be a difference in the amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biological techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above. Modifications (usually without changing the primary structure) include: chemical derivatization forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine), and polypeptides modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0057] The present invention also provides a polynucleotide sequence encoding the proteinase K mutant or its conservative variant protein of the present invention.

[0058] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0059] The polynucleotide encoding the mature protein of the mutant includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences.

[0060] A "polynucleotide encoding a protein" may be a polynucleotide that encodes the protein, or may be a polynucleotide that also includes additional coding and / or non-coding sequences.

[0061] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the proteinase K mutant coding sequence, and a method for producing the mutant enzyme of the present invention by recombinant technology.

[0062] The polynucleotide sequences of the present invention can be used to express or produce recombinant proteinase K mutants by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0063] (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a proteinase K mutant of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0064] (2) host cells cultured in a suitable culture medium;

[0065] (3) Isolate and purify proteins from culture medium or cells.

[0066] In the present invention, the proteinase K mutant polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0067] Methods well known to those skilled in the art can be used to construct expression vectors containing proteinase K mutant encoding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively connected to a suitable promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. The expression vector preferably includes one or more selectable marker genes to provide the phenotypic traits of the host cell for selection of transformation.

[0068] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0069] In the present invention, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a mold cell, a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: Escherichia coli, Bacillus subtilis, Streptomyces, Agrobacterium; eukaryotic cells such as yeast, plant cells, etc. In a specific embodiment of the present invention, yeast cells are used as host cells.

[0070] It is clear to those skilled in the art how to select appropriate vectors, promoters, enhancers and host cells.

[0071] In the present invention, multiple copies of the polynucleotide of the proteinase K mutant can be inserted into the host cell to increase the production of the proteinase K mutant, or the polynucleotide sequence can be integrated into a multi-copy site in the host cell genome to increase the copy number.

[0072] Recombinant expression of mutants

[0073] The recombinant cells (host cells) established by the present invention can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0074] When expressed, the proteinase K mutant of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, high-speed centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0075] The method of recombinantly expressing proteinase K mutants of the present invention solves the problem in the prior art that the activity of wild-type proteinase K in nucleic acid extraction lysate containing components such as isopropanol and guanidine isothiocyanate can only be maintained for a short time, generally not more than 5 minutes. This shortcoming brings unstable factors to the nucleic acid extraction process and affects the efficiency of nucleic acid extraction. After the outbreak of the new coronavirus in 2020, countries around the world have an increasing demand for proteinase K for molecular diagnosis. Wild-type proteinase K cannot maintain long-term activity in nucleic acid extraction lysate, which affects the stability of the nucleic acid extraction process.

[0076] Application of recombinant proteinase K

[0077] The modified proteinase K mutant of the present invention has various uses related to the properties of proteinase K, including but not limited to: specific recognition and cleavage of the carboxyl terminal peptide bonds of aliphatic amino acids and aromatic amino acids, enzymatic hydrolysis of proteins or denaturation of proteins.

[0078] As an embodiment, the proteinase K mutant can be applied to genomic DNA extraction and enzyme digestion and removal.

[0079] As an embodiment, the proteinase K mutant can be used to prepare chromosome DNA for pulsed electrophoresis, protein blotting, and remove nucleases in DNA and RNA preparation, etc.

[0080] As an embodiment, the proteinase K mutant can be used for pre-hybridization treatment in in situ hybridization technology, and it has the effect of digesting the protein surrounding the target DNA to increase the chance of the probe binding to the target nucleic acid and improve the hybridization signal.

[0081] As some industrial implementation methods, the proteinase K mutant can be used in leather, fur, silk, medicine, food, brewing and other aspects. Protease has been widely used in the dehairing and softening of the leather industry, which is time-saving and convenient. Protease can also be used for silk degumming, meat tenderization, and wine clarification. It can be used as medicine clinically, such as using pepsin to treat indigestion, using acid protease to treat bronchitis, using elastase to treat vasculitis, and using proteinase K and chymosin to purify surgical suppurative wounds and treat pleural adhesions. Enzyme washing powder is a new product in detergents, containing alkaline protease, which can remove blood stains and protein stains on clothes.

[0082] The proteinase K mutant provided by the present invention is more tolerant to high concentrations of guanidine isothiocyanate than wild-type proteinase K, and can maintain activity for a long time in a nucleic acid extraction lysis solution, thereby improving the stability of the nucleic acid extraction process.

[0083] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0084] Example 1: Recombinant fermentation and purification of proteinase K mutants

[0085] The present invention is based on the crystal structure of proteinase K, and the wild-type proteinase K is directed to be transformed by a rational design method, and the four amino acid sites of the wild-type proteinase K shown in the amino acid sequence as SEQ ID NO: 1 are directed to be mutated in a random combination manner. Wherein, the specific mutation sites include: the 151st tyrosine is mutated to alanine, the 208th lysine is mutated to histidine, the 273rd serine is mutated to threonine, and the 293rd glycine is mutated to alanine. And the 151st tyrosine is mutated to alanine as defined as type A mutation, the 208th lysine is mutated to histidine as defined as type B mutation, the 273rd serine is mutated to threonine as defined as type C mutation, and the 293rd glycine is mutated to alanine as defined as type D mutation. The point mutation random combination method is adopted (a total of 15 schemes, and the specific mutation forms are shown in Table 1 below).

[0086] Table 1

[0087] A B C D 1 √ ○ ○ ○ 2 ○ √ ○ ○ 3 ○ ○ √ ○ 4 ○ ○ ○ √ 5 √ √ ○ ○ 6 √ ○ √ ○ 7 √ ○ ○ √ 8 ○ √ √ ○ 9 ○ √ ○ √ 10 ○ ○ √ √ 11 √ √ √ ○ 12 √ √ ○ √ 13 √ ○ √ √ 14 ○ √ √ √ 15 √ √ √ √

[0088] Note: “√” means the mutant contains this type of point mutation, and “○” means it does not contain this type of point mutation.

[0089] The nucleotide sequences of proteinase K mutants 1-15, SEQ ID NOs: 13-27, were synthesized by gene synthesis, and the nucleotide sequences of NotI and CpoI restriction endonuclease cleavage sites were added on both sides of the sequences. Figure 1 ) and the nucleotides of the proteinase K mutant were simultaneously digested with NotI and CpoI enzymes, and the digestion products were purified and recovered and then connected with DNA ligase to obtain the recombinant expression vector.

[0090] The constructed recombinant Pichia pastoris was inoculated into a test tube containing 20 mL YDPA liquid medium and cultured overnight at 30°C until the bacterial concentration reached OD 600 =5-8; then the culture was inoculated into a conical flask containing 100mL YDPA liquid medium at a ratio of 1:100, and cultured at 30°C overnight with shaking until the bacterial solution concentration reached OD 600 =35-40, let the culture stand for 3-4, pour out the supernatant and resuspend in 100mL YDPA liquid medium; add 0.5% (v / v) methanol, induce expression at 25°C for 24h-72h, add 0.5% (v / v) methanol to the culture medium every 24h for induction, centrifuge at 4°C, 5000rpm for 5min, and collect the supernatant.

[0091] Proteinase K was purified by cation exchange chromatography column, and the steps were as follows: ① ultrafiltration and concentration of the centrifugal supernatant to a protein concentration of 5 mg / mL, and replacement with loading buffer (loading buffer: 10 mM NaAc, 25 mM NaCl, pH 5.0); ② balancing the cation exchange chromatography column with loading buffer: ③ loading the protein at a flow rate of 2 ml / min, and collecting the eluate; ④ washing with washing solution 1 (10 mM NaAC, 25 mM NaCl, pH 5.0), washing for 10 column volumes and collecting the eluate; ⑤ washing with washing solution 2 (10 mM NaAC, 50 mM NaCl, pH 5.0), washing for 10 column volumes and collecting the washing solution; ⑥ eluting with eluent (10 mM NaAC, 100 mM NaCl, pH 5.0), eluting for 5 column volumes, and collecting the eluate to obtain the purified enzyme; ⑦ washing with column washing solution (10 mM NaAC, 500mM NaCl, pH5.0), rinse for 10 column volumes; ⑧ 10 column volumes of ultrapure water to rinse the chromatography column; ⑨ 20% ethanol to preserve the cation exchange chromatography column.

[0092] Example 2: Recombinant fermentation and purification of wild-type proteinase K (PK)

[0093] The amino acid sequence of wild-type proteinase K (PK) is as follows (SEQ ID NO: 1):

[0094] APAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKV LDDNGSGQYSTIIAGMDFVASDKNRNRCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQALE

[0095] The coding sequence of the above wild-type proteinase K (PK) is as follows (SEQ ID NO: 2):

[0096]

[0097] The recombinant fermentation and purification methods are the same as those in Example 1.

[0098] Example 3: Comparison of tolerance of wild-type proteinase K and proteinase K mutants 1-15 to high concentrations of guanidine thiocyanate

[0099] First, the rapid detection method of residual proteinase K activity:

[0100] 1. Detection principle: Proteinase K is an important serine protease secreted by Tritirachium album Limber. It cuts the carboxyl terminal peptide bonds of aliphatic amino acids and aromatic amino acids and has a strong ability to degrade natural proteins. The Folin-phenol method is often used to determine the activity of proteinase K, but this method is complicated to operate and has a long reaction time, so it is not suitable for large-scale screening. Therefore, the following method is used for qualitative determination: Suc-(Ala)2-Pro-Phe-pNA is used as a substrate, reacted under certain temperature and pH conditions, terminated the reaction with glacial acetic acid, measured the absorbance at 405nm, and determined the amount of yellow nitroaniline released by the enzymatic reaction, thereby detecting the enzyme activity.

[0101] 2. Scope of application: This method is a qualitative activity measurement method, which is only used to compare the residual activity of proteins after being treated with lysis buffer. It is generally used to screen highly tolerant transformants or to determine the tolerance of proteins from different sources.

[0102] 3. Main reagents:

[0103] a) Substrate solution: accurately weigh 5 mg of Suc-(Ala)2-Pro-Phe-pNA (Sigma) and dissolve it in 200 μL of DMSO. Prepare and use immediately.

[0104] b) Enzyme diluent: 20 mM Tris-HCl, 2 mM CaCl2, sterilized by filtration and placed at room temperature;

[0105] 4. Sample determination: Pipette 200 μL enzyme diluent and 7 μL substrate solution into the ELISA plate, balance at 25°C for 5 min, mix well and add 10 μL enzyme solution sample, continue the reaction for 10 min, add glacial acetic acid to terminate the reaction, mix well and measure the absorbance at 405 nm;

[0106] 5. Result analysis: Residual activity (%) = Δ after treatment / Δ before treatment × 100%

[0107] ΔAfter treatment: absorbance of sample after treatment - absorbance of blank

[0108] ΔBefore treatment: absorbance of sample before treatment - absorbance of blank

[0109] Then, the residual activities of wild-type proteinase K and proteinase K mutants under treatment with different concentrations of guanidine isothiocyanate were tested and compared according to the above method. The specific treatment method is as follows:

[0110] 9 μL of 1.5 mg / mL protein solution was taken respectively and treated in 300 μL of guanidine isothiocyanate solution of different concentrations at 25°C for 5 min. The enzyme activity was then tested according to the above method to calculate the residual activity.

[0111] Table 2. Residual activity of proteinase K mutants 1-15 after treatment with guanidine thiocyanate at different concentrations

[0112]

[0113] The results are shown in Table 2 above. In a 3 mol / L guanidine thiocyanate solution, the wild-type proteinase K basically lost its residual activity, mutants 1-4, mutants 9 and mutant 13 also basically lost their residual activity, while the residual activity of mutants 5-8, mutants 10-12 and mutants 14-15 was greater than 40%.

[0114] Example 4. Comparison of resistance of wild-type proteinase K and proteinase K mutant 1-15 to nucleic acid extraction and lysis buffer containing high concentration of guanidine thiocyanate

[0115] 9 μL of 1.5 mg / mL protein solution was taken and treated in 300 μL of 3 mol / L guanidine isothiocyanate nucleic acid extraction and lysis solution. The enzyme activity was tested according to the above method at different treatment time points to calculate the residual activity.

[0116] Table 3. Residual activity of proteinase K mutants 1-15 after different treatment times in lysate

[0117]

[0118] The results are shown in the table above. At 25°C, the residual activity of the wild-type proteinase K was only 28% after 60 minutes of treatment with nucleic acid extraction lysis solution, the residual activity of proteinase K of mutants 1-4 and mutant 9 was between 0% and 30%, and mutants 5-8 and mutants 10-15 were still able to maintain more than 50% residual activity after 60 minutes of treatment with lysis solution.

[0119] Example 5: Comparison of the activities of wild-type proteinase K and proteinase K mutants

[0120] 1. Detection principle: Proteinase K is an important serine protease secreted by Tritirachium album Limber. It cuts the carboxyl terminal peptide bonds of aliphatic amino acids and aromatic amino acids and has a strong ability to degrade natural proteins. Hydrolysis of hemoglobin produces amino acids containing phenol groups, which can be reduced by Folin reagent under alkaline conditions to produce molybdenum blue and tungsten blue. The depth of its color is proportional to the content of phenolic amino acids. The activity of proteinase K can be calculated by detecting the absorbance at 750nm.

[0121] 2. Enzyme activity unit: The amount of proteinase K equivalent to 1 μM Folin-positive amino acid or polypeptide that can be produced within 1 minute at 37°C using hemoglobin as substrate is defined as one unit of proteinase K enzyme activity.

[0122] 3. Main reagents:

[0123] a) Folin phenol colorimetric solution (1M): purchased from Shanghai Biotechnology Co., Ltd.

[0124] b) 5% TCA solution: accurately weigh 4.9 g TCA and add 100 mL pure water to dissolve;

[0125] c) 2% hemoglobin solution: accurately weigh 2g hemoglobin (Diamond A002402-0005), add 40ml pure water, stir at 37℃ for 30min; add 8ml 1M NaOH solution, stir at 37℃ for 20min; add 36g urea, stir at 37℃ for 1h; add 10ml 1M Tris-HCl buffer, adjust pH to 7.5 with hydrochloric acid, filter at 0.22μm. Store at -20℃, valid for 3 months; store at 4℃, valid for 30 days.

[0126] d) Tyrosine standard solution (1.1 mM): accurately weigh 0.01812 g tyrosine and add 100 ml 0.2 M HCl to dissolve;

[0127] e) Enzyme diluent: dilute 1M Tris-HCl stock solution and 100mM CaCl2 stock solution to prepare 20mM Tris-HCl, 2mM CaCl2 enzyme diluent, filter, and place at room temperature.

[0128] 4. Sample determination:

[0129] a) diluting the purified protease solution to 1 mg / mL according to the protein concentration and then diluting it 200 times to measure the activity;

[0130] b) Pipette 50 μL of 2% hemoglobin solution into a 1.5 mL centrifuge tube, equilibrate at 37°C for 10 min, mix well, add 10 μL of diluted enzyme solution, continue the reaction at 37°C for 10 min, and then add 100 μL of 5% TCA to terminate the reaction;

[0131] c) At the same time, prepare a sample blank tube, add enzyme diluent after TCA termination;

[0132] d) After mixing, place at room temperature for 20 min and centrifuge at 12000 rpm for 5 min;

[0133] e) Take 60 μl of the supernatant after the previous centrifugation, add 120 μl of 0.5 M NaOH, mix well, add 36 μl of Folin phenol colorimetric solution, mix well, leave at room temperature for 30 min, and measure the change in absorbance at 750 nm.

[0134] 5. Vitality calculation:

[0135] Enzyme activity (U / mg) = 267×(Δ750nm×0.0256-0.00003) / (10×c)

[0136] c: Protein concentration after dilution

[0137] 6. The mutants 1-15 and wild-type proteinase K samples purified in Examples 1 and 2 were tested for enzyme activity according to this method. The test results are as follows:

[0138] Table 4

[0139]

[0140]

[0141] The results are shown in Table 4 above. The enzyme activity of the mutant of the present invention is comparable to that of the wild-type proteinase K, with a deviation within 5%;

[0142] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A proteinase K mutant having a prolonged tolerance to guanidine isothiocyanate in a nucleic acid extraction lysate, characterized in that: Its amino acid sequence is mutated from the sequence shown in SEQ ID NO.1, and the proteinase K mutant is selected from any one of the following: 1) The tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the lysine at position 208 is mutated to histidine; 2) the tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the serine at position 273 is mutated to threonine; 3) corresponding to the mutation of lysine at position 208 to histidine and serine at position 273 to threonine in SEQ ID NO.1; 4) corresponding to SEQ ID NO.1, the lysine at position 208 is mutated to histidine, the serine at position 273 is mutated to threonine, and the glycine at position 293 is mutated to alanine; The amino acid sequence of the mutant is shown in any one of SEQ ID NO. 3, 4, 6, and 11.

2. A polynucleotide encoding the proteinase K mutant according to claim 1, wherein the nucleotide sequence of the polynucleotide is shown in SEQ ID NOs. 17, 18, 20, and 26.

3. Use of a proteinase K mutant in extending the tolerance time of proteinase K to guanidine isothiocyanate in nucleic acid extraction lysis solution, characterized in that: Its amino acid sequence is mutated from the sequence shown in SEQ ID NO.1, and the proteinase K mutant is selected from any one of the following: 1) The tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the lysine at position 208 is mutated to histidine; 2) the tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the serine at position 273 is mutated to threonine; 3) corresponding to the mutation of tyrosine at position 151 to alanine and glycine at position 293 to alanine in SEQ ID NO.1; 4) corresponding to the mutation of lysine at position 208 to histidine and serine at position 273 to threonine in SEQ ID NO.1; 5) The serine at position 273 corresponding to SEQ ID NO.1 is mutated to threonine and the glycine at position 293 is mutated to alanine; 6) corresponding to SEQ ID NO.1, the tyrosine at position 151 is mutated to alanine, the lysine at position 208 is mutated to histidine, and the serine at position 273 is mutated to threonine; 7) corresponding to SEQ ID NO.1, the tyrosine at position 151 is mutated to alanine, the lysine at position 208 is mutated to histidine, and the glycine at position 293 is mutated to alanine; 8) The proteinase K mutant is based on the amino acid sequence shown in SEQ ID NO.1, with the tyrosine at position 151 mutated to alanine, the lysine at position 208 mutated to histidine, the serine at position 273 mutated to threonine, and the glycine at position 293 mutated to alanine; The amino acid sequence of the mutant is shown in any one of SEQ ID NOs. 3-7, 9, 11, and 12.

4. An expression vector, characterized in that: The vector contains the polynucleotide according to claim 2; the expression vector is selected from: Pichia pastoris expression vector pPinkα-HC, pPIC9K, pPICZα-C.

5. The expression vector according to claim 4, characterized in that The expression vector is pPinkα-HC.

6. A genetically engineered host cell, characterized in that The host cell contains the vector according to claim 4 or 5, or the polynucleotide according to claim 2 is integrated into its genome, and the host cell includes a prokaryotic cell or a eukaryotic cell.

7. The host cell according to claim 6, characterized in that The eukaryotic cell is selected from yeast cells, mold cells, insect cells, higher plant cells, fungal cells or mammalian cells.

8. The host cell according to claim 7, characterized in that The yeast cell is Pichia pastoris pPINK.

9. The host cell according to claim 7, characterized in that The prokaryotic cells include Escherichia coli cells and Bacillus subtilis cells.

10. A method for producing the proteinase K mutant according to claim 1, characterized in that: The genetically engineered host cell according to any one of claims 6 to 9 is propagated under suitable conditions and allowed to express the polynucleotide encoding the proteinase K mutant according to claim 2, and then the proteinase K mutant is isolated from the host cell culture.

11. A method for prolonging the tolerance time of proteinase K to guanidine isothiocyanate in nucleic acid extraction lysis solution, characterized in that: The method is to mutate the proteinase K of the amino acid sequence of Candida albicans derived from lincomycetes as shown in SEQ ID NO.1, wherein the proteinase K mutant is selected from any one of the following: 1) The tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the lysine at position 208 is mutated to histidine; 2) the tyrosine at position 151 corresponding to SEQ ID NO.1 is mutated to alanine and the serine at position 273 is mutated to threonine; 3) corresponding to the mutation of tyrosine at position 151 to alanine and glycine at position 293 to alanine in SEQ ID NO.1; 4) corresponding to the mutation of lysine at position 208 to histidine and serine at position 273 to threonine in SEQ ID NO.1; 5) The serine at position 273 corresponding to SEQ ID NO.1 is mutated to threonine and the glycine at position 293 is mutated to alanine; 6) corresponding to SEQ ID NO.1, the tyrosine at position 151 is mutated to alanine, the lysine at position 208 is mutated to histidine, and the serine at position 273 is mutated to threonine; 7) corresponding to SEQ ID NO.1, the tyrosine at position 151 is mutated to alanine, the lysine at position 208 is mutated to histidine, and the glycine at position 293 is mutated to alanine; 8) The proteinase K mutant is based on the amino acid sequence shown in SEQ ID NO.1, with the tyrosine at position 151 mutated to alanine, the lysine at position 208 mutated to histidine, the serine at position 273 mutated to threonine, and the glycine at position 293 mutated to alanine; The amino acid sequence of the mutant is shown in any one of SEQ ID NOs. 3-7, 9, 11, and 12.

12. A composition comprising the proteinase K mutant according to claim 1, characterized in that The composition contains an enzyme protectant or a substance for delaying enzyme decomposition; the composition, in addition to water, also includes the following components: Tris 30mmol / L, Ca 2+ 10mmol / L-50mmol / L, glycerol 5v%, use HCl to adjust pH to 7.5-8.

4.

13. Use of the proteinase K mutant of claim 1, the genetically engineered host cell of any one of claims 6 to 9, or the composition of claim 12 for enzymatic hydrolysis of proteins, which is used to decompose ester bonds or peptide bonds adjacent to the C-termini of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids.

14. A method for enzymatically hydrolyzing protein, characterized in that: The method comprises: performing an enzymatic hydrolysis reaction using the proteinase K mutant of claim 1, the genetically engineered host cell expressing the mutant of any one of claims 6 to 9 or its lysate, or the composition of claim 12.

15. A detection system or detection kit for enzymatic protein hydrolysis, comprising: the proteinase K mutant according to claim 1, the genetically engineered host cell expressing the mutant according to any one of claims 6 to 9, or a lysate thereof.

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