CN117025574A - Dipeptidase, dipeptidase mutant, encoding gene and application thereof - Google Patents
Dipeptidase, dipeptidase mutant, encoding gene and application thereof Download PDFInfo
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- CN117025574A CN117025574A CN202310795532.8A CN202310795532A CN117025574A CN 117025574 A CN117025574 A CN 117025574A CN 202310795532 A CN202310795532 A CN 202310795532A CN 117025574 A CN117025574 A CN 117025574A
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- dipeptidase
- amino acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/13—Dipeptidases (3.4.13)
- C12Y304/1302—Beta-Ala-His dipeptidase (3.4.13.20)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a dipeptidase and a mutant thereof, nucleic acid for encoding the dipeptidase, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid, a preparation method of the dipeptidase mutant and a method for synthesizing L-carnosine. Compared with the prior art, the dipeptidase disclosed by the invention has higher activity and better thermal stability. The enzyme is used for catalyzing beta-alanine and L-histidine to prepare L-carnosine through direct condensation, so that the protection, deprotection and harsh reaction conditions in the conventional method for chemically synthesizing carnosine are avoided, the process flow is simple, the reaction conditions are mild, the process is green and environment-friendly, and the method has good application prospect in the industrial production of L-carnosine.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a dipeptidase and a mutant thereof, nucleic acid for encoding the dipeptidase, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid, a preparation method of the dipeptidase mutant and a method for synthesizing L-carnosine.
Background
L-Carnosine (beta-alanyl-L-histidine), also known as beta-alanyl-L-histidine, of formula C 9 H 14 N 4 O 3 Molecular weight 226.23, CAS number 305-84-0, is a substance that is widely found in mammalian muscle tissue. Carnosine is readily soluble in water, HCl, naOH, but hardly soluble in methanol, absolute ethanol. Carnosine is resistant to most peptidases, but an enzyme called Carnosinase (Xaa-His dipeptidases) hydrolyzes carnosine to beta-alanine and L-histidine.
The studies in biochemistry, pharmacology, physiology and the like which have been published at present indicate that the biological activity of carnosine is mainly aimed at protecting cells and delaying senescence. However, the specific mechanism of action still needs to be studied more intensively. Current studies indicate that the protective effect of L-carnosine on cells is achieved by exerting various actions such as antioxidant action, physiological pH buffer action, inhibition of angiotensin converting enzyme, killing of transformed cells, etc., while its anti-aging effect on cells is mainly manifested in inhibition of carbonyl accumulation of proteins. In addition to the primary functions of protecting cells and anti-aging, L-carnosine also has many physiological regulatory functions. For example, carnosine is an important neuropeptide in vivo, and can stabilize cell membrane to control normal operation of cell membrane material exchange and maintain cell dynamic balance. Carnosine can also act as a regulator, inhibiting oxidative stress and the NF- κb pathway, thereby protecting kidney tissue with diabetic nephropathy. The mouse experiment also proves that the carnosine can reduce the aggregation capacity of the blood platelet, inhibit the activation state of the blood platelet and relieve the related symptoms caused by the blood platelet aggregation. While many potential mechanisms have not been explored, a number of studies have demonstrated a great potential for carnosine in the treatment and alleviation of a variety of diseases including cataracts, diabetes, myocardial damage, and the like.
At present, the industry mainly applies chemical synthesis methods to produce carnosine, such as phthalic anhydride method, uses beta-alanyl chloride and L-histidine protected by phthalic anhydride as substrates, and carries out synthesis reaction first, then carries out hydrazinolysis reaction for deprotection, and finally produces L-carnosine. The method is the most commonly used method at present, but the process of the method is complicated, meanwhile, the reaction condition of an ice salt bath is required, and the protection of L-carnosine is removed by using highly toxic hydrazine in the hydrazinolysis reaction, so that pollution can be caused or the product quality can be influenced. The chemical synthesis method is not environment-friendly due to the harsh reaction conditions, and does not accord with the development concept of green production. The enzymatic synthesis is widely focused as a more green chemical synthesis method because of the characteristics of being ecological and sustainable. There are many reports of related peptidases which can be used for enzymatic catalysis of L-carnosine synthesis, including beta-aminopeptidases such as carnosine synthase, bapA, dmpA, etc., hydrolases such as hCN1, hCN2 derived from mammals and PepV, pepD derived from prokaryotes.
The principle of carnosine synthesis by these enzymes is slightly different, wherein carnosine synthase is an enzyme used for naturally carrying out carnosine synthesis in mammals, and is used for catalyzing beta-Ala and L-His to synthesize carnosine under the energy of ATP, but is difficult to directly use in large-scale industrial application due to the consumption of ATP. The beta-aminopeptidase and the dipeptidase belong to hydrolases, and can catalyze the reversible hydrolysis of the carnosine, so that most of the currently reported methods are to realize the synthesis of the carnosine by optimizing a reaction system and pushing a reaction to the synthesis direction.
The reverse synthesis of carnosine by hydrolase has received much attention as a carnosine synthesis method having industrial application prospects because ATP is not required to be consumed. The dipeptidase SmPepD obtained by digging from Serratia marcescens is the dipeptidase with highest activity, which can be used for carnosine synthesis, and the total yield of carnosine can reach 60.2%. However, in the currently reported routes of the method, the activity of the enzyme is still low, and a large room for improvement exists.
Disclosure of Invention
Aiming at the defects of the prior art in the inverse hydrolysis synthesis of L-carnosine by an enzymatic method catalyzed by dipeptidase, the invention provides dipeptidase and a dipeptidase mutant with high catalytic activity, high thermal stability and good substrate tolerance, a recombinant expression vector and a recombinant expression transformant containing the dipeptidase or the dipeptidase mutant gene, a preparation method of the dipeptidase mutant and a method for synthesizing L-carnosine by using the dipeptidase or the dipeptidase mutant.
The aim of the invention can be achieved by the following technical scheme:
the invention adopts one of the technical schemes:
a dipeptidase is provided, the amino acid sequence of which is shown in SEQ ID No.2, also called BmPEPD.
The preparation method of the dipeptidase provided by the invention comprises the following steps: isolated from Bacillus megaterium Bacillus megaterium or from a transformant recombinantly expressing the carnosine hydrolase, or synthesized synthetically based on the amino acid sequence.
The invention adopts a second technical scheme that:
a dipeptidase mutant is provided, which is a protein with dipeptidase activity obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
The invention performs enzyme screening by a bioinformatics-assisted method. BLAST was performed in NCBI database with the highest activity of the dipeptidase with highest activity available for carnosine synthesis as probe reported in the literature. Screening sequences with 20% -80% of similarity with the probe sequences, constructing a evolutionary tree after performing multi-sequence alignment on the sequences, selecting 20 candidate enzymes from the sequences to perform heterologous expression in escherichia coli, and performing carnosine hydrolysis and synthesis reaction on the soluble expressed enzymes by using cell disruption liquid of the soluble expressed enzymes to verify the activity of the soluble expressed enzymes. The enzyme recombinant expression vector for heterologous expression in the process is obtained by PCR of a laboratory preservation strain or is artificially synthesized according to amino acid sequences in a database by the company of the Style biotechnology Co. Wherein the database is numbered WP_048020937.1, and the enzyme BmPepD derived from the bacillus megatherium Bacillus megaterium with the amino acid sequence shown in SEQ ID No.2 has higher carnosine hydrolysis and synthesis activity.
Based on the method, directed evolution transformation is carried out on BmPepD by adopting site-directed saturation mutation. Modeling is carried out according to BmPepD sequences by using alpha Fold, butt joint of the modeling model with substrate molecules and metal ions is carried out, so that substrate binding sites and metal binding sites are determined, modification and screening are carried out on selected sites, and a batch of dipeptidase mutants with improved activity, which are also called BmPepD mutants, are obtained.
In one embodiment of the invention, the amino acid sequence of the dipeptidase mutant is as follows:
(1) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with serine;
(2) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glutamine;
(3) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glycine;
(4) A 457 th valine of an amino acid sequence shown in SEQ ID No.2 is replaced by glycine;
(5) A substitution of valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with isoleucine;
(6) Substituting valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with cysteine;
(7) Substitution of valine at position 81 of the amino acid sequence shown in SEQ ID No.2 with cysteine;
(8) The 81 th valine of the amino acid sequence shown in SEQ ID No.2 is replaced by tryptophan;
(9) Threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 is replaced by serine, and valine at position 457 is replaced by glycine;
(10) Substitution of threonine at position 171 with glycine and valine at position 457 with isoleucine of the amino acid sequence shown in SEQ ID No. 2;
(11) Substituting valine at 457 th position of an amino acid sequence shown in SEQ ID No.2 with cysteine, and substituting valine at 81 th position with cysteine;
(12) A 457 th valine of an amino acid sequence shown in SEQ ID No.2 is replaced by isoleucine, and a 81 th valine is replaced by tryptophan;
(13) Threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 is replaced with serine, valine at position 457 is replaced with glycine, and valine at position 81 is replaced with cysteine.
The third technical scheme adopted by the invention is as follows:
a nucleic acid encoding the dipeptidase of claim one or the dipeptidase mutant of claim one.
Wherein the nucleotide sequence of the nucleic acid for encoding the dipeptidase according to the first technical scheme is shown as SEQ ID No. 1.
The preparation method of the nucleic acid of the present invention is a conventional preparation method in the art, and preferably comprises:
extracting naturally occurring nucleic acid molecules encoding dipeptides from wild type bacillus megatherium Bacillus megaterium thalli; or obtaining a gene nucleic acid molecule encoding the dipeptidase BmPepD and mutants thereof by a gene cloning technology; or by artificial total sequence synthesis to obtain nucleic acid molecules encoding dipepds and mutants thereof.
The method for obtaining the gene nucleic acid molecule for encoding the dipeptidase BmPepD and the mutants thereof by the gene cloning technology comprises the following steps: to be used for
Forward primer 5' -CCGGAATTCATGGTGCAAACAGTA-3’，
Reverse primer 5' -CCGCTCGAGTTATATCTTATTTGCCTC-3’，
Carrying out gene amplification on the DNA sequence for encoding the dipeptidase BmPepD and the mutants thereof obtained in the first technical scheme by utilizing a polymerase chain reaction technology:
PCR System (50. Mu.L): 2x Prime Star Mix 20. Mu.L of template plasmid was about 100ng, 1.5. Mu.L of each of the upstream and downstream primers, and ddH2O was made up to 50. Mu.L.
PCR reaction procedure: (1) pre-denaturation at 95℃for 3min; (2) denaturation at 98℃for 10s; (3) annealing at 60 ℃ for 15s; (4) extending at 72 ℃ for 1.5min; (5) And (3) performing 30 cycles in total in the steps (2) - (4), and finally extending at 72 ℃ for 10min and preserving at 4 ℃.
The invention adopts the technical scheme that:
a recombinant expression vector comprising the dipeptidase and mutant nucleic acid of the present invention is provided.
It can be constructed by ligating the dipeptidase gene nucleic acid sequence or mutant gene nucleic acid sequence of the present invention to various suitable vectors by conventional methods in the art. The vector may be any of a variety of conventional vectors in the art, preferably a plasmid, more preferably plasmid pET-28a. The dipeptidase gene may be operably linked downstream of regulatory sequences suitable for expression in the selected vector to achieve constitutive or inducible expression of the dipeptidase.
Preferably, the recombinant expression vector of the present invention can be produced by, for example, the following method: the gene sequence DNA fragment of the dipeptidase BmPepD obtained by PCR amplification is digested with restriction enzymes EcoR I and Xho I, simultaneously the empty plasmid pET-28a is digested with restriction enzymes EcoR I and Xho I, the digested gene DNA fragment and pET-28a plasmid are recovered by glue, and the recombinant expression plasmid pET-28a-BmPepD containing the dipeptidase BmPepD gene is obtained by connecting with T4DNA ligase.
The invention adopts the technical scheme that:
there is provided a recombinant expression transformant comprising the dipeptidase gene, the dipeptidase mutant gene or the recombinant expression vector thereof of the present invention. Which can be produced by transforming the recombinant expression vector of the present invention into a host cell. The host cell may be any of various conventional host cells in the art provided that it can stably self-replicate using the recombinant expression vector and that the dipeptidase gene or the dipeptidase mutant gene carried thereby can be efficiently expressed.
Preferably, the host cell of the invention is E.coli, more preferably E.coli BL21 (DE 3).
The invention adopts a sixth technical scheme that:
provided is a preparation method of a dipeptidase mutant, comprising the following steps: culturing the recombinant expression transformant of the present invention to obtain the dipeptidase mutant. Wherein the medium used for culturing the recombinant expression transformant may be selected from conventional media in the art, provided that the transformant can be grown and the dipeptidase mutant of the present invention can be produced. Specific operations for culturing the transformant can be performed according to conventional procedures in the art.
The recombinant escherichia coli constructed by the technical scheme is inoculated into a liquid culture medium containing 4mL of LB (containing 50 mu g mL) by taking 20 mu L of recombinant strain glycerol bacterial liquid -1 Kanamycin), 1mL of the bacterial liquid was transferred to 100mL of LB liquid medium (containing 50. Mu.g mL) after shaking culture at 200rpm for 12 hours in a shaking table at 37 ℃ -1 Kanamycin) was cultured in 500mL shake flasks at 200rpm in a shaking table at 37℃for 3-4h to OD 600 About 0.6, and IPTG (isopropyl-. Beta. -D-thiogalactoside) was added at a final concentration of 0.2mM using a syringe to induce the expression of the target protein. After induction, the cells were collected by centrifugation (8000 Xg, 5 min) using a large centrifuge after shaking culture at 200rpm for 24 hours in a shaking table at 16 ℃. The cells were resuspended in 10mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated in an ice-water bath using a sonicator at 30% power for 15min to obtain a crude enzyme solution.
The protein of the present invention contains a histidine Tag (His-Tag) at the N-terminus, and thus can be purified using Ni NTABeads6FF type nickel column. The enzyme protein purification buffers were: and (3) solution A: 50mM NaH 2 PO 4 (pH 8.0) containing 300mM NaCl and 20mM imidazole; and (2) liquid B: 50mM NaH 2 PO 4 (pH 8.0) containing 300mM NaCl and 500mM imidazole. And (3) washing the nickel column by using deionized water with the volume of five times of the column, loading crude enzyme solution onto the nickel column, washing by using the liquid A with the volume of ten times of the column, eluting by using the liquid B with the volume of fifteen to twenty times of the column, and collecting the eluent. SDS-PAGE protein electrophoresis is used to verify the purification condition of the purified protein, then an ultrafiltration tube is used for centrifugal replacement to remove imidazole, the concentration of the protein is measured by Nanodrop, and the protein is stored at-80 ℃ for standby after split charging.
The invention adopts a seventh technical scheme:
the invention provides application of the dipeptidase or the dipeptidase mutant in preparing L-carnosine by catalyzing beta-alanine and L-histidine to condense. The application is that the dipeptidase or the dipeptidase mutant is added into a buffer solution containing beta-alanine and L-histidine to catalyze the reverse hydrolysis reaction of the beta-alanine and the L-histidine to synthesize the L-carnosine.
The buffer salt system of the buffer solution is not limited as long as the pH range is between 6.0 and 9.0; a preferred buffer salt system is Tris-HCl, pH 8.0. The reaction temperature is 20-65℃and preferably 40 ℃. A suitable amount of water-soluble solvent or water-insoluble solvent may be added to the buffer solution. Other reaction conditions such as substrate concentration, enzyme amount, etc. may be selected according to the conventional conditions for such reactions in the art.
When the dipeptidase or the dipeptidase mutant is used as a catalyst, crude enzyme liquid containing the dipeptidase or the dipeptidase mutant or pure dipeptidase or the dipeptidase mutant is immobilized on a carrier to obtain immobilized enzyme, and the immobilized enzyme is used as the catalyst.
Intermittent sampling is carried out in the reaction process, and liquid chromatography is adopted for analysis. Using chiral crown ether columnsCR (+) 4.0 mm. Times.150 mm was used as a liquid chromatography column with a packing size of 5 μm, and perchloric acid (HClO) having a pH of 1.0 was selected 4 ) The aqueous solution was used as the mobile phase, the flow rate was 0.3mL min -1 Detection was performed at a wavelength of 210 nm.
Compared with the prior art, the invention has the beneficial technical effects that:
the dipeptidase or the dipeptidase mutant disclosed by the invention has the advantages of high activity, good thermal stability and strong substrate tolerance, can directly catalyze the condensation of beta-alanine and L-histidine to synthesize L-carnosine, avoids the steps of protecting and deprotecting a substrate in a chemical synthesis method, and is more environment-friendly. Compared with other reported L-carnosine synthesis methods, the method for preparing the L-carnosine has the advantages of mild reaction conditions, no need of pretreatment of a substrate, environment-friendly process, easy industrial amplification and the like, and has good industrial application and development prospects.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1 screening for dipeptidases.
BLAST was performed in NCBI database using the dipeptidase SmPepD reported in the literature as a probe. Screening sequences with 20-80% of sequence similarity with probes, constructing a evolutionary tree after performing multi-sequence comparison on the sequences, selecting 20 candidate enzymes from the sequences, obtaining corresponding dipeptidase recombinant plasmids through PCR or artificial synthesis, converting the corresponding dipeptidase recombinant plasmids into escherichia coli to obtain corresponding recombinant strains, inoculating the strains into a test tube containing 4mL of culture medium, shaking and culturing the strains in a shaking table at 37 ℃ for 12 hours at 200rpm, transferring 1mL of bacterial liquid into a shaking bottle containing 100mL of LB culture medium, and performing induction culture. After 24 hours, the cells were collected by centrifugation using a large centrifuge. The cells were resuspended in 10mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated in an ice-water bath using a sonicator at 30% power for 15min to obtain a crude enzyme solution.
The activity of the new enzyme crude enzyme solution on the L-carnosine is verified by a biocatalytic reaction. The total reaction system was 0.2mL and contained 100mM L-carnosine, 50mM Tris-HCl buffer (pH 8.0) and the appropriate concentration of crude enzyme. The reaction was carried out at 30℃with shaking at 1000rpm for 2h. After completion of the reaction, 10. Mu.L of the reaction solution was mixed with 990. Mu.L of perchloric acid to terminate the reaction, and after centrifugation at 13000 Xg for 2min, the mixture was filtered using a mixed cellulose filter membrane, and the mixture was analyzed by High Performance Liquid Chromatography (HPLC), and according to the result of the reaction, whether the screened novel enzyme had activity on L-carnosine was judged, and the results are shown in Table 1.
TABLE 1 comparison of carnosine hydrolytic Activity of different strains
Note that: "SmPepD" is the probe sequence; "++" indicates that the hydrolysis activity is higher than that of the probe; "+" indicates that the hydrolytic activity is lower than the probe, but there is hydrolytic activity towards L-carnosine; "-" indicates that no hydrolytic activity was detected for L-carnosine.
EXAMPLE 2 preparation of recombinant dipeptidase BmPepD
5' -CCG by forward primerGAATTCATGGTGCAAACAGTA-3’Reverse primer 5' -CCGCTCGAGTTATATCTTATTTGCCTC-3', the dipeptidase BmPepD coding gene selected in example 1 was amplified by the polymerase chain reaction technique, and the obtained amplified coding DNA fragment was digested with restriction endonucleases EcoR I and Xho I, and ligated with pET-28a plasmid which was digested with EcoR I and Xho I as well, to obtain recombinant plasmid pET-28a-BmPepD.
The obtained recombinant plasmid is transformed into E.coli BL21, the constructed recombinant strain is inoculated into a test tube containing 4mL of culture medium, 1mL of bacterial liquid is transferred into a shake flask containing 100mL of LB culture medium after shaking culture at 37 ℃ for 12h, IPTG is added after culture at 37 ℃ for 3h, induction culture is carried out at 16 ℃ for 24h, and bacterial bodies are collected centrifugally. The cells were resuspended in 10mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated in an ice-water bath using a sonicator at 30% power for 15min to obtain a crude enzyme solution. The nickel column is washed by deionized water with the volume of five times of the column, crude enzyme liquid is loaded on the nickel column, then washed by A liquid with the volume of ten times of the column, then eluted by B liquid with the volume of fifteen to twenty times of the column, and the eluent is collected. Removing imidazole by ultrafiltration tube replacement, packaging, adding glycerol, and storing at-80deg.C. The synthesis activity of BmPepD pure enzyme is 16.7. 16.7Umg -1 。
Example 3 molecular engineering of recombinant dipeptidase BmPepD
Constructing a structural model of BmPEPD by using alpha Fold, interfacing with substrate molecules and metal ions, and binding at a substrate binding or metal binding siteWithin the scope of 5 amino acid residues were selected as targets for site-directed saturation mutagenesis. Site-directed saturation mutation is carried out on a target site by designing NNK degenerate codons, the mutants are cultivated in a deep-hole plate, 50 mu L of the mutants are transferred to a secondary deep-hole plate containing 600 mu L after shaking culture at 37 ℃ for 3 hours, IPTG with the final concentration of 0.2mM is added for induction, and the mutants are cultivated at 16 ℃ for 24 hours. After centrifugation, the bacteria were collected, and 100. Mu.L of an enzyme solution was obtained after disruption by adding lysozyme, and mixed with 100. Mu.L of a substrate solution containing beta-alanine (2M) and L-histidine (200 mM) to carry out a synthesis reaction. The reaction solution passes throughLiquid chromatography analysis and preliminary screening were performed to find that twelve mutants had increased activity compared to the parent BmPepD. The purified twelve mutants were tested for L-carnosine synthesis activity by pure enzymes, and the results are shown in Table 2. The sequence numbers in table 2 correspond to the series of sequences following table 2, respectively. In the active column, one plus sign "+" indicates that the mutant vigor is improved by 1.2-5 times compared with the female parent BmPepD; two plus signs "++" indicate that mutant viability is improved by 5-10 times; three plus signs' ++ + + + is shown mutant viability was improved by more than 10-fold.
TABLE 2 dipeptidase BmPepD mutant sequences and corresponding list of L-carnosine synthetic activity improvement
The amino acid sequences of the corresponding numbered BmPepD mutants in the table are as follows:
(1) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with serine;
(2) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glutamine;
(3) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glycine;
(4) A 457 th valine of an amino acid sequence shown in SEQ ID No.2 is replaced by glycine;
(5) A substitution of valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with isoleucine;
(6) Substituting valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with cysteine;
(7) Substitution of valine at position 81 of the amino acid sequence shown in SEQ ID No.2 with cysteine;
(8) The 81 th valine of the amino acid sequence shown in SEQ ID No.2 is replaced by tryptophan;
(9) Threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 is replaced by serine, and valine at position 457 is replaced by glycine;
(10) Substitution of threonine at position 171 with glycine and valine at position 457 with isoleucine of the amino acid sequence shown in SEQ ID No. 2;
(11) Substituting valine at 457 th position of an amino acid sequence shown in SEQ ID No.2 with cysteine, and substituting valine at 81 th position with cysteine;
(12) A 457 th valine of an amino acid sequence shown in SEQ ID No.2 is replaced by isoleucine, and a 81 th valine is replaced by tryptophan;
(13) Threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 is replaced by serine, valine at position 457 is replaced by glycine, and valine at position 81 is replaced by cysteine;
example 4 dipeptidase mutant BmPepD M13 Is prepared from
Extraction of the recombinant plasmid pET-28a-BmPepD obtained as in example 3 M13 The recombinant strain is transformed into E.coli BL21, the constructed recombinant strain is inoculated into a test tube containing 4mL of culture medium, 1mL of bacterial liquid is transferred into a shake flask containing 100mL of LB culture medium after shaking culture at 37 ℃ for 12h, IPTG is added after culture at 37 ℃ for 3h, and bacterial cells are collected after induction culture at 16 ℃ for 24h by centrifugation. The cells were resuspended in 10mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated in an ice-water bath using a sonicator at 30% power for 15min to obtain a crude enzyme solution. The nickel column is washed by deionized water with the volume of five times of the column, crude enzyme liquid is loaded on the nickel column, then washed by A liquid with the volume of ten times of the column, then eluted by B liquid with the volume of fifteen to twenty times of the column, and the eluent is collected. Removing imidazole by ultrafiltration tube centrifugal displacement, subpackaging, adding glycerol, and storing at-80deg.C.
Example 5 Effect of temperature on dipeptidase BmPepD Activity
At different temperatures (20-65 ℃), 100. Mu.L of BmPEPD was taken M13 Pure enzyme (enzyme protein concentration 0.05 mg/mL) was added to 100. Mu.L of T containing beta-alanine (4M) and L-histidine (200 mM)The synthesis was performed in a ris-HCl substrate solution (pH 8.0), followed by shaking for 15min and HPLC analysis. The results are shown in Table 3. The enzyme exhibited the highest activity at 50 ℃, the enzyme activity at this temperature was defined as 100%, and the relative activities at the other temperatures were calculated. The enzyme activity increased with increasing temperature before 50℃and decreased rapidly above 50 ℃.
TABLE 3 Activity of dipeptidase BmPEPD at different temperatures
EXAMPLE 6 Effect of pH on dipeptidase BmPepD Activity
At a measured activation temperature of 40 ℃, 100 mu L of pure enzyme (enzyme protein concentration 0.05 mg/mL) is added into 100 mu L of substrate solution containing beta-alanine (4M), L-histidine (200 mM), PBS buffer (pH 6.0-7.5) or Tris-HCl buffer (pH 7.5-9.0) to carry out synthesis reaction, shake reaction is carried out for 15min, and HPLC analysis is carried out on the activity. The results are shown in Table 4. The relative activity of the enzyme was highest in Tris-HCl buffer at pH8.0, defined as 100%, and relative activities at other pH values were calculated.
TABLE 4 Activity of dipeptidases BmPEPD at different pH' s
Example 7 dipeptidase BmPEPD and mutant M13 thereof catalyzes a 10mL amplification reaction for L-carnosine synthesis
The carnosine synthesis reaction was performed in 50mL Erlenmeyer flasks, with a total system volume of 10mL containing final concentrations of saturated beta-Ala, 0.2M L-His and 50mM Tris-HCl buffer (pH 8.0), 0.05mg/mL BmPepD or mutant pure enzyme. The reaction was carried out at 40℃and 200rpm, samples were taken intermittently during the reaction and analyzed by HPLC. Reaction for 8h, using BmPEPD as catalyst, yielded 48.2mMThe specific yield of L-carnosine and the enzyme reaches 436g Car g catalyst -1 The method comprises the steps of carrying out a first treatment on the surface of the When mutant M13 was used, 61.3mM L-carnosine was produced, and the specific yield of the enzyme reached 554g Car g catalyst -1 。
The sequences involved in the present invention are as follows:
SEQ ID No.1
ATGGTGCAAACAGTAAATGAATTAATTAAACATCCGGTTTTCTATTTCTTTAACGAAATCTCAGCTATTCCTAGAGAATCTGGAAATGAAAAAGAAATTAGTAACTATTTAGTTTCTTTTGCAAAAGAAAGAAGCCTTGAGGTTATCCAAGACGAAGCTTTAAATGTAATCATTAAAAAGCCGGCAACTAAAGGATATGAACATGCTCCAGCTATCATTTTGCAAGGACATATGGACATGGTATGCGAGTTAAATAAAGGAACTGTCCATGACTTTGAAAAAGATCCTCTTCAACTGCGAATTGTTGAAGATATGCTTTATGCAAATGGTACTACGCTAGGAGCTGATAACGGGATTGCCGTTGCATATGCACTGGCTTTACTAGATGCTCAAAATATAGCGCACCCCTCTCTTGAAGTAGTCATTACCACTGAAGAAGAAACGACTATGGGCGGAGCCATTGCTGTAAATCCAGCTTATTTTGAAGGGAAAATCTTTATTAACCTTGATACAGAAGAGGACGGAAAGCTCCTTGTCAGCAGCGCAGGCGGGGTAAAAGGCGTGCTTCGTATTCCTATAAACTGGGAGTCGTCTTCCAATAACTCAGAAACATATAGCTTAAGCATCGGAGGACTGCGCGGCGGACATTCCGGAATGGAAATTGACAAAGAAAGAGGAAATGCGAATAAACTGCTAGGGAGAGTTTTATATGATTTACAACAAGAACTTCCCTATTCTTTAAGCAGCATTAGCGGAGGATTAAAATCAAATGCAATTCCCCGTGAATCAGAAGCAATTCTATCAGTTGAGCCTTCTGAGGTAGGAAAGCTAGAAAATAAGATTCGTGAGTGGAACGAAATAGTAAAAAATGAATTACAAGCGGCTGATCCGAGCGTATATGTAAAAATAAACAAATTTTCTTCCGTTGAGAAATGTTTTACAAGAGAAACGACAGAACGAATTGTACAAGCTATTATGCTAACACCAAACGGCGTTCAAAGCATGAGTATGAATATTGAAGGATTAGTAGAATCTTCTACCAACTTAGGTGTGATCACGACGACAGAATCTGAGGTTGTTTTTCAAAATGAAATTCGCAGCTCAGTAAAAAGCTTAAAAGAAAAAATAGTAAGTCAAGTGCGCATACTTGCTCAGGTAGTCGGGGGAAGAGTTGAAACAAAAGGTAATTATCCTGAATGGGCGTACAATGGAGATTCAAAAATTCGCGAATTATGTAAAAAGGTTTACAAAGAAAAGTATGGTGAAGAGGCGGAGATCATCGCCATCCATGCTGGAATTGAATGCGGTATTTTCTTAGAAAAAATCCCTGGATTAGATGCCATTTCGCTCGGACCCGATATGTACGACGTTCATACACCTGATGAACACCTGAGCATTCCGTCTACTCTTAAGACATGGGAATACTTATTAGCAGTATTAAAAGAGGCAAATAAGATATAA
SEQ ID No.2
MVQTVNELIKHPVFYFFNEISAIPRESGNEKEISNYLVSFAKERSLEVIQDEALNVIIKKPATKGYEHAPAIILQGHMDMVCELNKGTVHDFEKDPLQLRIVEDMLYANGTTLGADNGIAVAYALALLDAQNIAHPSLEVVITTEEETTMGGAIAVNPAYFEGKIFINLDTEEDGKLLVSSAGGVKGVLRIPINWESSSNNSETYSLSIGGLRGGHSGMEIDKERGNANKLLGRVLYDLQQELPYSLSSISGGLKSNAIPRESEAILSVEPSEVGKLENKIREWNEIVKNELQAADPSVYVKINKFSSVEKCFTRETTERIVQAIMLTPNGVQSMSMNIEGLVESSTNLGVITTTESEVVFQNEIRSSVKSLKEKIVSQVRILAQVVGGRVETKGNYPEWAYNGDSKIRELCKKVYKEKYGEEAEIIAIHAGIECGIFLEKIPGLDAISLGPDMYDVHTPDEHLSIPSTLKTWEYLLAVLKEANKI
the previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. A dipeptidase is characterized in that the amino acid sequence of the dipeptidase is shown as SEQ ID No. 2.
2. A dipeptidase mutant characterized in that the amino acid sequence of the dipeptidase mutant is as follows:
(1) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with serine;
(2) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glutamine;
(3) Substitution of threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glycine;
(4) A 457 th valine of an amino acid sequence shown in SEQ ID No.2 is replaced by glycine;
(5) A substitution of valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with isoleucine;
(6) Substituting valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with cysteine;
(7) Substitution of valine at position 81 of the amino acid sequence shown in SEQ ID No.2 with cysteine;
(8) The 81 th valine of the amino acid sequence shown in SEQ ID No.2 is replaced by tryptophan;
(9) Threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 is replaced by serine, and valine at position 457 is replaced by glycine;
(10) Substitution of threonine at position 171 with glycine and valine at position 457 with isoleucine of the amino acid sequence shown in SEQ ID No. 2;
(11) Substituting valine at 457 th position of an amino acid sequence shown in SEQ ID No.2 with cysteine, and substituting valine at 81 th position with cysteine;
(12) A 457 th valine of an amino acid sequence shown in SEQ ID No.2 is replaced by isoleucine, and a 81 th valine is replaced by tryptophan;
(13) Threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 is replaced with serine, valine at position 457 is replaced with glycine, and valine at position 81 is replaced with cysteine.
3. An isolated nucleic acid encoding the dipeptidase of claim 1 or encoding the dipeptidase mutant of claim 2.
4. A recombinant expression vector comprising the nucleic acid of claim 3.
5. A recombinant expression transformant comprising the recombinant expression vector according to claim 4.
6. A method for preparing the dipeptidase mutant according to claim 2, comprising the steps of: culturing the recombinant expression transformant according to claim 5, and isolating the dipeptidase mutant.
7. Use of the dipeptidase of claim 1 or the dipeptidase mutant of claim 2 for synthesizing L-carnosine.
8. The use according to claim 7, characterized by the steps of: the use of the dipeptidase enzyme according to claim 1 or the dipeptidase enzyme mutant according to claim 2 for catalyzing the reverse hydrolysis reaction of beta-alanine and L-histidine to produce L-carnosine, and then separating and extracting the product L-carnosine from the reaction mixture.
9. The use according to claim 8, wherein the reaction temperature is 20-65 ℃, the reaction pH is 6.0-9.0, the concentration of substrate β -alanine is 200mM to the saturation concentration, and the concentration of substrate L-histidine is 100-200mM.
10. The use according to claim 7, wherein when the dipeptidase or the dipeptidase mutant is used as a catalyst, the immobilized enzyme is obtained by immobilizing a crude enzyme solution or a pure dipeptidase or a dipeptidase mutant containing the dipeptidase or the dipeptidase mutant to a carrier, and the immobilized enzyme is used as a catalyst.
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| CN202310795532.8A CN117025574A (en) | 2023-06-30 | 2023-06-30 | Dipeptidase, dipeptidase mutant, encoding gene and application thereof |
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