CN113913413B

CN113913413B - Salt-tolerant RPK mutant and application thereof

Info

Publication number: CN113913413B
Application number: CN202110918233.XA
Authority: CN
Inventors: 刘晓; 赵致
Original assignee: SHANGHAI YAXIN BIOTECHNOLOGY CO Ltd
Current assignee: SHANGHAI YAXIN BIOTECHNOLOGY CO Ltd
Priority date: 2021-08-11
Filing date: 2021-08-11
Publication date: 2023-10-27
Anticipated expiration: 2041-08-11
Also published as: CN113913413A

Abstract

The application provides a salt-tolerant RPK mutant and application thereof. The inventor finds that some sites of proteinase K are closely related to salt tolerance and stability, and develops a proteinase K mutant on the basis, and compared with a wild type, the proteinase K mutant has better salt tolerance and stability. The application also provides an optimized method for expressing the recombinant proteinase K mutant, which has high yield and low production cost and is suitable for large-scale production.

Description

Salt-tolerant RPK mutant and application thereof

Technical Field

The application belongs to the field of biotechnology; more specifically, the application relates to a salt-tolerant RPK mutant and uses thereof.

Background

Proteinase K (PK) is a non-specific endoprotease belonging to the class of serine proteases, which cleaves ester and peptide bonds linked at the carboxyl terminus of aliphatic, hydrophobic and aromatic amino acids. The molecular weight is 29.3kDa (monomer), and the molecular weight is active under the condition of pH4 to pH12, and is stable in the presence of SDS, urea or EDTA.

Proteinase K is capable of cleaving the carboxyl-terminal peptide bonds of aliphatic and aromatic amino acids for degradation of proteins in biological samples. The enzyme was decolorized and purified to remove RNA and DNA, and no other enzyme activities were detected. Since proteinase K is stable in urea and SDS, it also has the ability to degrade natural proteins, the smallest polypeptide that it hydrolyzes is a tetrapeptide molecule.

However, the present inventors found that Recombinant Proteinase K (RPK) is prone to aggregation and precipitation during fermentation production, which brings about problems for practical production processes.

Disclosure of Invention

The application aims to provide a salt-tolerant RPK mutant and application thereof.

In a first aspect of the application, there is provided a proteinase K mutant which is: (a) An enzyme having an amino acid sequence corresponding to proteinase K shown in SEQ ID NO. 1, mutated at the following positions: 266 th bit and 279 th bit; the site mutation is hydrophilic or neutral amino acid.

In one or more embodiments, the proteinase K mutant has a mutation at position 266 to Tyr and a mutation at position 279 to Gly.

In one or more embodiments, the amino acid sequence of the proteinase K mutant is shown in SEQ ID NO. 2.

In another aspect of the application, there is provided an isolated polynucleotide encoding a proteinase K mutant as described in the previous aspect.

In one or more embodiments, the nucleotide sequence of the polynucleotide is set forth in SEQ ID NO. 4.

In another aspect of the application, there is provided a vector comprising said polynucleotide.

In another aspect of the application there is provided a genetically engineered host cell comprising said vector, or said polynucleotide integrated in the genome.

In one or more embodiments, the host cell comprises a prokaryotic cell or a eukaryotic cell.

In one or more embodiments, the eukaryotic cells include yeast cells, mold cells, insect cells, plant cells, fungal cells, mammalian cells, or the like.

In one or more embodiments, the prokaryotic cells include E.coli cells, bacillus subtilis cells, and the like.

In another aspect of the present application, there is provided a method for improving the salt tolerance or stability of proteinase K comprising mutating it to a position or combination of positions selected from the group consisting of: 266 th bit and 279 th bit; the site mutation is hydrophilic or neutral amino acid.

In another aspect of the present application, there is provided a method for producing a proteinase K mutant as described in any one of the preceding, the method comprising: (i) culturing said host cell; (ii) Collecting a culture containing said proteinase K mutant; (iii) Isolating the proteinase K mutant from the culture.

In a further aspect of the application there is provided the use of a proteinase K mutant, host cell expressing the mutant or lysate thereof as described in any one of the preceding claims for enzymatic hydrolysis of a protein; preferably, it is used to specifically recognize and cleave the carboxyl-terminal peptide bond of aliphatic and aromatic amino acids, enzymatic hydrolysis of proteins.

In another aspect of the application, there is provided a method of enzymatic hydrolysis of a protein comprising: the proteinase K mutant, the host cell expressing the mutant or the cleavage product thereof is used for enzymolysis reaction.

In another aspect of the present application, there is provided a detection system or a detection kit for enzymatic hydrolysis of proteins, comprising: a proteinase K mutant according to any one of the preceding claims, a host cell expressing the mutant or a lysate thereof.

Other aspects of the application will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, three-dimensional structural comparison of wild-type RPK and RPK mutant mutants.

FIG. 2, hydrophobicity plots of wild-type RPK and RPK mutant molecules.

FIG. 3, comparison of the stability of wild-type RPK and RPK mutant stocks.

Detailed Description

Through intensive research experiments, the inventor discovers that some sites of proteinase K are closely related to salt tolerance and stability. On the basis, the inventor obtains a proteinase K mutant, and compared with a wild type, the proteinase K mutant has better salt tolerance and stability. The application also provides an optimized method for expressing the recombinant proteinase K mutant, which has high yield and low production cost and is suitable for large-scale production.

Terminology

As used herein, unless otherwise indicated, the terms "mutant of proteinase K", "mutant proteinase K" are used interchangeably to refer to a protein corresponding to wild-type proteinase K (as set forth in SEQ ID NO: 1) that has undergone a mutation at a position selected from the group consisting of: 266 th and 279 th.

As used herein, unless otherwise indicated, the terms "mutant of proteinase K", "mutant proteinase K" and "mutant proteinase K" are used interchangeably to refer to the product of mutating wild-type proteinase K.

As used herein, if reference is made to wild-type proteinase K, it will be denoted "wild-type proteinase K", protein of the amino acid sequence shown in SEQ ID NO. 1 or WT.

As used herein, "isolated" refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the naturally occurring polynucleotide and protein are not isolated and purified within a living cell, the same polynucleotide or protein is isolated and purified if it is separated from other substances that are naturally occurring.

As used herein, an "isolated proteinase K mutant" refers to a proteinase K mutant that is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. The skilled person will be able to purify proteinase K mutants using standard protein purification techniques. Substantially pure proteins can produce a single main band on a non-reducing polyacrylamide gel.

As used herein, "Recombinant" refers to a protein, genetically engineered vector, cell, or the like obtained (or prepared in large quantities) by genetic engineering means.

As used herein, "improving salt tolerance or stability" refers to a statistically significant improvement in salt tolerance or stability of a mutated proteinase K, or referred to as a significant improvement, as compared to the wild-type proteinase K prior to modification. For example, the salt tolerance or stability of the mutant proteinase K having improved salt tolerance or stability is significantly improved by 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 70% or more, 80% or more, 100%,150% or more, or the like, as compared with the enzyme before modification under the same reaction conditions/environment.

As used herein, the term "comprising" or "including" includes "comprising," consisting essentially of … …, "and" consisting of … …. The term "consisting essentially of … …" means that in the composition/reaction system/kit, minor ingredients and/or impurities may be present in minor amounts and without affecting the active ingredient, in addition to the essential ingredients or components.

As used herein, the term "effective amount" refers to an amount that produces a function or activity for the reaction of interest in the present application that achieves the desired effect (accurate detection result).

Proteinase K mutant, its coding nucleic acid and construct

The proteinase K mutants of the application may be chemically synthesized products or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher plant, insect and mammalian cells).

The application also includes fragments, derivatives and analogues of the proteinase K mutant. As used herein, the terms "fragment," "derivative" and "analog" refer to proteins that retain substantially the same biological function or activity of the native proteinase K mutants of the present application. The protein fragments, derivatives or analogues of the application may be (i) proteins having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) proteins having a substituent in one or more amino acid residues, or (iii) proteins in which an additional amino acid sequence is fused to the protein sequence (such as a leader or secretory sequence or a sequence used to purify the protein or a proprotein sequence, or fusion proteins). Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known to those skilled in the art in view of the definitions herein. However, the conditions that need to be satisfied are: in the amino acid sequence of the proteinase K mutant and its fragments, derivatives and analogues there must be at least one mutation specifically indicated above in the present application, preferably the mutation corresponds to the amino acid sequence shown in SEQ ID No. 1, including the mutation at position 266 to Tyr and the mutation at position 279 to Gly.

In the present application, the term "proteinase K mutant" also includes (but is not limited to): deletion, insertion and/or substitution of several (usually 1-20, more preferably 1-10, still more preferably 1-8, 1-5, 1-3, or 1-2) amino acids, and addition or deletion of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of proteinase K mutants. However, in these variants, it is certain that the above-described mutations of the present application are present, preferably the mutation is an amino acid sequence corresponding to that shown in SEQ ID NO. 1, including the mutation at position 266 to Tyr and the mutation at position 279 to Gly.

In the present application, the term "proteinase K mutant" also includes (but is not limited to): a derivative protein having 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, such as 98% or more, 99% or more, sequence identity to the amino acid sequence of the proteinase K mutant, which retains its protein activity. Likewise, in these derived proteins, the mutations described above in the present application are certainly present, preferably the mutation is an amino acid sequence corresponding to that shown in SEQ ID NO. 1, including the mutation at position 266 to Tyr and the mutation at position 279 to Gly.

The application also provides analogues of the proteinase K mutant. These analogs may differ from the proteinase K mutant by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, by site-directed mutagenesis or other known techniques of molecular biology. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present application are not limited to the representative polypeptides exemplified above. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve their proteolytic resistance or to optimize solubility.

The application also provides polynucleotide sequences encoding the proteinase K mutants of the application or a conservative variant protein thereof.

The polynucleotides of the application may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

Polynucleotides encoding the mature proteins of the mutants include: a coding sequence encoding only the mature protein; coding sequences for mature proteins and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature protein, and non-coding sequences.

The "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, or may include additional coding and/or non-coding sequences.

The application also relates to vectors comprising the polynucleotides of the application, as well as host cells genetically engineered with the vectors or proteinase K mutant coding sequences of the application, and methods for producing the mutated enzymes of the application by recombinant techniques.

The polynucleotide sequences of the present application may be used to express or produce recombinant proteinase K mutants by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a proteinase K mutant of the application, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present application, the proteinase K mutant polynucleotide sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing proteinase K mutant encoding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

In the present application, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells such as mold cells, yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: coli, bacillus subtilis, streptomycete, agrobacterium; eukaryotic cells such as yeast, plant cells, and the like. In a specific embodiment of the application, yeast cells are used as host cells.

It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Recombinant expression of mutants

The recombinant cells (host cells) established by the present application can be cultured by conventional methods to express the polypeptide encoded by the gene of the present application. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The proteinase K mutants of the application may be expressed in cells, or on cell membranes, or secreted extracellularly, when expressed. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, sonication, 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.

The method for recombinant expression of proteinase K mutant solves the problems of low stability, unsatisfactory salt tolerance and easy generation of precipitation of the recombinant proteinase K in the prior art.

Application of recombinant proteinase K

RPK is obtained by fermentation of Pichia pastoris using a medium of about 1M high concentration of phosphate and sulfate and under acidic conditions at pH 5.0. The stability of RPK in this high salt medium becomes critical to the success of RPK fermentation.

The RPK expressed by Pichia pastoris is extremely easy to precipitate in higher salt concentration, and possible reasons for this are that the RPK is easy to aggregate or co-precipitate under higher ionic strength and hetero protein, and the higher the protein concentration, the more obvious the precipitation. Therefore, the problem that the RPK is easy to aggregate and precipitate brings problems to the actual production process. The prior art RPK or its modified polypeptides have not addressed this problem at high salt concentrations. The inventor analyzes the molecular structure of wild RPK, finds out the reason that aggregation and precipitation are easy to occur, carries out directional transformation on the primary structure of protein, and improves the solution stability of the protein. The application solves the problems of low salt tolerance, low stability and the like in the expression and purification of the existing recombinant proteinase K.

The engineered proteinase K mutants of the application have a variety of uses related to proteinase K properties including, but not limited to: specifically recognizes and cleaves the carboxyl-terminal peptide bond of aliphatic amino acids and aromatic amino acids, enzymatically hydrolyzes proteins or denatures proteins.

As one embodiment, the proteinase K mutant can be applied to extraction of genome DNA and digestion and removal of enzyme.

As one embodiment, the proteinase K mutant can be applied to preparing chromosomal DNA by pulse electrophoresis, western blotting, nuclease removal in DNA and RNA preparation and the like

As one embodiment, the proteinase K mutant may be used in an in situ hybridization technique for pre-hybridization treatment, which has the effect of digesting surrounding target DNA proteins to increase the chance of binding of the probe to the target nucleic acid and enhance hybridization signals.

As some industrial embodiments, the proteinase K mutant can be applied to leather, fur, silk, medicine, food, brewing and the like. The dehairing and softening of leather industry have largely utilized protease, and have the characteristics of time saving and convenience. The protease can also be used for silk degumming, meat tenderization and wine clarification. Clinically, it can be used for treating dyspepsia with pepsin, bronchitis with acid protease, vasculitis with elastase, and purification of suppurative wound and intrathoracic serosa adhesion with proteinase K and chymotrypsin. The enzyme-added washing powder is a new product in the detergent, contains alkaline protease and can remove blood stains and protein dirt on clothes.

The proteinase K mutant has the advantages of high yield, good stability, high enzyme activity, low cost and suitability for large-scale industrial production. Meanwhile, the proteinase K mutant can be stored for a long time, and meets the requirements of industrial production.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

EXAMPLE 1 recombinant fermentation of wild-type Proteinase K (PK)

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

AAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA

the coding sequence of the wild-type Proteinase K (PK) is as follows (SEQ ID NO: 3):

gcggcgcagaccaacgcgccgtggggcctggcgcgcattagcagcaccagcccgggcaccagcacctattattatgatgaaagcgcgggccagggcagctgcgtgtatgtgattgataccggcattgaagcgagccatccggaatttgaaggccgcgcgcagatggtgaaaacctattattatagcagccgcgatggcaacggccatggcacccattgcgcgggcaccgtgggcagccgcacctatggcgtggcgaaaaaaacccagctgtttggcgtgaaagtgctggatgataacggcagcggccagtatagcaccattattgcgggcatggattttgtggcgagcgataaaaacaaccgcaactgcccgaaaggcgtggtggcgagcctgagcctgggcggcggctatagcagcagcgtgaacagcgcggcggcgcgcctgcagagcagcggcgtgatggtggcggtggcggcgggcaacaacaacgcggatgcgcgcaactatagcccggcgagcgaaccgagcgtgtgcaccgtgggcgcgagcgatcgctatgatcgccgcagcagctttagcaactatggcagcgtgctggatatttttggcccgggcaccagcattctgagcacctggattggcggcagcacccgcagcattagcggcaccagcatggcgaccccgcatgtggcgggcctggcggcgtatctgatgaccctgggcaaaaccaccgcggcgagcgcgtgccgctatattgcggataccgcgaacaaaggcgatctgagcaacattccgtttggcaccgtgaacctgctggcgtataacaactatcaggcg

the above coding sequence was inserted into plasmid pPIC9K and recombinantly integrated into pichia pastoris GS115.

FermentationAnd (3) liquid: 85% H ₃ PO ₄ 26.7ml/L, KOH 4.13g/L, glycerol 40g/L, K ₂ SO ₄ 18.2g/L，CaSO ₄ 0.93g/L，MgSO ₄ ·7H ₂ O14.9 g/L, salt concentration about 1M.

Fermentation process (including fermentation conditions): the whole fermentation is divided into three stages of glycerol batch fermentation, glycerol feed fermentation and methanol induction fermentation. In glycerol batch fermentation, higher dissolved oxygen levels are maintained in the fermentation broth due to lower bacterial concentration. As the bacterial concentration increases, DO begins to gradually decrease to the point where glycerol metabolism in the medium is complete, and depletion of glycerol will lead to a slow down of bacterial growth, with a steep rise in DO. The initiation of the induction expression phase starts when the bacterial concentration OD600 reaches a higher value, and methanol induction expression is performed. Wherein the fermentation is controlled to be maintained at 20-30% mainly according to the change of dissolved oxygen parameters.

Monitoring the progress of fermentation, the inventors found that pichia pastoris-expressed Recombinant Proteinase K (RPK) is highly susceptible to precipitation at higher salt concentrations (salt concentrations of about 1M or higher than 1M), probably because RPK is susceptible to aggregation or co-precipitation at higher protein concentrations at higher ionic strengths. Moreover, as the fermentation proceeds, the higher the protein concentration, the more pronounced the precipitation that can be observed. The yield of active RPK protein is severely affected by the large amount of precipitate generated.

Therefore, in fermentation production, the problem of aggregation and precipitation of RPK is easy to occur, and the problem is brought to the actual production process.

Example 2 optimized engineering of RPK proteins

Based on the wild-type Proteinase K (PK), the inventors analyzed its three-dimensional structure, surface hydrophilicity and hydrophobicity, accessibility to amino acid solvents, and the like.

Results repeated studies and analyses, the present inventors located the engineered sites at Phe266 and Ala279, which are hydrophobic amino acids on the surface of the three-dimensional structure of the protein, which contributed to the tendency to aggregate precipitation under conditions of higher protein concentration and ionic strength, resulting in precipitation of RPK zymogen solution and thus a decrease in protein concentration.

The inventor adopts a site-directed mutagenesis method to modify the amino acid into hydrophilic and neutral amino acids with similar side chain group structures, thereby reducing the hydrophobicity of the site. The amino acid analysis shows that Phe266 is mutated into Tyr, one more hydroxyl group is used as a hydrophilic group on the R group side chain of Tyr compared with Phe, ala279 is mutated into Gly, the R group of the Phe is changed into a hydrophobic methyl group, and the hydrophobicity of the hydrogen atom is reduced.

As in FIG. 1, wild-type RPK (left panel) and mutant RPK (right panel) were compared, and amino acid residues 266 and 279 in the figures were marked. The asterisks scattered in wild-type RPK are indicated as water molecules, with few surrounding water molecules and far away, as Phe266 and Ala279 are on the molecular surface and are more hydrophobic. The right panel shows the simulated three-dimensional structure of the RPK mutant, and therefore no water molecules are shown around. The 266 th amino acid residue of PK mutant is changed into Tyr, and the side chain benzene ring increases the hydrophilic hydroxyl, so the hydrophilicity is increased. Ala at position 279 is mutated into Gly, and the hydrophobic liquid is reduced.

As shown in FIG. 2, the inventors analyzed the hydrophobicity of the molecular surfaces of wild-type RPK (left panel) and RPK mutant (right panel), in which blue represents hydrophilic region, red represents hydrophobic region, and white represents neutral region. The region of amino acid residues 226 and 279 on the surface of the molecule is marked with black ellipses and amino acid numbers. From the comparison of the two, the RPK mutant presents stronger hydrophobicity at 266 and 279 amino acid residues, the right-hand RPK mutant is changed into a hydrophilic group at 266, and the 266 amino acid is changed into a neutral region from a hydrophobic region, so that the unstable phenomena such as aggregation and precipitation in aqueous solution and the like are relieved by reducing the surface hydrophobic region of the RPK mutant.

According to the modification described above, the mutated amino acid sequence is as follows (SEQ ID NO: 2):

AAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPYGTVNLLAYNNYQG

the coding sequence of the mutant Proteinase K (PK) is as follows (SEQ ID NO: 4):

gcggcgcagaccaacgcgccgtggggcctggcgcgcattagcagcaccagcccgggcaccagcacctattattatgatgaaagcgcgggccagggcagctgcgtgtatgtgattgataccggcattgaagcgagccatccggaatttgaaggccgcgcgcagatggtgaaaacctattattatagcagccgcgatggcaacggccatggcacccattgcgcgggcaccgtgggcagccgcacctatggcgtggcgaaaaaaacccagctgtttggcgtgaaagtgctggatgataacggcagcggccagtatagcaccattattgcgggcatggattttgtggcgagcgataaaaacaaccgcaactgcccgaaaggcgtggtggcgagcctgagcctgggcggcggctatagcagcagcgtgaacagcgcggcggcgcgcctgcagagcagcggcgtgatggtggcggtggcggcgggcaacaacaacgcggatgcgcgcaactatagcccggcgagcgaaccgagcgtgtgcaccgtgggcgcgagcgatcgctatgatcgccgcagcagctttagcaactatggcagcgtgctggatatttttggcccgggcaccagcattctgagcacctggattggcggcagcacccgcagcattagcggcaccagcatggcgaccccgcatgtggcgggcctggcggcgtatctgatgaccctgggcaaaaccaccgcggcgagcgcgtgccgctatattgcggataccgcgaacaaaggcgatctgagcaacattccgtatggcaccgtgaacctgctggcgtataacaactatcagggc

using the same method as in example 1, the present inventors introduced the coding sequence of the above-described modified enzyme into a Pichia pastoris expression vector, and transformed Pichia pastoris.

Using the same fermentation method as in example 1, the present inventors used the recombinant Pichia pastoris to ferment and express mutant proteinase K.

Monitoring the progress of fermentation, the inventors found that pichia pastoris-expressed mutant Recombinant Proteinase K (RPK) is less prone to precipitate at higher salt concentrations (salt concentrations of about 1M or above 1M) and no significant aggregation or co-precipitation was observed.

EXAMPLE 3 comparison of stability of RPK stock before and after mutation

By carrying out the fermentation by the fermentation method of example 1, the present inventors quantitatively counted and compared the activity of the fermentation broth of the active protein of RPK of the wild type as well as the mutant.

The activity of the fermentation broth was measured 72 hours after methanol induction, and the measurement method was as follows: casein is used as a substrate. Definition of Activity: the amount of proteinase K which hydrolyzes casein substrates to 1. Mu. Mol L-tyrosine per minute at 37℃pH 7.5 is defined as one unit (U).

The results of the comparison of the activities of the fermentation broths are shown in Table 1.

TABLE 1 Activity of fermentation broths

According to Table 1, the wild type RPK produced precipitation during fermentation in a high salt environment of the fermentation broth, so that the fermentation broth activity was only 530U/ml. In contrast, the activity of the RPK mutant fermentation broth can reach 716U/ml.

Therefore, the RPK mutant has good salt tolerance.

Example 4 comparison of RPK purification Processes and recovery rates before and after mutagenesis

By carrying out the fermentation by the fermentation method of example 1, the present inventors quantitatively counted and compared the recovery rates of the active protein of the wild-type and mutant RPKs.

After fermentation was completed for 72 hours, the fermentation was terminated, and the fermentation supernatant was purified.

The purification method comprises the following steps: the fermentation liquor is subjected to microfiltration, the supernatant is collected, ultrafiltration and liquid exchange are carried out on the supernatant to low-concentration 0.1M Tris-HCL buffer solution, the solution is directly purified by a nickel column, imidazole elution is carried out, the activity peaks are collected, and the obtained products are combined after activity measurement.

The presence of the active protein during and after purification is shown in Table 2.

TABLE 2

As a result, it was found that the wild-type RPK had a 46% loss of activity during microfiltration and ultrafiltration, and the activity after final nickel column purification was only 45% of the total activity of the fermentation broth. In contrast, the mutant RPK has reduced activity loss during microfiltration and ultrafiltration, activity recovery of 90%, and further purification by nickel column to obtain an RPK mutant with activity recovery of 85%.

Comparing the specific activities of the purified stock solutions to be similar, the mutant is proved to not change the catalytic property.

Example 5 comparison of stock stability before and after mutation

Fermentation was performed using the fermentation method of example 1 to produce wild-type RPK and RPK mutants, and purified concentrated stock solutions thereof (example 3) were obtained to compare the stability of the RPK of the wild-type and mutant.

Concentrating the protein concentration to 30mg/ml, taking 1ml of wild RPK and RPK mutant stock solutions (three in each group) respectively, placing the wild RPK and RPK mutant stock solutions at the room temperature of 25 ℃, respectively carrying out activity measurement at the 0 th, 6 th, 12 th, 24 th and 48 th hours, calculating the average activity of the three groups, calculating the residual rate of the activity of each point by taking the activity of the stock solution at 0h as 100%, and plotting the relation between the activity and time.

As a result, as shown in FIG. 3, the activity of the wild-type RPK was continuously decreased during the standing, and it was observed that the precipitation formed by aggregation was gradually generated under the bottom of the centrifuge tube, and the precipitation was gradually increased, with the 48 th h activity remaining at 68%. In contrast, the activity of the RPK mutant optimized by the application is more stable in the placing process, almost no precipitation occurs, and the activity residue at 48h is 93%.

Conclusion(s)

The mutant improves the dissolution stability of RPK, the activity of fermentation liquor is improved in the production process, the recovery rate in the whole production and purification process is increased, the storage stability of stock solution is compared, the RPK mutant stock solution has no obvious precipitation in the storage process, and the activity of the solution is hardly reduced.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Shanghai Endocarpium biotechnology Co., ltd

<120> a salt-tolerant RPK mutant and use thereof

<130> 211440

<160> 4

<170> SIPOSequenceListing 1.0

<210> 2

<211> 279

<212> PRT

<213> Candida albicans (Candida albicans)

<400> 2

Ala Ala Gln Thr Asn Ala Pro Trp Gly Leu Ala Arg Ile Ser Ser Thr

1 5 10 15

Ser Pro Gly Thr Ser Thr Tyr Tyr Tyr Asp Glu Ser Ala Gly Gln Gly

20 25 30

Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ala Ser His Pro Glu

35 40 45

Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Tyr Ser Ser Arg

50 55 60

Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Val Gly Ser Arg

65 70 75 80

Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Phe Gly Val Lys Val Leu

85 90 95

Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr Ile Ile Ala Gly Met Asp

100 105 110

Phe Val Ala Ser Asp Lys Asn Asn Arg Asn Cys Pro Lys Gly Val Val

115 120 125

Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala

130 135 140

Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly

145 150 155 160

Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser

165 170 175

Val Cys Thr Val Gly Ala Ser Asp Arg Tyr Asp Arg Arg Ser Ser Phe

180 185 190

Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Gly Pro Gly Thr Ser Ile

195 200 205

Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser

210 215 220

Met Ala Thr Pro His Val Ala Gly Leu Ala Ala Tyr Leu Met Thr Leu

225 230 235 240

Gly Lys Thr Thr Ala Ala Ser Ala Cys Arg Tyr Ile Ala Asp Thr Ala

245 250 255

Asn Lys Gly Asp Leu Ser Asn Ile Pro Phe Gly Thr Val Asn Leu Leu

260 265 270

Ala Tyr Asn Asn Tyr Gln Ala

275

<210> 2

<211> 837

<212> DNA

<213> Candida albicans (Candida albicans)

<400> 2

gcggcgcaga ccaacgcgcc gtggggcctg gcgcgcatta gcagcaccag cccgggcacc 60

agcacctatt attatgatga aagcgcgggc cagggcagct gcgtgtatgt gattgatacc 120

ggcattgaag cgagccatcc ggaatttgaa ggccgcgcgc agatggtgaa aacctattat 180

tatagcagcc gcgatggcaa cggccatggc acccattgcg cgggcaccgt gggcagccgc 240

acctatggcg tggcgaaaaa aacccagctg tttggcgtga aagtgctgga tgataacggc 300

agcggccagt atagcaccat tattgcgggc atggattttg tggcgagcga taaaaacaac 360

cgcaactgcc cgaaaggcgt ggtggcgagc ctgagcctgg gcggcggcta tagcagcagc 420

gtgaacagcg cggcggcgcg cctgcagagc agcggcgtga tggtggcggt ggcggcgggc 480

aacaacaacg cggatgcgcg caactatagc ccggcgagcg aaccgagcgt gtgcaccgtg 540

ggcgcgagcg atcgctatga tcgccgcagc agctttagca actatggcag cgtgctggat 600

atttttggcc cgggcaccag cattctgagc acctggattg gcggcagcac ccgcagcatt 660

agcggcacca gcatggcgac cccgcatgtg gcgggcctgg cggcgtatct gatgaccctg 720

ggcaaaacca ccgcggcgag cgcgtgccgc tatattgcgg ataccgcgaa caaaggcgat 780

ctgagcaaca ttccgtttgg caccgtgaac ctgctggcgt ataacaacta tcaggcg 837

<210> 3

<211> 279

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (1)..(279)

<223> mutant

<400> 3

Ala Ala Gln Thr Asn Ala Pro Trp Gly Leu Ala Arg Ile Ser Ser Thr

1 5 10 15

Ser Pro Gly Thr Ser Thr Tyr Tyr Tyr Asp Glu Ser Ala Gly Gln Gly

20 25 30

Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ala Ser His Pro Glu

35 40 45

Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Tyr Ser Ser Arg

50 55 60

Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Val Gly Ser Arg

65 70 75 80

Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Phe Gly Val Lys Val Leu

85 90 95

Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr Ile Ile Ala Gly Met Asp

100 105 110

Phe Val Ala Ser Asp Lys Asn Asn Arg Asn Cys Pro Lys Gly Val Val

115 120 125

Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala

130 135 140

Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly

145 150 155 160

Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser

165 170 175

Val Cys Thr Val Gly Ala Ser Asp Arg Tyr Asp Arg Arg Ser Ser Phe

180 185 190

Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Gly Pro Gly Thr Ser Ile

195 200 205

Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser

210 215 220

Met Ala Thr Pro His Val Ala Gly Leu Ala Ala Tyr Leu Met Thr Leu

225 230 235 240

Gly Lys Thr Thr Ala Ala Ser Ala Cys Arg Tyr Ile Ala Asp Thr Ala

245 250 255

Asn Lys Gly Asp Leu Ser Asn Ile Pro Tyr Gly Thr Val Asn Leu Leu

260 265 270

Ala Tyr Asn Asn Tyr Gln Gly

275

<210> 4

<211> 837

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(837)

<223> mutant

<400> 4

gcggcgcaga ccaacgcgcc gtggggcctg gcgcgcatta gcagcaccag cccgggcacc 60

agcacctatt attatgatga aagcgcgggc cagggcagct gcgtgtatgt gattgatacc 120

ggcattgaag cgagccatcc ggaatttgaa ggccgcgcgc agatggtgaa aacctattat 180

tatagcagcc gcgatggcaa cggccatggc acccattgcg cgggcaccgt gggcagccgc 240

acctatggcg tggcgaaaaa aacccagctg tttggcgtga aagtgctgga tgataacggc 300

agcggccagt atagcaccat tattgcgggc atggattttg tggcgagcga taaaaacaac 360

cgcaactgcc cgaaaggcgt ggtggcgagc ctgagcctgg gcggcggcta tagcagcagc 420

gtgaacagcg cggcggcgcg cctgcagagc agcggcgtga tggtggcggt ggcggcgggc 480

aacaacaacg cggatgcgcg caactatagc ccggcgagcg aaccgagcgt gtgcaccgtg 540

ggcgcgagcg atcgctatga tcgccgcagc agctttagca actatggcag cgtgctggat 600

atttttggcc cgggcaccag cattctgagc acctggattg gcggcagcac ccgcagcatt 660

agcggcacca gcatggcgac cccgcatgtg gcgggcctgg cggcgtatct gatgaccctg 720

ggcaaaacca ccgcggcgag cgcgtgccgc tatattgcgg ataccgcgaa caaaggcgat 780

ctgagcaaca ttccgtatgg caccgtgaac ctgctggcgt ataacaacta tcagggc 837

Claims

1. A proteinase K mutant has an amino acid sequence shown in SEQ ID No. 3.

2. An isolated polynucleotide encoding the proteinase K mutant of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A genetically engineered host cell comprising the vector of claim 3, or having integrated into its genome the polynucleotide of claim 2; the host cell is eukaryotic; the eukaryotic cell is a yeast cell.

5. The genetically engineered host cell of claim 4, wherein the yeast cell is a pichia cell.

6. A method for improving the salt tolerance or stability of proteinase K comprises mutating proteinase K to obtain proteinase K mutant, wherein the amino acid sequence of the mutant is shown as SEQ ID NO. 3.

7. A method for preparing the proteinase K mutant according to claim 1, comprising: (i) culturing the host cell of claim 4 or 5; (ii) Collecting a culture containing said proteinase K mutant; (iii) Isolating the proteinase K mutant from the culture.

8. Use of a proteinase K mutant according to claim 1, a host cell according to claim 4 or 5 or a lysate thereof for enzymatic hydrolysis of a protein.

9. The use according to claim 8 for the specific recognition and cleavage of carboxyl-terminal peptide bonds of aliphatic and aromatic amino acids, enzymatic hydrolysis of proteins.

10. A method of enzymatic hydrolysis of a protein comprising: enzymatic hydrolysis using the proteinase K mutant according to claim 1, the host cell according to claim 4 or 5 or a lysate thereof.

11. A detection system or detection kit for enzymatic hydrolysis of a protein, comprising: proteinase K mutant according to claim 1, host cell according to claim 4 or 5 or a lysate thereof.