A protease and its application in L-carnosine synthesis

Abstract

The present application discloses a protease having carnosine hydrolase function and its use in L-carnosine synthesis, wherein the protease comprises the amino acid sequence shown in SEQ ID NO: 6. The protease described herein can utilize either β-alanine methyl ester hydrochloride and L-histidine as substrates, or β-alanine and L-histidine as substrates, thereby reducing costs and utilizing β-alanine produced due to the instability of β-alanine methyl ester hydrochloride.

Description

Technical Field

[0001] The present application relates to the technical field of carnosine, and in particular to a protease having carnosine hydrolase function and its application in L-carnosine synthesis. Background Art

[0002] L-carnosine, a dipeptide composed of β-alanine and L-histidine, is one of the most widely discovered bioactive peptides. L-carnosine is widely present in the brain, muscles, and other tissues of mammals.

[0003] Research has shown that L-carnosine possesses multiple biological activities, including antioxidant activity, intracellular free radical scavenging, and anti-aging properties. It also has therapeutic effects on hypertension, heart disease, senile cataracts, and ulcers. Due to its strong antioxidant activity, low toxicity and side effects, and diverse physiological activities, this active peptide has promising applications in medicine, healthcare, sanitation, and cosmetics.

[0004] Currently reported methods for synthesizing L-carnosine primarily include chemical synthesis and enzymatic catalysis. Enzymatic methods are more widely used due to their environmentally friendly, low-cost, and short synthesis time. However, the enzymatic synthesis of L-carnosine, typically using β-alanine methyl ester hydrochloride and histidine as substrates, presents the following challenges: First, the preparation of β-alanine methyl ester hydrochloride is complex and expensive; second, β-alanine methyl ester hydrochloride is highly acidic and corrosive to metal products such as stainless steel tanks; and third, β-alanine methyl ester hydrochloride is susceptible to moisture and is unstable when exposed to moisture. Summary of the Invention

[0005] In response to the above technical problems, the present application provides a protease with carnosine hydrolase function and its application in L-carnosine synthesis. The protease can prepare carnosine using β-alanine methyl ester hydrochloride and L-histidine or β-alanine and L-histidine as substrates, and the enzymatic properties of the protease are stable, which is conducive to industrialization.

[0006] The specific technical solutions of this application are as follows:

[0007] 1. A protease having carnosine hydrolase function, characterized in that the protease comprises the amino acid sequence shown in SEQ ID NO: 6.

[0008] 2. Biomaterial, characterized in that the biomaterial is any one of the following:

[0009] B1) a nucleic acid molecule encoding the protease according to item 1;

[0010] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0011] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0012] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0013] 3. The biomaterial according to item 2, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 2.

[0014] 4. Use of any of the following materials in preparing the protease described in item 1 or in regulating the production of the protease described in item 1 in microorganisms:

[0015] C1) a substance that regulates the expression of the gene encoding the protease;

[0016] C2) A substance that regulates the activity or content of the protease.

[0017] 5. The use according to item 4, characterized in that it is achieved by using any one or more of the following:

[0018] D1) a nucleic acid molecule encoding the protein enzyme described in item 1;

[0019] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0020] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0021] D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3).

[0022] 6. A method for recombining a microorganism, the method comprising:

[0023] Introducing a gene encoding the protease described in item 1 into the microorganism;

[0024] Optionally, the gene encoding the protease of item 1 can be regulated to increase the activity or yield of the protease.

[0025] 7. The biomaterial according to item 2 or 3, the use according to item 4, or the method according to item 6, wherein the microorganism is any one of the following: Escherichia coli; Bacillus subtilis; or Saccharomyces cerevisiae.

[0026] 8. A method for preparing L-carnosine, characterized in that the method comprises producing L-carnosine using the protease described in item 1, the biomaterial described in item 2 or 3, or the recombinant microorganism prepared by the method described in item 6.

[0027] 9. The method according to item 8, wherein the substrate is β-alanine methyl ester hydrochloride and L-histidine or β-alanine and L-histidine.

[0028] Effects of the Invention

[0029] The protease described in the present application can utilize β-alanine methyl ester hydrochloride and L-histidine as substrates, or can utilize β-alanine and L-histidine as substrates, which can reduce costs and can also utilize β-alanine produced due to the instability of β-alanine methyl ester hydrochloride.

[0030] The protease described in the present application has stable enzymatic properties, that is, the conversion conditions are wide, which is conducive to industrialization.

[0031] The present application adopts protease to catalyze the synthesis of L-carnosine, avoiding the tedious process of conventional chemical methods, effectively reducing production costs and improving economic benefits; at the same time, the synthesis process is simple, green and environmentally friendly, meets the requirements of clean production, and has broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Agarose electrophoresis diagram of protease.

[0033] Figure 2 This is the SDS-PAGE protein electrophoresis diagram of the protease.

[0034] Figure 3 This is a schematic diagram of the enzymatic properties of proteases versus temperature.

[0035] Figure 4 Schematic diagram of the enzymatic properties of proteases-pH.

[0036] Figure 5 The diagram shows the stability-temperature relationship of proteases.

[0037] Figure 6 Schematic diagram of enzyme stability-pH of protease. DETAILED DESCRIPTION

[0038] The present application is described in detail below in conjunction with the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0039] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

[0040] As used herein, the terms "polynucleotide," "nucleotide sequence," and "nucleic acid molecule" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as guide RNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0041] As used herein, the term "vector" is used to describe a nucleic acid molecule that can be engineered to contain a polynucleotide or multiple polynucleotides that can be cloned and 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, no free ends (e.g., circular); nucleic acid molecules 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 fragments can be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome.

[0042] In addition, certain vectors are capable of directing the expression of 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 the nucleic acid of the present application in a form suitable for expressing the nucleic acid in a host cell, meaning that the recombinant expression vector includes one or more regulatory elements, which may be selected based on the host cell used for expression and to which the nucleic acid sequence to be expressed may be operably linked.

[0043] As used herein, the term "recombinant microorganism" includes a microorganism (e.g., bacteria, yeast, algae, fungi, etc.) or microbial strain that has been genetically altered, modified, or engineered (e.g., genetically engineered) so that it exhibits an altered, modified, or different genotype and / or phenotype (e.g., when the genetic modification affects the encoding nucleic acid sequence of the microorganism) compared to the naturally occurring microorganism or "parent" microorganism from which it was derived.

[0044] The term "expression cassette" refers to DNA capable of expressing the protease having carnosine hydrolase function of the present application in a microorganism. The DNA includes not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0045] The present application provides a protease having carnosine hydrolase function, which comprises the amino acid sequence shown in SEQ ID NO: 6 or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0046] In the present application, the protease is an enzyme that hydrolyzes a substrate into carnosine.

[0047] The amino acid sequence of SEQ ID NO:6 is as follows:

[0048] MTSQTPTRKPRARDLGLPFTGVTGPYNAITDVDGVGVGFQTIIENEPRPGRKRPARSGVTAILPH

[0049] KQSETPVPVYAGVHRFNGNGEMTGTHWIEDGGYFLGPVVITNTHGIGMAHHATVRWMVDRYAST

[0050] YQTDDFLWIMPVVAETYDGALNDINGFPVTEADVRKALDNVASGPVQEGNCGGGTGMITYGFKG

[0051] GTGTASRVVEFGGRSFTIGALVQANHGQRDWLTIAGVPVGQHMRDGTPQSQLQERGSIIVVLATDL

[0052] PLMPHQLKRLARRASIGIGRNGTPGGNNSGDIFIAFSTANQRPMQHRSAPFLDVEMVNDEPLDTVYL

[0053] AAVDSVEEAVVNAMIAAEDMGGTPFDRLLVQAIDHERLRAVLRQYGRLA

[0054] Those skilled in the art will understand that polypeptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, but having similar activity to the amino acid sequence shown in SEQ ID NO: 6 also fall within the scope of the protease of the present application.

[0055] Polypeptides that have been modified, and / or substituted with one or several amino acids, and / or deleted and / or added with one or several amino acids based on the amino acid sequence shown in SEQ ID NO: 6 and have 95% or 96% or 97% or 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 6 and have the same function are also within the scope of protection of the protease of this application.

[0056] Polypeptides having the same function as those obtained by modification of the amino acid sequence shown in SEQ ID NO: 6, and / or substitution of one or several amino acids, and / or deletion and / or addition of one or several or dozens of amino acids are also within the protection scope of the protease of this application.

[0057] Furthermore, a fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 6 is also within the protection scope of the protease of the present application.

[0058] The present application also provides a biomaterial, wherein the biomaterial can be any of the following:

[0059] B1) a nucleic acid molecule encoding the above-mentioned protease;

[0060] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0061] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0062] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0063] Furthermore, the nucleotide sequence of the nucleic acid molecule SEQ ID NO: 2 is as follows:

[0064]

[0065] In the present application, the nucleic acid sequence shown in SEQ ID NO: 2 is obtained by codon optimization of the nucleic acid sequence of SEQ ID NO: 1. Preferably, in the present application, there is no limitation on the method of codon optimization, which can be optimized by a codon optimization method commonly used in the art. For example, in the present application, codon optimization is performed by Kingrise.

[0066] In the present application, the nucleic acid sequence shown in SEQ ID NO: 1 is obtained from Ochrobactrum sp. that can produce L-carnosine. The Ochrobactrum sp. was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on August 26, 2022, with the deposit number CGMCC No. 25590. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101.

[0067] In this application, the nucleic acid sequence described in SEQ ID NO: 1 is:

[0068]

[0069] Various polynucleotide and control sequence can be linked together to produce recombinant vectors, and this recombinant vector can comprise one or more suitable restriction sites to allow the polynucleotide of inserting or replacing coding variant at this type of site.Alternately, can be by polynucleotide or the nucleic acid construct that comprises this polynucleotide be inserted in the suitable carrier for expression and express this polynucleotide.When producing expression vector, coding sequence is so positioned in carrier so that coding sequence is operably connected with the suitable control sequence for expression.Recombinant vector can be any carrier (for example, plasmid or virus) that can easily stand recombinant DNA program and can cause polynucleotide expression.

[0070] The choice of 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 that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication.

[0071] Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicates along with the chromosome or chromosomes into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome, or a transposon may be used. The vector preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, or transduced cells, among others. A selectable marker is a gene whose product provides biocide resistance or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, and the like. In a specific embodiment, the recombinant vector is a plasmid comprising the nucleotide sequence shown in SEQ ID NO: 2.

[0072] The present application provides a vector comprising the nucleic acid molecule described above. In some embodiments, the vector is a recombinant vector. In some embodiments, the recombinant vector is a pET vector, preferably pET28a(+).

[0073] For example, one or more nucleic acids encoding the above-mentioned protease are cloned into suitable one or more recombinant vectors, which can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for breeding and amplification or for expression or for both, such as plasmids and viruses.

[0074] The vector may contain regulatory sequences (such as transcriptional and translational start and stop codons) that are specific for the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate and taking into account whether the vector is DNA-based or RNA-based.

[0075] In the present application, the vector is digested with a restriction endonuclease, and the nucleic acid molecule shown in SEQ ID NO: 2 is ligated to the digested vector using a ligase to obtain a vector containing the nucleic acid molecule.

[0076] The biological material of the present application may also include any one of the above-mentioned nucleic acid molecules, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the nucleic acid molecules, recombinant vectors containing the expression cassettes, recombinant microorganisms containing nucleic acid molecules, recombinant microorganisms containing expression cassettes, or recombinant microorganisms containing recombinant vectors.

[0077] The construct or vector comprising the polynucleotide is introduced into the microorganism so that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector. The term "microorganism" encompasses any parent cell progeny that is not identical to the parent cell due to a mutation that occurs during replication. The selection of microorganisms will depend to a great extent on the gene encoding the variant and its source.

[0078] The microorganism of the present application can be any Gram-positive or Gram-negative bacteria, yeast, mold, amoeba and more general unicellular organisms, which can be manipulated and operated in the laboratory. Gram-positive bacteria include but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Thuringia, Neisseria, Pseudomonas, Salmonella and Ureaplasma. Yeast includes but is not limited to: Candida, Cryptococcus, Saccharomyces and Trichosporo. Mold includes but is not limited to: Aspergillus, Penicillium, Cladosporium.

[0079] In some embodiments, the microorganism is Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae.

[0080] The present application provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector, wherein the host cell expresses the aforementioned protease.

[0081] In order to produce protease, the nucleic acid molecules encoding the protease can be separated and inserted into one or more vectors to further clone and / or express in a host cell. Conventional techniques can be used (e.g., by using oligonucleotide probes that can be specifically combined with the gene encoding the protease) to easily separate and order-check this nucleic acid.

[0082] The host cell refers to a host cell into which an exogenous nucleic acid has been introduced, including the progeny of such a host cell.

[0083] Methods for introducing vectors into host cells are well known, for example, vectors can be introduced into host cells by heat shock.

[0084] 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 Escherichia coli.

[0085] Examples of Escherichia coli include Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076), Escherichia coli K5 strain (ATCC 23506), Escherichia coli B strains such as BL21 (DE3) strain, and derivatives thereof.

[0086] The present application provides a method for expressing the above-mentioned protease, comprising expressing the above-mentioned host cell and isolating and purifying it.

[0087] The expression of the host cell refers to culturing the host cell. The culture medium and culture conditions are well known to those skilled in the art. For example, LB, YPD, YNB and other culture media are used for culturing.

[0088] The present invention does not impose any limitation on the expression mode, which can be confirmed as needed, for example, the expression can be constitutive expression or induced expression or a combination of the two. For induced expression, the inducer can be IPTG, β-galactoside, methanol, ethanol, etc.

[0089] In the present application, there is no limitation on the separation and purification method, which can be separated and purified according to conventional methods in the art, for example, after the cells are expressed, they are broken and centrifuged.

[0090] The present application also provides the use of any of the following materials in preparing the above-mentioned protease and regulating the production of the above-mentioned protease in microorganisms:

[0091] C1) a substance that regulates the expression of the gene encoding the protease;

[0092] C2) A substance that regulates the activity or content of the protease.

[0093] Among them, the substance that regulates the expression of the gene encoding the protease refers to a substance that can regulate and control the expression of the gene encoding the protease with carnosine hydrolase function of the present application, including promoters, enhancers, terminators, increasing gene copy number, inducing expression, mutant sequences, fusion protein expression, etc.

[0094] As used herein, the term "promoter" refers to a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for specific RNA polymerase binding and transcription initiation. Most promoters are located upstream of the transcription start site of a structural gene; the promoter itself is not transcribed. However, some promoters (such as tRNA promoters) are located downstream of the transcription start site; these DNA sequences can be transcribed. The properties of promoters were initially identified through mutations that increase or decrease the rate of gene transcription. Promoters are generally located upstream of the transcription start site.

[0095] The term "enhancer" refers to a small region of DNA that can bind to proteins, thereby enhancing gene transcription. Enhancers can be located upstream or downstream of a gene and are not necessarily located close to the gene they affect. This is because the coiled structure of chromatin allows distant sites in the sequence to come into contact.

[0096] The term "terminator (T)" is a DNA sequence that signals RNA polymerase to terminate transcription. Within an operon, there is at least one terminator following the last gene in the structural gene group. The term "multi-copy gene" refers to the large number of duplications of a specific gene, either naturally or through artificial means.

[0097] The term "induced expression" means that the expression of a gene is activated or enhanced under the action of an inducer (such as a metabolite).

[0098] The term "mutation" refers to a change in the composition or order of base pairs in a gene structure.

[0099] The substance that regulates the activity or content of the protease refers to a substance that can regulate and control the activity or content of the protease encoding the carnosine hydrolase function of the present application, including promoters, enhancers, terminators, increasing gene copy number, inducing expression, mutant sequences, fusion protein expression, etc.

[0100] In the above, the regulation may be up-regulation, enhancement, or increase, or down-regulation, attenuation, or reduction.

[0101] Wherein, upregulating, enhancing or increasing the expression of the gene encoding the protein or the activity or content of the protein can upregulate, enhance or increase the production of the protease having the carnosine hydrolase function of the microorganism.

[0102] Down-regulating, weakening or reducing the expression of the gene encoding the protein or the activity or content of the protein can down-regulate, weaken or reduce the production of the protease having the function of carnosine hydrolase in the microorganism.

[0103] In the above, regulating the expression of the gene encoding the protein (referred to as gene) may be at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the cell nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0104] In a specific embodiment, the above-mentioned use can be achieved by using any one or more of the above-mentioned biomaterials of the present application.

[0105] The present application also provides a method for recombining a microorganism, the method comprising:

[0106] A gene encoding the protease of the present application is introduced into a microorganism.

[0107] Furthermore, when the coding gene for expressing the protease of the present application is introduced into a microorganism, the coding gene can be regulated to increase the activity or yield of the protease.

[0108] The present application provides the use of the above-mentioned protease in the preparation of carnosine. In some embodiments, the substrate is β-alanine methyl ester hydrochloride and L-histidine or β-alanine and L-histidine.

[0109] The protease described in the present application can use either β-alanine methyl ester hydrochloride and L-histidine as substrates, or β-alanine and L-histidine as substrates, thereby reducing costs.

[0110] The present application provides a method for preparing carnosine, comprising:

[0111] Carnosine is obtained by using the above-mentioned protease to catalyze the substrate to react. In some embodiments, the substrate is β-alanine methyl ester hydrochloride and L-histidine or β-alanine and L-histidine.

[0112] In the present application, the reaction temperature is 20-60°C, preferably 50-60°C.

[0113] For example, the reaction temperature can be 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc.

[0114] In the present application, the pH value of the reaction is 7.0-11.0, preferably 7.0-8.0.

[0115] For example, the pH value of the reaction is 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, etc.

[0116] The enzymatic properties of the protease described in the present application are stable, and the yield of carnosine obtained by enzymatically hydrolyzing a substrate using the protease is high.

[0117] The present application provides the use of the above-mentioned protease in the preparation of medicines or cosmetics containing carnosine.

[0118] Example

[0119] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0120] Example 1 Construction of protease engineering bacteria

[0121] 1. Plasmid construction:

[0122] After a large amount of strain screening, a strain that can produce L-carnosine was obtained from the fish intestine, and its 16sRNA sequence is shown in SEQ ID NO: 8. The strain was identified as Ochrobactrum sp., which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Committee (CGMCC) on August 26, 2022, with a deposit number of CGMCC No. 25590, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101. The conserved sequence of protease in NCBI (as shown in SEQ ID NO: 7) was selected as a template to design primers (as shown in SEQ ID NO: 3 and SEQ ID NO: 4), and the protease nucleic acid sequence in Ochrobactrum was amplified to obtain the sequence shown in SEQ ID NO: 8. The nucleic acid sequence shown in IDNO: 1, the protease nucleic acid sequence amplification conditions are as follows: 98°C, 1s; 98°C, 10s; 53°C, 15s; 72°C, 15s) for 30 cycles; 72°C, 5min; and insulation at 4°C.

[0123] The nucleic acid sequence shown in SEQ ID NO:1 was optimized based on the codon preference of Escherichia coli. Codon optimization was performed by GenScript Biotechnology, resulting in the nucleotide sequence shown in SEQ ID NO:2. The amino acid sequence encoded by the optimized nucleotide sequence is shown in SEQ ID NO:6, which is identical to the amino acid sequence encoded by the wild-type nucleotide sequence. The codon-optimized carnosine hydrolase sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0124] The plasmid pET28a(+) was digested with restriction endonucleases BamHI and SmiI, and then the nucleotide sequence described in SEQ ID NO: 2 was ligated thereto using T4 DNA ligase to obtain the recombinant vector pET28a(+)-JT.

[0125] 2. Construction of recombinase engineering bacteria:

[0126] (1) Gently mix the recombinant vector pET28a(+)-JT aqueous solution with the competent E. coli BL21 and incubate on ice for 30 minutes.

[0127] (2) Heat shock in a 42°C water bath for 90 seconds, and then quickly place in an ice bath for 3 minutes.

[0128] (3) Add 500 μL of fresh LB liquid medium and resuscitate at 37°C for 1 h.

[0129] (4) The revived bacterial suspension is spread on LB screening plates with antibiotics.

[0130] (5) Incubate the LB screening plate upside down at 37°C overnight.

[0131] 3. Verification:

[0132] (1) Pick a single colony, re-plate it, and incubate the LB screening plate upside down at 37°C overnight.

[0133] (2) Pick 2-5 mm bacterial lawns and inoculate them into fresh LB liquid medium containing 50 mg / mL kanamycin. Culture them at 37°C and 200 rpm overnight.

[0134] (3) Collect the bacteria and extract the plasmid according to the instructions of plasmid extraction (Shanghai Bioengineering).

[0135] (4) Prepare 1% agarose gel and perform electrophoresis to detect the size of the plasmid. The results are as follows: Figure 1 shown.

[0136] from Figure 1 It can be seen that the plasmid length is in line with expectations.

[0137] Example 2 Shake flask fermentation to produce protease

[0138] (1) Seed preparation: 2-5 mm bacterial lawn prepared in Example 1 (formed by continuous colonies) was picked and inoculated into fresh LB liquid culture medium containing 50 mg / mL kanamycin, and cultured at 37° C., 200 rpm overnight.

[0139] (2) Shake flask fermentation: The protease engineered bacteria described in Example 1 were inoculated into LB liquid medium containing 50 mg / mL kanamycin at a 1% inoculum size. The initial culture temperature was 30-40°C and cultured until the OD 600 =0.5-0.8, add IPTG with a final concentration of 0.1-0.8 mM as an inducer, set the induction temperature at 15-30°C, and induce culture at a rotation speed of 200-300 rpm for 10-24 h to obtain a fermentation broth.

[0140] (3) Cell wall disruption: The fermentation broth was centrifuged at 8000-15000 rpm for 5-20 min to collect the cells. Purified water (20-80% of the volume of the fermentation broth) was added to the cells to resuspend the cells. The cells were then disrupted using an ultrasonic disruptor at 30 Hz for 5-20 min to obtain cell wall disruption solution.

[0141] (4) Crude extraction: The cell wall disruption solution is centrifuged at 8000-15000 rpm for 5-20 min, and the supernatant is collected as the protease solution, wherein the amino acid sequence of the protease is shown in SEQ ID NO: 6.

[0142] The protease solution was subjected to SDS-PEGA electrophoresis detection, and the detection results were as follows: Figure 2 shown.

[0143] from Figure 2 It can be seen that the size of the protein band in the enzyme solution is in line with expectations.

[0144] Example 3 Determination of protease activity

[0145] 1. Enzyme activity determination reaction process

[0146] Add the reaction substrate according to Table 1 below, along with 200 μL of the enzyme solution obtained in Example 2, and then dilute to 10 mL with purified water. Incubate the reaction system in a 25°C thermomixer for 30 min. Add approximately 180 μL of concentrated hydrochloric acid to the reaction solution to adjust the pH to 3-4 to inactivate the enzyme. Calculate enzyme activity: 1 μmol of product produced per minute is defined as 1 unit of activity.

[0147] Table 1 Enzyme activity reaction system (10mL)

[0148] Reagents Addition amount (mL) 0.2M L-histidine 5 0.5Mβ-Alanine methyl ester hydrochloride / 0.5Mβ-Alanine 1 Sodium carbonate buffer 2

[0149] 2. Sample testing-derivation

[0150] Add the reaction reagents according to Table 2.

[0151] Table 2 Derivatization reaction system (50 mL, volumetric flask)

[0152] Components Volume / mL sample 5 Sodium bicarbonate solution 5 2,4-Dinitrofluorobenzeneacetonitrile solution 2.5

[0153] After adding the above components, mix well and place in a 60°C water bath for 1 hour. After the reaction is completed, cool to room temperature and dilute to 50 mL with 0.01 M potassium dihydrogen phosphate solution.

[0154] 3. Sample testing - liquid phase testing

[0155] Each sample was filtered through a 0.22 μm filter and loaded onto a C18 PLUS column. The mobile phase consisted of a 3:7 ratio of acetonitrile to 0.05 M sodium acetate (1 L plus 100 μL of glacial acetic acid), a flow rate of 0.8 mL / min, an injection volume of 20 μL, a column temperature of 35°C, and a UV detector at 360 nm. The peak area of ​​carnosine in the sample was recorded, and the carnosine content was calculated using an external standard method, thereby calculating the enzyme activity. Enzyme activity is expressed as the micromoles of product produced per mL of enzyme solution per minute.

[0156] The formula for carnosine production is as follows:

[0157] Carnosine content:

[0158] C: Carnosine concentration in the sample, mg / mL

[0159] Cr: Carnosine standard concentration, mg / mL

[0160] A: Carnosine peak area in the sample

[0161] Ar: peak area of ​​standard carnosine

[0162] D: Sample dilution multiple

[0163] Among them, β-alanine methyl ester hydrochloride and L-histidine are used as substrates, carnosine hydrolase is used as catalyst, and L-carnosine is synthesized catalytically, and the enzymatic activity of carnosine hydrolase is 0.4U / mL; β-alanine and L-histidine are used as substrates, and protease is used as catalyst to catalyze the synthesis of L-carnosine, and the enzymatic activity of protease is 12.3U / mL.

[0164] Example 4 Enzymatic property identification of protease

[0165] 1. Effective temperature and optimum temperature of protease produced by protease engineering bacteria

[0166] Set 10 temperature gradients: 20, 25, 30, 35, 40, 45, 50, 60, 80, 100 ° C, add the reaction substrate according to Table 1, add 200 μL of the enzyme solution obtained in Example 2, and then dilute the system to 10 mL with purified water. Place the reaction system in a constant temperature mixer for 30 minutes, add about 180 μL of concentrated hydrochloric acid to the reaction solution, and adjust the pH to 3-4 to inactivate the enzyme. Calculate the enzyme activity: 1 μmol of product generated per minute is defined as 1 enzyme activity unit. The enzyme activity is determined in the same way as in Example 3, and the results are as follows: Figure 3 shown.

[0167] from Figure 3 It can be seen that the effective temperature range of the protease is 20-60°C, and the optimum temperature is 60°C.

[0168] 2. Effective pH and optimal pH of proteases produced by protease engineering bacteria

[0169] Set up 14 pH gradients: 4, 5, 6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13. Add the reaction substrate according to Table 1, add appropriate amount of NaOH or hydrochloric acid to adjust the pH to the specified value, add 200 μL of the enzyme solution obtained in Example 2, and then dilute the system to 10 mL with purified water. Place the reaction system in a constant temperature mixer and react at 25°C for 30 minutes. Add appropriate amount of concentrated hydrochloric acid to the reaction solution to adjust the pH to 3-4, thereby inactivating the enzyme. The enzyme activity is measured in the same way as in Example 3. The results are as follows: Figure 4 shown.

[0170] from Figure 4 It can be seen that the effective pH range of the protease is 7-11, and the optimum pH is 7.5.

[0171] 3. Temperature stability of proteases produced by protease engineering bacteria

[0172] Seven temperature gradients were set: 25, 40, 45, 50, 60, 80, and 100°C. 1 mL of enzyme solution was taken and incubated in metal baths at different temperatures for 1 hour. Then, reaction substrates were added according to Table 1, the pH was adjusted to 9.0, 200 μL of the enzyme solution obtained in Example 2 was added, and the system was then diluted to 10 mL with purified water. The reaction system was placed in a shaker at 25°C for 30 minutes. About 180 μL of concentrated hydrochloric acid was added to the reaction solution to adjust the pH to 3-4, thereby inactivating the enzyme. The enzyme activity was measured in the same manner as in Example 3. The results are shown in FIG. Figure 5 shown.

[0173] from Figure 5 It can be seen that the enzymatic activity of the protease is relatively stable at a temperature of 25-50°C.

[0174] 4. pH stability of carnosine hydrolase produced by protease engineered bacteria

[0175] Set up 5 pH gradients: 7, 7.5, 8, 8.5, 9, take 1 mL of enzyme solution in a test tube, add appropriate amount of NaOH or hydrochloric acid to adjust the pH to different pH values ​​and maintain for 1 hour, then add reaction substrate according to Table 1, adjust the pH to 9.0, add 200 μL of enzyme solution obtained in Example 2, and then dilute the system to 10 mL with purified water. Place the reaction system in a shaker at 25°C for 30 minutes, add about 180 μL of concentrated hydrochloric acid to the reaction solution, adjust the pH to 3-4, thereby inactivating the enzyme, and measure the enzyme activity in the same way as in Example 3. The results are as follows: Figure 6 shown.

[0176] from Figure 6 It can be seen that when the pH is 7-9, the enzymatic activity of the protease is relatively stable.

[0177] Example 5 Carnosine production verification

[0178] The reaction substrates according to Table 3 and the enzyme solution obtained in Example 2 were added, and the reaction was carried out for 24 hours under the following pH and temperature conditions. After the reaction was completed, an appropriate amount of concentrated hydrochloric acid was added to inactivate the enzyme. The carnosine content was determined according to the method in Example 3, and the measured carnosine production was 1.36 g / L, 68 times that of the Ochrobacterium strain.

[0179] Table 3 Transformation conditions

[0180] pH temperature L-histidine β-Alanine Example 2 Enzyme solution obtained Transformation system 8.0 30℃ 0.465g (15.5g / L) 1.335g (44.5g / L) 10mL 30mL

[0181] Example 6 Carnosine Output Verification

[0182] The reaction substrates listed in Table 4 and the enzyme solution obtained in Example 2 were added, and the reaction was carried out for 24 hours under the following pH and temperature conditions. After the reaction was completed, an appropriate amount of concentrated hydrochloric acid was added to inactivate the enzyme. The carnosine content was determined according to the method in Example 3, and the carnosine yield was 15.80 g / L.

[0183] Table 4 Conversion conditions

[0184] pH temperature L-histidine β-Alanine methyl ester hydrochloride Example 2 Enzyme solution obtained Transformation system 8.0 30℃ 0.465g (15.5g / L) 2.094g (69.8g / L) 10mL 30mL

[0185] Sequence Listing

[0186]

[0187]

[0188]

[0189] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. A method for preparing L-carnosine, characterized in that, The method comprises producing L-carnosine using a protease as shown in SEQ ID NO:6; The substrates are β-alanine and L-histidine.

2. A method for preparing L-carnosine, characterized in that, The method comprises producing L-carnosine using the following biological materials: The biological material is any one of the following: B1) a nucleic acid molecule encoding the protease shown in SEQ ID NO: 6; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); The substrates are β-alanine and L-histidine.

3. The method according to claim 2, wherein the microorganism is any one of the following: Escherichia coli; Bacillus subtilis; Saccharomyces cerevisiae.

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