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CN113913413A - Salt-tolerant RPK mutant and application thereof - Google Patents

Salt-tolerant RPK mutant and application thereof Download PDF

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CN113913413A
CN113913413A CN202110918233.XA CN202110918233A CN113913413A CN 113913413 A CN113913413 A CN 113913413A CN 202110918233 A CN202110918233 A CN 202110918233A CN 113913413 A CN113913413 A CN 113913413A
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proteinase
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rpk
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CN113913413B (en
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刘晓
赵致
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SHANGHAI YAXIN BIOTECHNOLOGY CO Ltd
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Abstract

The invention provides a salt-tolerant RPK mutant and application thereof. The inventor finds that some sites of proteinase K are closely related to the salt tolerance and stability of proteinase K, and develops a proteinase K mutant on the basis that the mutant has better salt tolerance and stability compared with the wild type. The invention 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 invention belongs to the field of biotechnology; more specifically, the invention relates to a salt-tolerant RPK mutant and application thereof.
Background
Proteinase K (PK) is a non-specific endoprotease, which belongs to serine proteases and can cut ester bonds and peptide bonds connected at the carboxyl terminals of aliphatic amino acids, hydrophobic amino acids and aromatic amino acids. The molecular weight is 29.3kDa (monomer), and the compound has activity under the conditions of pH4 to pH12, and is also stable in the presence of SDS, urea or EDTA.
Proteinase K is capable of cleaving the carboxyl-terminal peptide bond of aliphatic and aromatic amino acids for degradation of proteins in biological samples. The enzyme was decolourised and chromatographically purified to remove RNA and DNA and no other miscellaneous enzyme activity could be detected. Since proteinase K is stable in urea and SDS and also has the ability to degrade native proteins, the smallest polypeptide it hydrolyses is a tetrapeptide molecule.
However, the inventors found that Recombinant Proteinase K (RPK) is prone to aggregation and precipitation during fermentation production, which causes problems in the actual production process.
Disclosure of Invention
The invention aims to provide a salt-tolerant RPK mutant and application thereof.
In a first aspect of the invention, there is provided a protease K mutant which is: (a) the amino acid sequence corresponds to proteinase K shown in SEQ ID NO. 1, and the following sites are mutated enzymes: 266 th, 279 th; the site mutation is a 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 invention 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 invention, there is provided a vector comprising said polynucleotide.
In another aspect of the invention, there is provided a genetically engineered host cell comprising said vector, or having said polynucleotide integrated into its genome.
In one or more embodiments, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In one or more embodiments, the eukaryotic cell includes a yeast cell, a mold cell, an insect cell, a plant cell, a fungal cell, or a mammalian cell, and the like.
In one or more embodiments, the prokaryotic cells include E.coli cells, B.subtilis cells, and the like.
In another aspect of the present invention, there is provided a method for improving the salt tolerance or stability of proteinase K, comprising mutating a site or a combination of sites selected from the group consisting of the sites corresponding to proteinase K as shown in SEQ ID NO. 1: 266 th, 279 th; the site mutation is a hydrophilic or neutral amino acid.
In another aspect of the present invention, there is provided a method for producing a proteinase K mutant as described in any one of the above, the method comprising: (i) culturing said host cell; (ii) collecting a culture containing the proteinase K mutant; (iii) isolating the proteinase K mutant from the culture.
In another aspect of the invention, there is provided the use of a proteinase K mutant, a host cell expressing the mutant, or a lysate thereof, as defined in any one of the preceding claims, for the enzymatic cleavage of a protein; preferably, it is used to specifically recognize and cleave the carboxy-terminal peptide bond of aliphatic and aromatic amino acids, enzymatically hydrolyzing the protein.
In another aspect of the present invention, there is provided a method for enzymatic hydrolysis of a protein, comprising: carrying out enzymolysis reaction by using any one of the proteinase K mutant, the host cell expressing the mutant or the lysate thereof.
In another aspect of the present invention, there is provided a detection system or a detection kit for enzymatically hydrolyzing a protein, comprising: a proteinase K mutant as described in any of the preceding, a host cell expressing the mutant or a lysate thereof.
Other aspects of the invention 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 mutant RPK mutants.
FIG. 2, hydrophobic surface map of wild type RPK and RPK mutant molecules.
FIG. 3, comparison of stock stability of wild type RPK and RPK mutant.
Detailed Description
Through intensive research and experiments, the inventor finds that some sites of the proteinase K are closely related to the salt tolerance and the stability of the proteinase K. On the basis, the inventor obtains a protease K mutant which has better salt tolerance and stability compared with the wild type. The invention 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.
Term(s) for
As used herein, unless otherwise indicated, the terms "mutant of proteinase K" and "mutant proteinase K" are used interchangeably and refer to a protein which is made by mutation at a position corresponding to wild-type proteinase K (as shown in SEQ ID NO:1) selected from the group consisting of: 266 th position and 279 th position.
As used herein, unless otherwise indicated, the terms "mutant of proteinase K" and "mutant proteinase K" are used interchangeably and refer to the product of a mutation in wild-type proteinase K.
As used herein, wild-type proteinase K, if desired, will be designated "wild-type proteinase K", a protein having 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 (which, if it is a natural substance, is the natural environment). If the polynucleotide or protein in the natural state in the living cell is not isolated or purified, the same polynucleotide or protein is isolated or purified if it is separated from other substances coexisting in the natural state.
As used herein, "isolated proteinase K mutant" means that the proteinase K mutant is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify proteinase K mutants using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.
As used herein, "Recombinant" refers to a protein, genetically engineered vector or cell, etc., obtained (or prepared in large quantities) by means of genetic engineering.
As used herein, "increased salt tolerance or stability" refers to a statistically significant, or referred to as a significant, increase in salt tolerance or stability of a mutated proteinase K as compared to the wild-type proteinase K prior to alteration. For example, the salt tolerance or stability of the mutant proteinase K having improved salt tolerance or stability under the same reaction conditions/environment 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% or more, 150% or more, etc. as compared with the enzyme before modification.
As used herein, the terms "comprising" or "including" include "comprising," consisting essentially of … …, "and" consisting of … …. The term "consisting essentially of … …" means that minor ingredients and/or impurities which do not affect the effective ingredients may be contained in small amounts in addition to the essential ingredients or essential components in the composition/reaction system/kit.
The term "effective amount" as used herein refers to an amount that produces a function or activity to achieve the desired effect (accurate test result) on the reaction of interest of the present invention.
Proteinase K mutant, and coding nucleic acid and construct thereof
The proteinase K mutants of the invention can be chemically synthesized products or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).
The invention also includes fragments, derivatives and analogues of the proteinase K mutants. As used herein, the terms "fragment," "derivative," and "analog" refer to a protein that retains substantially the same biological function or activity as the native proteinase K mutant of the invention. A protein fragment, derivative or analog of the invention may be (i) a protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substituent group in one or more amino acid residues, or (iii) a protein in which an additional amino acid sequence is fused to the protein sequence (e.g., a leader or secretory sequence or a sequence used to purify the protein or a pro-protein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art, as defined herein. However, the conditions to be satisfied are: in the amino acid sequences of the proteinase K mutant and the fragments, derivatives and analogs thereof, at least one mutation specifically indicated above is necessarily present, and preferably, the mutation is an amino acid sequence corresponding to SEQ ID NO. 1, including a mutation at the 266 th position of Tyr and a mutation at the 279 th position of Gly.
In the present invention, the term "proteinase K mutant" also includes (but is not limited to): deletion, insertion and/or substitution of several (usually 1 to 20, more preferably 1 to 10, still more preferably 1 to 8, 1 to 5, 1 to 3, or 1 to 2) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of proteinase K mutants. However, in these variants, the above-described mutations of the present invention are certainly present, and preferably correspond to the amino acid sequence shown in SEQ ID NO. 1, including a mutation at position 266 to Tyr and a mutation at position 279 to Gly.
In the present invention, the term "proteinase K mutant" also includes (but is not limited to): and a derivative protein having more than 80%, preferably more than 85%, more preferably more than 90%, further more preferably more than 95%, such as more than 98% or more than 99% sequence identity with the amino acid sequence of the proteinase K mutant and retaining the protein activity. Similarly, the above-mentioned mutation of the present invention is positively present in these derived proteins, and preferably, the mutation is an amino acid sequence corresponding to SEQ ID NO. 1, including a mutation at position 266 to Tyr and a mutation at position 279 to Gly.
The invention also provides analogues of the proteinase K mutants. These analogs may differ from the proteinase K mutant by amino acid sequence differences, by modifications 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, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (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 invention are not limited to the representative polypeptides exemplified above. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a 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 increase their resistance to proteolysis or to optimize solubility.
The invention also provides a polynucleotide sequence for encoding the proteinase K mutant or the conservative variant protein thereof.
The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
The polynucleotides encoding the mature proteins of the mutants include: a coding sequence that encodes only the mature protein; the coding sequence for the mature protein and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature protein.
A "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, and may also include additional coding and/or non-coding sequences.
The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells engineered with the vector or proteinase K mutant coding sequences of the invention, and methods for producing the mutated enzymes of the invention by recombinant techniques.
The polynucleotide sequences of the invention may be used to express or produce recombinant proteinase K mutants by conventional recombinant DNA techniques. Generally, the following steps are performed:
(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a proteinase K mutant, or with a recombinant expression vector comprising the polynucleotide;
(2) a host cell cultured in a suitable medium;
(3) isolating and purifying the protein from the culture medium or the cells.
In the present invention, the proteinase K mutant polynucleotide sequence may 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 general, any plasmid or vector can be used as long as it can replicate 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 translation control elements.
Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding proteinase K mutants and appropriate transcription/translation 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 a suitable 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 described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
In the present invention, 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: escherichia coli, Bacillus subtilis, Streptomyces, and Agrobacterium; eukaryotic cells such as yeast, plant cells, and the like. In a specific embodiment of the invention, yeast cells are used as host cells.
It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.
Recombinant expression of the mutant
The recombinant cells (host cells) established in the present invention can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
When expressed, the proteinase K mutant of the invention can be expressed intracellularly or on the cell membrane or secreted extracellularly. If necessary, the recombinant protein can be isolated 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 such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell disruption by osmosis, 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 thereof.
The method for recombinant expression of the proteinase K mutant solves the problems of low stability, unsatisfactory salt tolerance and easy generation of precipitate of the recombinant proteinase K in the prior art.
Application of recombinant proteinase K
RPK is obtained by fermentation of Pichia pastoris with a medium of high phosphate and sulfate concentration of about 1M under acidic conditions of pH 5.0. The stability of RPK in this high salt medium becomes the key to the success of RPK fermentation.
The possible reasons that the RPK expressed by pichia pastoris is easy to precipitate in higher salt concentration are that the RPK is easy to aggregate or coprecipitate under higher ionic strength and hybrid protein, and the precipitate is more obvious when the protein concentration is higher. Therefore, the RPK is easy to aggregate and precipitate, which brings problems to the practical production process. RPK or its engineered polypeptides in the prior art have not addressed this problem at high salt concentrations. The inventor analyzes the molecular structure of wild RPK, finds out the reason of easy aggregation and precipitation, carries out directional modification on the primary structure of protein, and improves the solution stability. The invention solves the problems of low salt tolerance, low stability and the like in the expression and purification of the prior recombinant proteinase K.
The engineered proteinase K mutants of the invention have a variety of uses related to proteinase K properties, including but not limited to: specifically recognizing and cutting carboxyl terminal peptide bonds of aliphatic amino acids and aromatic amino acids, and hydrolyzing proteins or denaturing proteins.
As an embodiment, the proteinase K mutant can be applied to extraction of genome DNA and digestion and removal of enzyme.
As an embodiment, the proteinase K mutant can be applied to preparation of chromosome DNA of pulse electrophoresis, western blotting, removal of nuclease and the like in preparation of DNA and RNA
As an embodiment, the proteinase K mutant can be used in situ hybridization techniques for treatment prior to hybridization, which has the effect of digesting the proteins surrounding the target DNA to increase the chance of binding of the probe to the target nucleic acid and increase the hybridization signal.
As some industrial embodiments, the proteinase K mutant can be applied to leather, fur, silk, medicine, food, brewing and the like. The unhairing and softening of leather industry has made use of protease in large quantities, and has the characteristics of time saving and convenience. The protease can also be used for degumming silk, tenderizing meat, and clarifying wine. The medicine can be used for clinical medicine, such as treating dyspepsia with pepsin, treating bronchitis with acid protease, treating angiitis with elastase, and cleaning surgical suppurative wound and treating pleural effusion adhesion with proteinase K and chymotrypsin. The enzymatic washing powder is a new product in detergent, contains alkaline protease and can remove bloodstain 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 requirement of industrial production.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
Example 1 recombinant fermentation of wild-type Protease K (PK)
The amino acid sequence of the wild-type Proteinase K (PK) is as follows (SEQ ID NO: 1):
AAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA
the coding sequence of the wild-type Protease K (PK) is as follows (SEQ ID NO: 3):
gcggcgcagaccaacgcgccgtggggcctggcgcgcattagcagcaccagcccgggcaccagcacctattattatgatgaaagcgcgggccagggcagctgcgtgtatgtgattgataccggcattgaagcgagccatccggaatttgaaggccgcgcgcagatggtgaaaacctattattatagcagccgcgatggcaacggccatggcacccattgcgcgggcaccgtgggcagccgcacctatggcgtggcgaaaaaaacccagctgtttggcgtgaaagtgctggatgataacggcagcggccagtatagcaccattattgcgggcatggattttgtggcgagcgataaaaacaaccgcaactgcccgaaaggcgtggtggcgagcctgagcctgggcggcggctatagcagcagcgtgaacagcgcggcggcgcgcctgcagagcagcggcgtgatggtggcggtggcggcgggcaacaacaacgcggatgcgcgcaactatagcccggcgagcgaaccgagcgtgtgcaccgtgggcgcgagcgatcgctatgatcgccgcagcagctttagcaactatggcagcgtgctggatatttttggcccgggcaccagcattctgagcacctggattggcggcagcacccgcagcattagcggcaccagcatggcgaccccgcatgtggcgggcctggcggcgtatctgatgaccctgggcaaaaccaccgcggcgagcgcgtgccgctatattgcggataccgcgaacaaaggcgatctgagcaacattccgtttggcaccgtgaacctgctggcgtataacaactatcaggcg
the coding sequence was inserted into the plasmid pPIC9K and recombinantly integrated into Pichia pastoris GS 115.
Fermentation liquor: 85% H3PO426.7ml/L, KOH 4.13g/L, glycerol 40g/L, K2SO4 18.2g/L,CaSO40.93g/L,MgSO4·7H2O14.9 g/L, salt concentration about 1M.
Fermentation process (including fermentation conditions): the whole fermentation is divided into three stages of glycerol batch fermentation, glycerol fed-batch fermentation and methanol induced fermentation. In glycerol batch fermentation, higher dissolved oxygen levels are maintained in the fermentation broth due to lower microbial concentrations. Along with the increase of the concentration of the bacteria, the DO begins to gradually decrease until the glycerol in the culture medium is completely metabolized, the depletion of the glycerol leads to the slow growth of the bacteria, and the DO shows a sharp rising trend. The induction expression stage begins when the OD600 of the bacterial concentration reaches a higher value, and methanol induction expression is carried out. Wherein the fermentation is controlled by 20-30% according to the change of dissolved oxygen parameter.
Monitoring the progress of fermentation, the inventors have found that Recombinant Proteinase K (RPK) expressed by Pichia pastoris is highly susceptible to precipitation at higher salt concentrations (salt concentrations around 1M or above 1M), probably due to the tendency of RPK to aggregate or co-precipitate at higher protein concentrations at higher ionic strengths. Moreover, as the fermentation process continues, the higher the protein concentration, the more pronounced the observed precipitation. The yield of active RPK protein is severely affected by the massive production of precipitates.
Therefore, in the fermentation production, RPK is easy to aggregate and precipitate, which brings problems to the actual production process.
Example 2 optimized engineering of RPK protein
On the basis of wild type Proteinase K (PK), the inventor analyzes the three-dimensional structure, the surface hydrophilicity and hydrophobicity, the accessibility of amino acid solvent and the like.
As a result of repeated research and analysis, the inventors of the present invention have localized the engineered positions to Phe266 and Ala279, which are located on the surface of the three-dimensional structure of the protein and are hydrophobic amino acids, which promote easy aggregation and precipitation under the conditions of high protein concentration and high ionic strength, resulting in the precipitation of RPK zymogen liquid, and thus, the protein concentration is reduced.
The inventor modifies the site-directed mutagenesis method into hydrophilic and neutral amino acids with similar side chain group structures, thereby reducing the hydrophobicity of the site. According to amino acid analysis, Phe266 is mutated into Tyr, the side chain of the R group of the Tyr is added with one more hydroxyl group as a hydrophilic group compared with Phe, Ala279 is mutated into Gly, and the hydrophobic methyl group on the R group of the Gly is changed into a hydrogen atom, so that the hydrophobicity is reduced.
As shown in FIG. 1, the wild-type RPK (left panel) and the mutant RPK (right panel) were compared, and the amino acid residues at positions 266 and 279 were labeled. The asterisks interspersed in wild-type RPK indicate water molecules, which are less and more distant because Phe266 and Ala279 are on the surface of the molecule and more hydrophobic. The right panel shows the three-dimensional structure simulated by the mutant RPK, so no water molecules are shown around. The amino acid residue at the 266 position of the PK mutant is changed into Tyr, and the side chain benzene ring increases the hydrophilic hydroxyl group, so that the hydrophilicity is increased. Mutation of Ala to Gly at position 279 with reduced hydrophobicity.
As shown in FIG. 2, the present inventors analyzed the hydrophobicity of the molecular surface of wild-type RPK (left panel) and RPK mutant (right panel), wherein blue represents a hydrophilic region, red represents a hydrophobic region, and white represents a neutral region. The regions of amino acid residues 226 and 279 on the surface of the molecule are marked with black ovals and amino acid numbers. From the comparison of the two, it is known that the RPK mutant has stronger hydrophobicity at the 266 and 279 amino acid residues, and the RPK mutant in the right picture 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 phenomenon of aggregation and precipitation in aqueous solution, and the like, of the RPK mutant is relieved due to the reduction of the hydrophobic region on the surface.
According to the above modification scheme, the amino acid sequence after mutation 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
the present inventors introduced the above-modified enzyme coding sequence into a pichia pastoris expression vector, and transformed pichia pastoris, using the same method as in example 1.
The present inventors used the recombinant Pichia pastoris to perform fermentation to express mutant proteinase K by the same fermentation method as in example 1.
Monitoring the fermentation process, the inventors found that pichia pastoris expression of mutant form of recombinant proteinase k (rpk) is not prone to precipitation at higher salt concentrations (about 1M or above 1M), and no significant aggregation or co-precipitation is observed.
Example 3 comparison of RPK stock stability before and after mutagenesis
By performing fermentation by the fermentation method of example 1, the present inventors quantitatively counted and compared the fermentation broth activities of the active proteins of RPK of the wild type and the mutant.
And (3) carrying out fermentation liquor activity determination 72 hours after the methanol induction, wherein the determination method comprises the following steps: casein is used as a substrate. Definition of Activity: the amount of proteinase K which hydrolyzes a casein substrate to form 1. mu. mol L-tyrosine per minute at 37 ℃ under 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 fermentation broth Activity
Figure BDA0003206398140000121
As shown in Table 1, the wild type RPK precipitates in the fermentation process under the high-salt environment of the fermentation liquor, so that the activity of the fermentation liquor can only reach 530U/ml. Compared with the prior art, the activity of the RPK mutant fermentation liquor can reach 716U/ml.
Therefore, the RPK mutant has good salt resistance.
Example 4 comparison of RPK purification before and after mutation and recovery
By performing fermentation using the fermentation method of example 1, the present inventors quantitatively counted and compared the recovery rates of active protein of RPK of wild type as well as mutant.
After fermentation for 72 hours, the fermentation was terminated and the fermentation supernatant was purified.
The purification method comprises the following steps: microfiltering the fermentation liquor, collecting the supernatant, ultrafiltering the supernatant, changing the filtrate into a low-concentration 0.1M Tris-HCL buffer solution, directly purifying by a nickel column, eluting by imidazole, collecting the activity peak, detecting the activity peak, and combining the active peaks.
The presence of active protein during and after purification is shown in table 2.
TABLE 2
Figure BDA0003206398140000131
As a result, the activity loss of the wild RPK in the micro-filtration and ultra-filtration process is 46%, and the activity of the final purified nickel column can only reach 45% of the total activity of the fermentation liquor. Compared with the mutant RPK, the loss of activity of the mutant RPK in the micro-filtration and ultrafiltration processes is reduced, the activity recovery is 90 percent, and the activity recovery of the RPK mutant is 85 percent finally after further purification by a nickel column.
The specific activities of the purified stock solutions are similar, which indicates that the mutant does not change the catalytic property.
Example 5 comparison of stock stability before and after mutagenesis
The fermentation procedure of example 1 was used to produce wild-type RPK and RPK mutants and to obtain purified concentrated stock solutions (as in example 3) to compare the stability of RPK in wild-type and mutant.
Concentrating the protein concentration to 30mg/ml, taking 1ml of stock solutions of wild type RPK and RPK mutant respectively (three parallel in each group), placing the stock solutions at 25 ℃ and room temperature, performing activity determination at 0 th, 6 th, 12 th, 24 th and 48 th hours respectively, calculating the average activity of the three groups, calculating the residual rate of activity at each point by taking the activity of the stock solution at 0h as 100%, and plotting the relationship between the activity and the time.
As shown in FIG. 3, the activity of wild-type RPK was decreased during the standing process, and the precipitate formed by aggregation was observed to gradually grow under the bottom of the centrifuge tube, and gradually increased, and 68% of activity remained at 48 h. In contrast, the activity of the optimized RPK mutant is stable during the standing process, almost no precipitation occurs, and the activity residue at 48h is 93%.
Conclusion
The mutant improves the dissolving stability of the RPK, shows that the activity of the fermentation liquor is increased in the production process, the recovery rate in the whole production and purification process is increased, the stability of the stock solution in placement is compared, the stock solution of the RPK mutant has no obvious precipitate in the placement process, and the activity of the solution is hardly reduced.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai Yaxin Biotechnology Ltd
<120> salt-tolerant RPK mutant and application 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 (10)

1. A protease K mutant which is: (a) the amino acid sequence corresponds to proteinase K shown in SEQ ID NO. 1, and the following sites are mutated enzymes: 266 th, 279 th; the site mutation is a hydrophilic or neutral amino acid.
2. The proteinase K mutant of claim 1 wherein the mutation at position 266 is Tyr and the mutation at position 279 is Gly; preferably, the amino acid sequence of the proteinase K mutant is shown as SEQ ID NO. 2.
3. An isolated polynucleotide encoding the proteinase K mutant of any one of claims 1 to 2.
4. A vector comprising the polynucleotide of claim 3.
5. A genetically engineered host cell comprising the vector of claim 4, or having the polynucleotide of claim 3 integrated into its genome; preferably, the host cell comprises 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, and the like; preferably, the prokaryotic cells include Escherichia coli cells, Bacillus subtilis cells, and the like.
6. A method for improving the salt tolerance or stability of proteinase K, which comprises mutating a site or a combination of sites corresponding to proteinase K shown as SEQ ID NO. 1: 266 th, 279 th; the site mutation is a hydrophilic or neutral amino acid.
7. The method for producing the proteinase K mutant according to any one of claims 1 to 2, which comprises: (i) culturing the host cell of claim 5; (ii) collecting a culture containing the proteinase K mutant; (iii) isolating the proteinase K mutant from the culture.
8. Use of the proteinase K mutant, a host cell expressing the mutant, or a lysate thereof according to any one of claims 1-2, for enzymatic hydrolysis of proteins; preferably, the carboxyl terminal peptide bond used for specifically recognizing and cutting the aliphatic amino acid and the aromatic amino acid is used for enzymolysis of the protein.
9. A method of enzymatically hydrolyzing a protein, comprising: carrying out enzymolysis reaction by using any one of the proteinase K mutant, the host cell expressing the mutant or the lysate thereof.
10. A detection system or kit for enzymatically hydrolyzing a protein, comprising: the proteinase K mutant according to any one of claims 1 to 2, a host cell expressing the mutant or a lysate thereof.
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