CN115838713A

CN115838713A - Protease and application thereof in L-carnosine synthesis

Info

Publication number: CN115838713A
Application number: CN202211726298.5A
Authority: CN
Inventors: 乔莉苹; 于鲁孟; 周宁; 吴佳鹏; 李帆; 郭学平
Original assignee: Huaxi Biotechnology Tianjin Co ltd; Bloomage Biotech Co Ltd
Current assignee: Huaxi Biotechnology Tianjin Co ltd; Bloomage Biotech Co Ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-03-24

Abstract

The application discloses a protease with a carnosine hydrolase function and application thereof in L-carnosine synthesis, wherein the protease comprises an amino acid sequence shown as SEQ ID NO. 6. The protease can use beta-alanine methyl ester hydrochloride and L-histidine as substrates, can also use beta-alanine and L-histidine as substrates, can reduce the cost, and can also use beta-alanine generated by instability of beta-alanine methyl ester hydrochloride.

Description

Protease and application thereof in L-carnosine synthesis

Technical Field

The application relates to the technical field of carnosine, in particular to a protease with a carnosine hydrolase function and application thereof in L-carnosine synthesis.

Background

L-carnosine, a dipeptide consisting of beta-alanine and L-histidine, is one of the most widely biologically active peptides found today. L-carnosine is widely found in the brain, muscle and other tissues of mammals.

Research shows that the L-carnosine has multiple biological activities of resisting oxidation, eliminating intracellular free radicals, resisting aging and the like, and has treatment effects on hypertension, heart disease, senile cataract, ulcer and the like. The active peptide has good application prospect in the fields of medicine, health care, sanitation, cosmetics and the like due to strong antioxidant activity, low toxic and side effect and multiple physiological activities.

The currently reported synthesis method of the L-carnosine mainly comprises chemical synthesis and biological enzyme catalysis, wherein the enzyme method has the advantages of environmental protection, low preparation cost and short synthesis time, so that the application of the enzyme method is wider. However, when the enzymatic method is used for synthesizing L-carnosine, the beta-alanine methyl ester hydrochloride and histidine are generally used as substrates, and the following problems are also caused: firstly, the preparation method of beta-alanine methyl ester hydrochloride is complex and expensive; secondly, the beta-alanine methyl ester hydrochloride has strong acidity and strong corrosivity on metal products such as stainless steel tanks and the like; thirdly, the beta-alanine methyl ester hydrochloride is easy to be affected with damp and is unstable after being affected with damp.

Disclosure of Invention

In view of the above technical problems, the present application provides a protease with carnosine hydrolase function and its application in L-carnosine synthesis, wherein the protease can prepare carnosine by using beta-alanine methyl ester hydrochloride and L-histidine or beta-alanine and L-histidine as substrates, and the enzymatic properties of the protease are stable, which is beneficial for industrial production.

The specific technical scheme of the application is as follows:

1. a protease having a carnosine hydrolase function, characterized in that the protease comprises an amino acid sequence shown in SEQ ID NO 6.

2. A biomaterial, characterized in that the biomaterial is any one of the following:

b1 A nucleic acid molecule encoding the protease of item 1;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3).

3. The biomaterial according to claim 2, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO. 2.

4. Use of any one of the following materials for the preparation of the protease of item 1, for the regulation of the production of the protease of item 1 in a microorganism:

c1 Substances regulating the expression of genes encoding said proteases;

c2 Substances which regulate the activity or content of the protease.

5. The use according to item 4, characterized in that it is achieved by using any one or two or more of the following:

d1 A nucleic acid molecule encoding the protease of item 1;

d2 An expression cassette comprising a nucleic acid molecule according to D1);

d3 A recombinant vector containing the nucleic acid molecule according to D1) or a recombinant vector containing the expression cassette according to D2);

d4 A recombinant microorganism containing a nucleic acid molecule according to D1), or a recombinant microorganism containing an expression cassette according to D2), or a recombinant microorganism containing a recombinant vector according to D3).

6. A method of recombining a microorganism, said method comprising:

introducing a gene encoding the protease according to item 1 into the microorganism;

optionally, the gene encoding the protease of claim 1 is regulated for expression to increase the activity or yield of the protease.

7. The biomaterial of

item

2 or 3, the use of item 4, the method of item 6, wherein the microorganism is any one of: escherichia coli; b, bacillus subtilis; and (4) saccharomyces cerevisiae.

8. A method for producing L-carnosine, which comprises producing L-carnosine using the protease of item 1 or the biological material of

item

2 or 3 or the recombinant microorganism produced by the method of item 6.

9. The method of claim 8, wherein the substrate is beta-alanine methyl ester hydrochloride and L-histidine or beta-alanine and L-histidine.

ADVANTAGEOUS EFFECTS OF INVENTION

The protease can use beta-alanine methyl ester hydrochloride and L-histidine as substrates, can also use beta-alanine and L-histidine as substrates, can reduce the cost, and can also use beta-alanine generated by instability of beta-alanine methyl ester hydrochloride.

The protease described in the application has stable enzymatic properties, namely the conversion conditions are wide, and the industrial implementation is facilitated.

According to the method, the L-carnosine is synthesized by adopting protease catalysis, so that the complex process of the conventional chemical method is avoided, the production cost is effectively reduced, and the economic benefit is improved; meanwhile, the synthesis process is simple, green and environment-friendly, meets the requirement of clean production, and has wide industrial application prospect.

Drawings

FIG. 1 is an agarose nucleic acid electrophoresis picture of protease.

FIG. 2 is an SDS-PAGE protein electrophoresis of protease.

FIG. 3 is a diagram showing the enzymatic properties of a protease-temperature.

FIG. 4 is a diagram showing the pH and the enzymatic properties of a protease.

FIG. 5 is a schematic of the enzyme stability versus temperature for proteases.

FIG. 6 is a schematic of the enzymatic stability-pH of proteases.

Detailed Description

The present application will be described in detail with reference to the embodiments described below. While specific embodiments of the present application have been illustrated, it should be understood that the present application may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the application, however, the description is made for the purpose of illustrating the general principles of the application and is not intended to limit the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

The terms "polynucleotide", "nucleotide sequence" and "nucleic acid molecule" are used interchangeably herein. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, and the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as a gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

The term "vector" is used herein to describe a nucleic acid molecule that can be engineered to contain a cloned polynucleotide or polynucleotides that can be amplified in a host cell. Vectors include, but are not limited to: single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, without free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other polynucleotide species known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

In addition, certain vectors are capable of directing the expression of those genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or "recombinant vectors". A recombinant vector may comprise a nucleic acid of the present application in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements, which may be selected on the basis of the host cell used for expression, which may be operably linked to the nucleic acid sequence to be expressed.

As used herein, the term "recombinant microorganism" includes a microorganism (e.g., bacteria, yeast, algae, fungi, etc.) or strain of microorganism that has been genetically altered, modified, or engineered (e.g., genetically engineered) such that it exhibits an altered, modified, or different genotype and/or phenotype (e.g., when the genetic modification affects the coding nucleic acid sequence of the microorganism) as compared to the naturally-occurring microorganism or "parent" microorganism from which it is derived.

The term "expression cassette" refers to a DNA capable of expressing a protease having a carnosine hydrolase function of the present application in a microorganism, which includes not only a promoter that initiates transcription of a gene of interest but also a terminator that terminates transcription of the gene of interest. Further, the expression cassette may also include an enhancer sequence.

The application provides a protease with a carnosine hydrolase function, which comprises an amino acid sequence shown as SEQ ID NO. 6 or consists of the amino acid sequence shown as SEQ ID NO. 6.

In the present application, the protease is an enzyme that hydrolyses a substrate to carnosine.

The amino acid sequence of SEQ ID NO 6 is as follows:

MTSQTPTRKPRARDLGLPFTGVTGPYNAITDVDGVGVGFQTIIENEPRPGRKRPARSGVTAILPH

KQSETPVPVYAGVHRFNGNGEMTGTHWIEDGGYFLGPVVITNTHGIGMAHHATVRWMVDRYAST

YQTDDFLWIMPVVAETYDGALNDINGFPVTEADVRKALDNVASGPVQEGNCGGGTGMITYGFKG

GTGTASRVVEFGGRSFTIGALVQANHGQRDWLTIAGVPVGQHMRDGTPQSQLQERGSIIVVLATDL

PLMPHQLKRLARRASIGIGRNGTPGGNNSGDIFIAFSTANQRPMQHRSAPFLDVEMVNDEPLDTVYL

AAVDSVEEAVVNAMIAAEDMGGTPFDRLLVQAIDHERLRAVLRQYGRLA

as will be appreciated by those skilled in the art, the sequences shown in SEQ ID NOs: 6, but has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:6 have similar activity also fall within the scope of the proteases of the present application.

In SEQ ID NO:6, and/or one or more amino acid is/are substituted, and/or one or more amino acid is deleted and/or added, and the amino acid sequence is similar to the amino acid sequence shown in SEQ ID NO:6, and polypeptides having the same function and 95% or 96% or 97% or 98% or 99% identity are also within the scope of the protease of the present application.

In SEQ ID NO:6, and/or one or more amino acid substitutions, and/or deletion and/or addition of one or more or tens of amino acids, and polypeptides having the same function are also within the protection scope of the protease of the present application.

Furthermore, fusion proteins with the same function obtained by connecting tags to the N-terminal and/or C-terminal of the amino acid sequence shown in SEQ ID NO. 6 are also within the protection scope of the protease of the present application.

The present application also provides a biomaterial, wherein the biomaterial can be any one of the following:

b1 Nucleic acid molecules encoding the above proteases;

Further, the nucleotide sequence SEQ ID NO of the nucleic acid molecule 2 is as follows:

ATGACTTCACAAACGCCAACAAGGAAACCCAGAGCCCGTGACCTGGGCCTGCCGTTTACGGGAGTAACTGGACCGTATAACGCGATCACCGACGTGGACGGTGTTGGTGTGGGTTTCCAGACC ATCATCGAGAACGAACCGCGTCCGGGTCGCAAGCGCCCAGCGCGTTCCGGCGTCACCGCGATCCTGCCGCACAAACAGTCCGAAACCCCGGTCCCAGTCTACGCTGGTGTTCATCGTTTTAATGGTAACGGCGAAATGACCGGCACGCATTGGATCGAGGATGGTGGCTATTTCCTGGGTCCGGTCGTTATTACCAATACCCACGGCATCGGTATGGCACATCATGCAACCGTTCGCTGGATGGTGGACCGCTATGCAAGCACCTACCAGACCGATGATTTTCTGTGGATTATGCCGGTGGTTGCTGAAACCTACGATGGCGCATTGAATGACATCAACGGCTTCCCGGTGACCGAGGCCGATGTTCGTAAAGCGTTGGACAACGTGGCTTCTGGTCCTGTGCAAGAGGGCAACTGCGGTGGCGGTACGGGCATGATTACGTACGGCTTCAAGGGTGGCACCGGCACTGCGTCTCGCGTTGTAGAGTTCGGCGGTAGGTCCTTTACCATCGGTGCTTTGGTGCAGGCGAATCATGGTCAACGTGACTGGCTGACCATTGCGGGTGTTCCGGTGGGCCAACACATGCGTGACGGCACCCCGCAGTCGCAACTCCAAGAGCGTGGTAGCATTATCGTGGTGCTGGCGACTGACTTACCGCTGATGCCGCACCAGCTGAAACGTCTTGCGCGCCGTGCGAGCATTGGCATTGGTCGTAATGGTACGCCGGGTGGCAACAACAGCGGTGATATTTTTATCGCGTTTAGCACCGCGAACCAGCGTCCGATGCAGCACCGTAGCGCACCGTTCCTCGACGTCGAGATGGTTAATGATGAACCGCTGGACACGGTTTACCTGGCGGCTGTTGACAGCGTCGAGGAAGCAGTGGTGAACGCCATGATCGCTGCGGAAGATATGGGTGGCACCCCATTCGATCGCTTGCTGGTTCAGGCGATTGACCACGAACGCCTGCGCGCCGTGCTGCGTCAATATGGACGTTTGGCCTAA

in the present application, the nucleic acid sequence shown as SEQ ID NO.2 is obtained by codon optimization of the nucleic acid sequence shown as SEQ ID NO. 1, and preferably, in the present application, the codon optimization method is not limited in any way, and can be performed by a codon optimization method commonly used in the art, for example, in the present application, the codon optimization is performed by the King Raiss company.

In the application, the nucleic acid sequence shown in SEQ ID NO. 1 is obtained from Ochrobactrum capable of producing L-carnosine, wherein the Ochrobactrum sp is preserved in China general microbiological culture Collection center (CGMCC for short) at 26.08.2022, with the preservation number of CGMCC No.25590 and the preservation address: the microbial research institute of the national academy of sciences No. 3, xilu No. 1, beijing, chaoyang, china, zip code: 100101.

in the present application, the nucleic acid sequence shown in SEQ ID NO. 1 is:

atgacttcacaaacacctacacgtaaacCGCGCGCTCGCGATCTTGGGCTTCCTTTCACTGGTGTGACCGGTCCGTACAACGCGATCACCGATGTCGATGGCGTTGGCGTCGGCTTTCAGACCATTATCGAGAACGAGCCGCGCCCCGGCCGCAAGCGTCCGGCGCGTAGCGGCGTGACCGCCATTCTGCCGCATAAGCAGTCTGAAACCCCGGTTCCGGTTTATGCAGGCGTCCATCGCTTCAACGGCAATGGTGAGATGACCGGAACGCACTGGATCGAAGATGGCGGCTACTTCCTGGGCCCTGTCGTTATCACCAACACGCACGGTATCGGCATGGCACATCATGCGACAGTGCGCTGGATGGTTGACCGCTATGCCTCGACCTACCAGACCGACGATTTCCTCTGGATCATGCCGGTTGTCGCAGAAACTTATGACGGTGCACTCAACGACATCAACGGCTTTCCTGTGACGGAAGCGGATGTGCGCAAGGCGCTCGACAATGTTGCATCCGGCCCGGTGCAGGAAGGCAATTGCGGCGGCGGCACCGGTATGATCACCTATGGCTTCAAGGGCGGTACAGGCACGGCATCGCGCGTCGTGGAGTTCGGCGGTCGCAGTTTCACCATCGGTGCGCTGGTGCAGGCCAATCACGGGCAGCGCGATTGGCTGACCATTGCCGGTGTGCCGGTGGGGCAGCATATGCGGGATGGCACGCCGCAGAGCCAGTTGCAGGAGCGCGGCTCGATCATCGTCGTGCTGGCGACCGATCTGCCACTGATGCCGCACCAGCTGAAGCGCCTAGCGCGTCGTGCAAGCATCGGCATCGGCCGTAACGGAACGCCGGGCGGTAACAATTCGGGCGATATTTTTATCGCTTTTTCCACCGCCAACCAGAGACCTATGCAGCATCGTTCCGCGCCCTTTCTGGACGTCGAGATGGTGAATGACGAGCCGCTTGATACCGTCTATCTGGCGGCGGTCGATAGTGTGGAAGAGGCAGTGGTTAATGCGATGATCGCGGCTGAGGATATGGGTGGAACACCCTTTGACCGGCTGCTTGTTCAGGCCATAGATCACGAACGTCTTCGTGCCgtgctgcgccaatatgggcgtcttgcctga

the various polynucleotides and control sequences may be joined together to produce a recombinant vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide.

The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication.

Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome or chromosomes into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell may be used, or a transposon may be used. The vector preferably contains one or more selectable markers that allow for convenient selection of transformed cells, transfected cells, transduced cells, and the like. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. In a specific embodiment, the recombinant vector is a vector comprising SEQ ID NO:2 in the sequence table 2.

The present application provides a vector comprising a nucleic acid molecule as described above. In some embodiments, the vector is a recombinant vector. In some embodiments, the recombinant vector is a pET vector, preferably pET28a (+).

For example, one or more nucleic acids encoding the proteases described above are cloned into a suitable recombinant vector or vectors, which may be any suitable recombinant expression vector, and which may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.

The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific for the type of host into which the vector is to be introduced (e.g., bacterial, fungal, plant or animal), as appropriate and taking into account whether the vector is DNA-based or RNA-based.

In the application, the vector is subjected to enzyme digestion by using restriction endonuclease, and the nucleic acid molecule containing the sequence shown in SEQ ID NO.2 is connected with the vector subjected to enzyme digestion by using ligase to obtain the vector containing the nucleic acid molecule.

The biological material of the present application may also be a recombinant microorganism comprising any one of the above-described nucleic acid molecules, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the expression cassettes, recombinant microorganisms containing the nucleic acid molecules, recombinant microorganisms containing the expression cassettes, or recombinant microorganisms containing the recombinant vectors.

A construct or vector comprising a polynucleotide is introduced into a microorganism such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector. The term "microorganism" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of microorganism will depend to a large extent on the gene encoding the variant and its source.

The microorganism of the present application can be any gram-positive or gram-negative bacteria, yeasts, molds, amoebae, and more generally unicellular organisms, which can be manipulated and manipulated in the laboratory. Gram-positive bacteria include, but are not limited to: bacillus, clostridium, enterococcus, geobacillus, lactobacillus, lactococcus, paenibacillus, staphylococcus, streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to: campylobacter, escherichia, flavobacterium, clostridium, helicobacter, citrobacter, neisseria, pseudomonas, salmonella, and Urethania. Yeasts include, but are not limited to: candida (Candida), cryptococcus (Cryptococcus), saccharomyces cerevisiae (Saccharomyces) and Trichosporon (Trichosporo). Molds include, but are not limited to: aspergillus (Aspergillus), penicillium (Penicillium), and Mycobacterium (Cladosporum).

In some embodiments, the microorganism is escherichia coli, bacillus subtilis, or saccharomyces cerevisiae.

The present application provides a host cell comprising the nucleic acid molecule or the recombinant expression vector, wherein the host cell expresses the protease.

To produce the protease, the nucleic acid molecule encoding the protease may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that are capable of binding specifically to a gene encoding a protease).

The host cell refers to a host cell into which an exogenous nucleic acid has been introduced, including progeny of such a host cell.

Methods for introducing a vector into a host cell are well known, and for example, a vector is introduced into a host cell by heat shock.

In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell, preferably a prokaryotic cell. In some embodiments, the prokaryotic cell is e.

Examples of Escherichia coli include Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076), escherichia coli B strains such as Escherichia coli K5 strain (ATCC 23506) and BL21 (DE 3) strains, and derivatives thereof.

The application provides a method for expressing the protease, which comprises the steps of expressing the host cell, and separating and purifying the host cell.

The expression of the host cell refers to the culture of the host cell, and the culture medium and the culture conditions are well known to those skilled in the art, and for example, the culture is performed using a medium such as LB, YPD, YNB or the like.

The expression mode is not limited, and the expression mode can be confirmed according to requirements, such as constitutive expression or inducible expression or combination of the constitutive expression and the inducible expression, the inducer can be IPTG, beta-galactoside, methanol, ethanol and the like.

In the present application, the method for separation and purification is not limited in any way, and the separation and purification can be performed according to the conventional method in the art, for example, after the cells are expressed, wall-breaking centrifugation is performed.

The application also provides the use of any one of the following materials in the preparation of the protease and the regulation and control of the yield of the protease in microorganisms:

c1 Substances regulating the expression of genes encoding said proteases;

c2 Substances which regulate the activity or content of the protease.

The substance which regulates the expression of the gene encoding the protease refers to a substance which can regulate and control the expression of the gene encoding the protease having a carnosine hydrolase function of the present application, and includes a promoter, an enhancer, a terminator, an increase in the copy number of the gene, inducible expression, a mutant sequence, expression of a fusion protein, and the like.

In this context, the term "promoter" is a DNA sequence recognized, bound and transcribed by RNA polymerase, which contains conserved sequences required for RNA polymerase specific binding and transcription initiation, most of which is located upstream of the transcription initiation point of a structural gene, and which is not transcribed per se. However, promoters, such as tRNA promoters, are located downstream of the transcription initiation point and these DNA sequences can be transcribed. The nature of the promoter was originally identified by mutations that increase or decrease the transcription rate of the gene. Promoters are generally located upstream of the transcription start site.

The term "enhancer" is a short segment of DNA that binds to a protein, and transcription of a gene is enhanced after binding to the protein. Enhancers may be located upstream or downstream of a gene. And not necessarily close to the gene to be affected, because of the winding structure of chromatin, giving the sequences an opportunity to come into contact at positions that are far apart.

The term "terminator (terminator T)" is a DNA sequence that gives a transcription termination signal to RNA polymerase. In an operon there is at least one terminator following the last gene of the structural gene group. The term "multicopy gene" is a gene whose repeats occur in large numbers in its natural state or by artificial means.

The term "inducible expression" means that the expression of a gene is initiated or enhanced by the action of an inducer (e.g., a metabolite).

The term "mutation" refers to a change in the base pair composition or order of arrangement of a gene in structure.

The substance for regulating the activity or content of the protease refers to a substance capable of regulating and controlling the activity or content of the protease having a carnosine hydrolase function encoding the present application, and includes a promoter, an enhancer, a terminator, an increase in gene copy number, inducible expression, a mutant sequence, expression of a fusion protein, and the like.

As above, the modulation may be up-regulation or enhancement or increase, or down-regulation or attenuation or decrease.

Wherein the up-regulation or enhancement or improvement of the expression of a gene encoding said protein or the activity or content of said protein is capable of up-regulating or enhancing or improving the production of a protease having a carnosine hydrolase function by a microorganism.

Downregulating or attenuating or reducing the expression of a gene encoding said protein or the activity or amount of said protein is capable of downregulating or attenuating or reducing the production of a protease with carnosine hydrolase function by the microorganism.

As described above, the expression of the gene encoding the protein (abbreviated as gene) can be regulated by at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In a specific embodiment, the above uses can be achieved by any one or more of the above biomaterials of the present application.

The present application also provides a method of recombining a microorganism, the method comprising:

a gene encoding a protease of the present invention is introduced into a microorganism to express the protease.

Further, when a gene encoding for expressing a protease of the present application is introduced into a microorganism, the encoding gene may be regulated to increase the activity or yield of the protease.

The application provides an application of the protease in preparing carnosine. In some embodiments, the substrate is beta-alanine methyl ester hydrochloride and L-histidine or is beta-alanine and L-histidine.

The protease can take beta-alanine methyl ester hydrochloride and L-histidine as substrates, and also can take beta-alanine and L-histidine as substrates, so that the cost is reduced.

The present application provides a method of preparing carnosine, comprising:

and (3) catalyzing a substrate by using the protease to perform reaction to obtain the carnosine. In some embodiments, the substrate is beta-alanine methyl ester hydrochloride and L-histidine or is beta-alanine and L-histidine.

In the present application, the temperature of the reaction is 20 to 60 ℃ and preferably 50 to 60 ℃.

For example, the reaction temperature can be 20 ℃,25 ℃,30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃,60 ℃ and so on.

In the present application, the pH of the reaction is from 7.0 to 11.0, preferably from 7.0 to 8.0.

For example, the pH of the reaction is 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, etc.

The protease has stable enzymatic properties, and the yield of carnosine obtained by using the protease to carry out enzymolysis on a substrate is high.

The application provides the application of the protease in preparing medicines or cosmetics containing carnosine.

Examples

The materials used in the tests and the test methods are generally and/or specifically described herein, and in the examples below,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1 protease engineering bacteria construction

1. Plasmid construction:

a strain capable of producing L-carnosine is obtained from fish intestinal tracts through a large amount of strain screening work, the 16sRNA sequence of the strain is shown as SEQ ID NO:8, the strain is identified to be Ochrobactrum sp, and the strain is preserved in China general microbiological culture Collection center (CGMCC for short) in 26 months at 2022, 08 and has the preservation number: CGMCC No.25590, preservation Address: the microbial research institute of the national academy of sciences No. 3, xilu No. 1, beijing, chaoyang, china, zip code: 100101, selecting a conserved sequence of protease in NCBI (as shown in SEQ ID NO: 7) as a template to design a primer (as shown in SEQ ID NO:3 and SEQ ID NO: 4), amplifying a protease nucleic acid sequence in Ochrobactrum anthropi to obtain a nucleic acid sequence as shown in SEQ ID NO:1, wherein the amplification conditions of the protease nucleic acid sequence are as follows: 1s at 98 ℃; 10s at 98 ℃; at 53 ℃ for 15s;72 ℃,15 s) for 30 times; 72 ℃ for 5min; keeping the temperature at 4 ℃.

The nucleic acid sequence shown in SEQ ID NO. 1 was optimized according to the codon preference of E.coli, and the codon optimization was accomplished by the Gireas company to obtain the nucleotide sequence shown in SEQ ID NO. 2. The amino acid sequence encoded by the optimized nucleotide sequence is shown as SEQ ID NO. 6, and is consistent with the amino acid sequence encoded by a wild type nucleotide sequence. The codon optimized carnosine hydrolase sequence was entrusted to the whole-gene synthesis by Nanjing Kinsrui Biotech, inc.

The plasmid pET28a (+) is cut by restriction endonucleases BamH I and Smii I, and then the nucleotide sequence shown in SEQ ID NO:2 is connected with the restriction endonucleases BamH I and Smii I through T4DNA ligase, so as to obtain the recombinant vector pET28a (+) -JT.

2. Constructing recombinant enzyme engineering bacteria:

(1) The recombinant vector pET28a (+) -JT aqueous solution is mildly and uniformly mixed with escherichia coli BL21 competence, and ice bath is carried out for 30min.

(2) The water bath was heat-shocked at 42 ℃ for 90s and rapidly iced for 3min.

(3) 500. Mu.L of fresh LB liquid medium was added and revived at 37 ℃ for 1 hour.

(4) And (4) coating an LB screening plate with antibiotics on the recovered bacterial suspension.

(5) LB screening plates were cultured in an inverted format at 37 ℃ overnight.

3. And (3) verification:

(1) Single colonies were picked, re-plated, and LB-screened plates were grown upside down at 37 ℃ overnight.

(2) 2-5mm lawn is selected and inoculated into a fresh LB liquid culture medium containing 50mg/mL kanamycin and cultured overnight at 37 ℃ and 200 rpm.

(3) The cells were collected and plasmids were extracted according to the plasmid extraction protocol (Shanghai Biotech).

(4) A1% agarose gel was prepared, and the size of the plasmid was checked by electrophoresis, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the plasmid length was as expected.

EXAMPLE 2 Shake flask fermentation to produce protease

(1) Preparing seeds: 2-5mm of the lawn (formed by the colonies connected to the plate) prepared in example 1 was picked up, inoculated into fresh LB liquid medium containing 50mg/mL kanamycin, and cultured overnight at 37 ℃ and 200 rpm.

(2) And (3) shake flask fermentation: inoculating the protease engineering bacteria described in example 1 into LB liquid medium containing 50mg/mL kanamycin at an initial culture temperature of 30-40 deg.C to OD ₆₀₀ Adding IPTG with final concentration of 0.1-0.8mM as inducer when the concentration is 0.5-0.8, inducing at 15-30 deg.C, and performing induced culture at rotation speed of 200-300rpm for 10-24h to obtain fermentation broth.

(3) Wall breaking: centrifuging the fermentation liquid at 8000-15000rpm for 5-20min to collect thallus, adding purified water 20-80% of the volume of the fermentation liquid into the thallus, resuspending the thallus, breaking cell wall with ultrasonication instrument, and treating at 30Hz frequency for 5-20min to obtain cell wall-broken liquid.

(4) Crude extraction: centrifuging the cell wall breaking solution at 8000-15000rpm for 5-20min, and collecting supernatant to obtain protease solution, wherein the amino acid sequence of the protease is shown in SEQ ID NO: and 6, respectively.

The protease solution was subjected to SDS-PEGA electrophoresis, and the results are shown in FIG. 2.

As can be seen from FIG. 2, the size of the protein band in the enzyme solution was as expected.

Example 3 determination of enzyme Activity of protease

1. Enzyme activity determination reaction process

The reaction substrate was added as in Table 1 below, and 200. Mu.L of the enzyme solution obtained in example 2 was added, followed by diluting the system to 10mL with purified water. The reaction system is placed in a constant temperature mixer at 25 ℃ for reaction for 30min, about 180 mu L of concentrated hydrochloric acid is added into the reaction liquid, and the pH is adjusted to 3-4, so that the enzyme is inactivated. Calculating enzyme activity: the production of 1. Mu. Mol of product per minute was defined as 1 enzyme activity unit.

TABLE 1 enzyme activity reaction System (10 mL)

Reagent	Addition amount (mL)
		0.2M L-histidine	5
0.5M beta-alanine methyl ester hydrochloride/0.5M beta-alanine	1
		Sodium carbonate buffer	2

2. Sample detection-derivatization

The reaction reagents were added as in table 2,

TABLE 2 derivatization reaction system (50 mL, volumetric flask)

Components	volume/mL
		Sample(s)	5
Sodium bicarbonate solution	5
		2, 4-dinitrofluorobenzeneacetonitrile solution	2.5

Adding the components, uniformly mixing, carrying out water bath at 60 ℃ for 1h, cooling to room temperature after the reaction is finished, and carrying out constant volume to 50mL by using 0.01M potassium dihydrogen phosphate solution.

3. Sample detection-liquid phase detection

Each sample was filtered through a 0.22 μm filter membrane under liquid phase conditions: c18PLUS column, mobile phase acetonitrile: 0.05M sodium acetate (1L with 100 μ L glacial acetic acid) =3:7, the flow rate is 0.8mL/min, the sample injection amount is 20 mu L, the column temperature is 35 ℃, and the ultraviolet detector is 360nm. And recording the peak area of the carnosine in the sample, and calculating the content of the carnosine by using an external standard method so as to calculate the enzyme activity. The enzyme activity is the micromole amount of the product produced per minute per milliliter of enzyme solution.

The formula for carnosine production is as follows:

carnosine content:

c: carnosine concentration in the sample, mg/mL

Cr: concentration of standard myopeptides, mg/mL

A: area of carnosine peak in sample

Ar: peak area of standard carnosine

D: dilution factor of sample

Wherein, beta-alanine methyl ester hydrochloride and L-histidine are taken as substrates, carnosine hydrolase is taken as a catalyst, L-carnosine is catalytically synthesized, and the enzyme activity of the carnosine hydrolase is 0.4U/mL; the beta-alanine and the L-histidine are used as substrates, the protease is used as a catalyst, the L-carnosine is catalytically synthesized, and the enzyme activity of the protease is 12.3U/mL.

Example 4 enzymatic characterization of proteases

1. Effective temperature and optimum temperature of protease produced by protease engineering bacteria

10 temperature gradients were set: 20. reaction substrates were added at 25, 30, 35, 40, 45, 50, 60, 80, and 100 ℃ as shown in Table 1, 200. Mu.L of the enzyme solution obtained in example 2 was added, and the system was made to 10mL with purified water. The reaction system is placed in a constant temperature mixing instrument for reaction for 30min, about 180 mu L of concentrated hydrochloric acid is added into the reaction solution, and the pH is adjusted to 3-4, so that the enzyme is inactivated. Calculating enzyme activity: the production of 1. Mu. Mol of product per minute was defined as 1 unit of enzyme activity, and the enzyme activity was measured in the same manner as in example 3, and the results are shown in FIG. 3.

As can be seen from FIG. 3, the effective temperature range of the protease is 20-60 ℃ and the optimum temperature is 60 ℃.

2. Effective pH and optimum pH of protease produced by protease engineering bacteria

Set 14 pH gradients: 4. 5, 6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, the reaction substrate was added as shown in Table 1, an appropriate amount of NaOH or hydrochloric acid was added to adjust the pH to a predetermined value, 200. Mu.L of the enzyme solution obtained in example 2 was added, and the volume of the system was adjusted to 10mL with purified water. The reaction system was placed in a homothermal mixer, reacted at 25 ℃ for 30min, an appropriate amount of concentrated hydrochloric acid was added to the reaction solution to adjust the pH to 3 to 4, thereby inactivating the enzyme, and the enzyme activity was measured in the same manner as in example 3, the results of which are shown in fig. 4.

As can be seen from FIG. 4, the effective pH range of the protease is 7-11, and the optimum pH is 7.5.

3. Temperature stability of protease produced by protease engineering bacteria

7 temperature gradients were set: 25. 40, 45, 50, 60, 80 and 100 ℃, respectively taking 1mL of enzyme solution, incubating in metal baths at different temperatures for 1h, adding reaction substrates according to the table 1, adjusting the pH to 9.0, adding 200 μ L of the enzyme solution obtained in example 2, and then adding purified water to the system to a constant volume of 10mL. The reaction system was placed in a shaker at 25 ℃ for 30min, and about 180. Mu.L of concentrated hydrochloric acid was added to the reaction solution to adjust the pH to 3 to 4, thereby inactivating the enzyme, and the enzyme activity was measured in the same manner as in example 3, and the results are shown in FIG. 5.

As can be seen from FIG. 5, the enzyme activity of the protease is relatively stable at a temperature of 25-50 ℃.

4. pH stability of carnosine hydrolase produced by protease engineering bacteria

5 pH gradients were set: 7. 7.5, 8, 8.5 and 9, respectively taking 1mL of the enzyme solution, adding a proper amount of NaOH or hydrochloric acid to adjust the pH value to different pH values, maintaining for 1h, adding a reaction substrate according to the table 1, adjusting the pH value to 9.0, adding 200 mu L of the enzyme solution obtained in the example 2, and then fixing the volume of the system to 10mL by using purified water. The reaction system was placed in a shaker at 25 ℃ for 30min, and about 180. Mu.L of concentrated hydrochloric acid was added to the reaction solution to adjust the pH to 3 to 4, thereby inactivating the enzyme, and the enzyme activity was measured in the same manner as in example 3, and the results are shown in FIG. 6.

As can be seen from FIG. 6, the enzyme activity of the protease is relatively stable at pH 7-9.

Example 5 verification of carnosine production

The reaction substrate and the enzyme solution obtained in example 2 were added as shown in Table 3, and the reaction was carried out for 24 hours under the following pH and temperature conditions. After the conversion is finished, a proper amount of concentrated hydrochloric acid is added to inactivate the enzyme, the content of the carnosine is measured according to the method in the example 3, and the yield of the carnosine is measured to be 1.36g/L and is 68 times of the yield of the ochrobactrum strain.

TABLE 3 transformation conditions

pH	Temperature of	L-histidine	Beta-alanine	Example 2 enzyme solution	Transformation system
						8.0	30℃	0.465g(15.5g/L)	1.335g(44.5g/L)	10mL	30mL

Example 6 verification of carnosine production

The reaction substrate and the enzyme solution obtained in example 2 were added as shown in Table 4, and the reaction was carried out for 24 hours under the following pH and temperature conditions. After the conversion was completed, a proper amount of concentrated hydrochloric acid was added to inactivate the enzyme, and the carnosine content was measured by the method of example 3, whereby the yield of carnosine was 15.80g/L.

TABLE 4 transformation conditions

pH	Temperature of	L-histidine	Beta-alanine methyl ester hydrochloride	Example 2 enzyme solution	Transformation system
						8.0	30℃	0.465g(15.5g/L)	2.094g(69.8g/L)	10mL	30mL

Sequence listing

The foregoing is directed to preferred embodiments of the present application, other than the limiting examples of the present application, and variations of the present application may be made by those skilled in the art using the foregoing teachings. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.

Claims

b1 A nucleic acid molecule encoding the protease of claim 1;

4. Use of any one of the following materials in the preparation of the protease of claim 1 for modulating the production of the protease of claim 1 in a microorganism:

c1 Substances regulating the expression of genes encoding said proteases;

c2 Substances which regulate the activity or content of the protease.

5. Use according to claim 4, by using any one or two or more of:

d1 A nucleic acid molecule encoding the protease of claim 1;

d2 An expression cassette containing the nucleic acid molecule according to D1);

d4 A recombinant microorganism containing the nucleic acid molecule according to D1), or a recombinant microorganism containing the expression cassette according to D2), or a recombinant microorganism containing the recombinant vector according to D3).

6. A method of recombining a microorganism, said method comprising:

introducing a gene encoding the protease of claim 1 into the microorganism;

7. The biomaterial according to claim 2 or 3, the use according to claim 4, the method according to claim 6, wherein the microorganism is any one of: e.coli; b, bacillus subtilis; and (3) saccharomyces cerevisiae.

8. A method for producing L-carnosine comprising producing L-carnosine using the protease of claim 1 or the biological material of claim 2 or 3 or the recombinant microorganism produced by the method of claim 6.