Dipeptidase, dipeptidase mutants, encoding genes and their applications

Abstract

This invention belongs to the field of bioengineering technology, and specifically relates to a dipeptidase and its mutant, a nucleic acid encoding the dipeptidase, a recombinant expression vector containing the nucleic acid and a recombinant expression transformant, a method for preparing the dipeptidase mutant, and a method for using it in the synthesis of L-carnosine. Compared with existing technologies, the dipeptidase disclosed in this invention has higher activity and better thermal stability. Using this enzyme to catalyze the direct condensation of β-alanine and L-histidine to prepare L-carnosine avoids the protection and deprotection processes and harsh reaction conditions required in conventional chemical synthesis of carnosine. The process is simple, the reaction conditions are mild, and the process is environmentally friendly, showing good application prospects in the industrial production of L-carnosine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a dipeptidase and its mutant, a nucleic acid encoding the dipeptidase, a recombinant expression vector containing the nucleic acid and a recombinant expression transformant, a method for preparing the dipeptidase mutant, and a method for using it in L-carnosine synthesis. Background Technology

[0002] L-Carnosine (β-alanyl-L-histidine), also known as β-alanyl-L-histidine, has the molecular formula C9H. 14 N4O3, with a molecular weight of 226.23 and CAS number 305-84-0, is a substance widely found in mammalian muscle tissue. Carnosine is readily soluble in water, HCl, and NaOH, but almost insoluble in methanol and anhydrous ethanol. Carnosine is resistant to most peptidases, but an enzyme called carnosinase (Xaa-His dipeptidases) can hydrolyze carnosine into β-alanine and L-histidine.

[0003] Current research in biochemistry, pharmacology, and physiology indicates that the bioactivity of carnosine primarily lies in protecting cells and delaying aging. However, the specific mechanisms of action still require further investigation. Current research suggests that L-carnosine's protective effect on cells is achieved through various mechanisms, including antioxidant activity, physiological pH buffering, inhibition of angiotensin-converting enzyme, and killing of transformed cells. Its anti-aging effect on cells is mainly manifested in its inhibition of protein carbonyl accumulation. Besides its main functions of protecting cells and anti-aging, L-carnosine also possesses many physiological regulatory functions. For example, as an important neuropeptide in the body, carnosine can stabilize cell membranes, controlling the normal operation of cell membrane substance exchange and maintaining cellular homeostasis. Carnosine can also act as a regulator, inhibiting oxidative stress and the NF-κB pathway, thereby protecting kidney tissue in diabetic nephropathy. Mouse experiments have also demonstrated that carnosine can reduce platelet aggregation, inhibit platelet activation, and alleviate symptoms related to platelet aggregation. Although many underlying mechanisms remain to be explored, numerous studies have demonstrated the great potential of carnosine in treating and alleviating a variety of diseases, including cataracts, diabetes, and myocardial damage.

[0004] Currently, the main industrial method for producing carnosine is chemical synthesis, such as the phthalic anhydride method. This method uses phthalic anhydride-protected β-alanyl chloride and L-histidine as substrates, first undergoing a synthetic reaction, followed by hydrazolysis to deprotect the compounds and ultimately produce L-carnosine. This is the most widely used method, but it is cumbersome, requires ice-salt bath conditions, and the hydrazolysis reaction uses highly toxic hydrazine to remove the protection of L-carnosine, potentially causing pollution or affecting product quality. Chemical synthesis, due to its stringent reaction conditions, is environmentally unfriendly and does not align with the principles of green production. Enzymatic synthesis, due to its eco-friendly and sustainable characteristics, has received widespread attention as a greener method for chemical synthesis. Several peptidases that can be used for enzymatic catalysis of L-carnosine synthesis have been reported, including carnosine synthase, β-amino peptidases such as BapA and DmpA, and hydrolases such as hCN1 and hCN2 derived from mammals and PepV and PepD derived from prokaryotes.

[0005] These enzymes synthesize carnosine using slightly different mechanisms. Carnosine synthase is a naturally occurring enzyme used in mammals to synthesize carnosine. It catalyzes the synthesis of carnosine from β-Ala and L-His cells using ATP. However, because it requires ATP, it is difficult to use directly for large-scale industrial applications. β-aminopeptidase and dipeptidase, on the other hand, are hydrolases that catalyze the reversible hydrolysis of carnosine. Therefore, most reported methods currently involve optimizing the reaction system to drive the reaction towards synthesis, thereby achieving carnosine synthesis.

[0006] The reverse synthesis of carnosine using hydrolytic enzymes has attracted widespread attention as a promising industrial method for carnosine synthesis because it does not require ATP consumption. SmPepD, a dipeptidase discovered from *Serratia marcescens*, is currently the highest-activity dipeptidase reported for carnosine synthesis, achieving a total carnosine yield of 60.2%. However, the enzyme activity in currently reported routes of this type remains relatively low, indicating significant room for improvement. Summary of the Invention

[0007] To address the shortcomings of existing technologies in the enzymatic reverse hydrolysis synthesis of L-carnosine catalyzed by dipeptidase, this invention provides a dipeptidase with high catalytic activity, high thermal stability, and good substrate tolerance, a dipeptidase mutant, a recombinant expression vector containing the dipeptidase or dipeptidase mutant gene, a recombinant expression transformant, a method for preparing the dipeptidase mutant, and a method for synthesizing L-carnosine using the dipeptidase or dipeptidase mutant.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] One of the technical solutions adopted in this invention:

[0010] A dipeptidase is provided, the amino acid sequence of which is shown in SEQ ID No.2, also known as BmPepD.

[0011] The dipeptidase preparation method provided by the present invention is as follows: it is isolated from Bacillus megaterium, or from a transformant that recombinantly expresses the carnosine hydrolase, or it can be artificially synthesized according to its amino acid sequence.

[0012] The second technical solution adopted in this invention:

[0013] A dipeptidase mutant is provided, which is a protein with dipeptidase activity obtained by substituting, deleting or adding one or more amino acids from the amino acid sequence shown in SEQ ID No. 2.

[0014] This invention utilizes bioinformatics-assisted methods for enzyme screening. Using SmPepD, a dipeptidase with the highest reported activity for carnosine synthesis, as a probe, a BLAST analysis was performed in the NCBI database. Sequences with 20%-80% sequence similarity to the probe were screened, and a phylogenetic tree was constructed after multiple sequence alignment. Twenty candidate enzymes were selected for heterologous expression in *E. coli*. The activity of soluble enzymes was verified by hydrolyzing and synthesizing carnosine using cell lysate. The recombinant expression vectors used for heterologous expression were obtained by PCR from laboratory-preserved strains or synthesized artificially by Genscript Biotech Co., Ltd. based on amino acid sequences from the database. Among them, the enzyme BmPepD, with database number WP_048020937.1 and amino acid sequence as shown in SEQ ID No. 2, derived from *Bacillus megaterium*, exhibits high carnosine hydrolysis and synthesis activity.

[0015] Based on this, site-directed saturation mutagenesis was used to perform directed evolutionary modification of BmPepD. AlphaFold was used to model the BmPepD sequence, and the model was docked with substrate molecules and metal ions to determine the substrate binding site and metal binding site. Sites were selected for modification and screening to obtain a batch of dipeptidase mutants with improved activity, also known as BmPepD mutants.

[0016] In one embodiment of the present invention, the amino acid sequence of the dipeptidase mutant is as follows:

[0017] (1) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with serine;

[0018] (2) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with glutamine;

[0019] (3) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with glycine;

[0020] (4) Replace the valine at position 457 of the amino acid sequence shown in SEQ ID No. 2 with glycine;

[0021] (5) Replace the valine at position 457 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0022] (6) Replace valine at position 457 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0023] (7) Replace the valine at position 81 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0024] (8) Replace valine at position 81 of the amino acid sequence shown in SEQ ID No. 2 with tryptophan;

[0025] (9) Replace threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with serine and valine at position 457 with glycine;

[0026] (10) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glycine and the valine at position 457 with isoleucine;

[0027] (11) Replace the valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with cysteine, and replace the valine at position 81 with cysteine;

[0028] (12) Replace valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with isoleucine and valine at position 81 with tryptophan;

[0029] (13) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with serine, the valine at position 457 with glycine, and the valine at position 81 with cysteine.

[0030] The third technical solution adopted in this invention:

[0031] A nucleic acid encoding a dipeptidase as described in technical solution 1 or a dipeptidase mutant as described in technical solution 1.

[0032] The nucleotide sequence of the nucleic acid encoding the dipeptidase as described in technical solution one is shown in SEQ ID No. 1.

[0033] The nucleic acid preparation method described in this invention is a conventional preparation method in the art, and the preparation method preferably includes:

[0034] Nucleic acid molecules encoding the dipeptidase BmPepD can be extracted from wild-type Bacillus megaterium cells; or nucleic acid molecules encoding the dipeptidase BmPepD and its mutants can be obtained through gene cloning technology; or nucleic acid molecules encoding the dipeptidase BmPepD and its mutants can be obtained through artificial full-sequence synthesis.

[0035] The method for obtaining the gene nucleic acid molecule encoding dipeptidase BmPepD and its mutants by gene cloning technology as described in this invention is as follows:

[0036] Forward primer 5'-CCG GAATTC ATGGTGCAAACAGTA-3',

[0037] Reverse primer 5'-CCG CTCGAG TTATATCTTATTTGCCTC-3',

[0038] The DNA sequences encoding the dipeptidase BmPepD and its mutants obtained in Technical Scheme 1 were amplified using polymerase chain reaction (PCR) technology.

[0039] PCR system (50μL): 2x Prime Star Mix 20μL, template plasmid approximately 100ng, forward and reverse primers 1.5μL each, ddH2O to bring the total to 50μL.

[0040] PCR reaction procedure: (1) 95℃ pre-denaturation for 3 min; (2) 98℃ denaturation for 10 s; (3) 60℃ annealing for 15 s; (4) 72℃ extension for 1.5 min; (5) 30 cycles of steps (2)-(4) are performed, and the final extension is performed at 72℃ for 10 min. Store at 4℃.

[0041] The fourth technical solution adopted in this invention:

[0042] A recombinant expression vector comprising the dipeptidase and mutant nucleic acid described in this invention is provided.

[0043] The dipeptidase gene nucleic acid sequence or mutant gene nucleic acid sequence of the present invention can be constructed by linking it to various suitable vectors using conventional methods in the art. The vector can be any conventional vector in the art, preferably a plasmid, more preferably plasmid pET-28a. The dipeptidase gene can be operatively linked downstream of a suitable regulatory sequence in the selected vector to achieve constitutive or inducible expression of the dipeptidase.

[0044] Preferably, as an example, the recombinant expression vector of the present invention can be prepared by the following method: the DNA fragment of the dipeptidase BmPepD gene sequence obtained by PCR amplification is digested with restriction endonucleases EcoRI and XhoI, and the empty vector plasmid pET-28a is also digested with restriction endonucleases EcoRI and XhoI. The digested gene DNA fragment and pET-28a plasmid are recovered by gel extraction and ligated using T4 DNA ligase to obtain the recombinant expression plasmid pET-28a-BmPepD containing the dipeptidase BmPepD gene.

[0045] The fifth technical solution adopted in this invention:

[0046] A recombinant expression transformant comprising the dipeptidase gene, dipeptidase mutant gene, or recombinant expression vector thereof described in this invention is provided. The recombinant expression transformant can be prepared by transforming the recombinant expression vector described in this invention into a host cell. The host cell can be any conventional host cell in the art, provided that it can stably replicate spontaneously using the recombinant expression vector and that the dipeptidase gene or dipeptidase mutant gene it carries can be effectively expressed.

[0047] The preferred host cell of this invention is Escherichia coli, more preferably E. coli BL21(DE3).

[0048] The sixth technical solution adopted in this invention:

[0049] A method for preparing a dipeptidase mutant is provided, comprising the following steps: culturing the recombinant expression transformant of the present invention to obtain the dipeptidase mutant. The culture medium used for culturing the recombinant expression transformant may be selected from conventional culture media in the art, provided that it allows the transformant to grow and produce the dipeptidase mutant of the present invention. The specific operations for culturing the transformant can be performed according to conventional operations in the art.

[0050] The recombinant Escherichia coli constructed using the above technical solution was inoculated with 20 μL of glycerol bacterial suspension into 4 mL of LB liquid medium (containing 50 μg / mL glycerol). -1 In test tubes containing kanamycin, the bacteria were cultured at 37°C with shaking at 200 rpm for 12 hours. Then, 1 mL of the bacterial culture was transferred to 100 mL of LB liquid medium (containing 50 μg / mL kanamycin).-1 In a 500 mL shake flask containing kanamycin, incubate at 37°C with shaking at 200 rpm for 3-4 hours until OD reaches zero. 600 Approximately 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.2 mM using a syringe to induce the expression of the target protein. After induction, the cells were cultured at 200 rpm in a shaker at 16°C for 24 h, and then collected by centrifugation (8000×g, 5 min). The cells were resuspended in 10 mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated at 30% power for 15 min in an ice-water bath to obtain the crude enzyme solution.

[0051] The protein in this invention contains a histidine tag at its N-terminus, therefore a Ni NTABeads6FF nickel column can be used for protein purification. The enzyme-protein purification buffers are as follows: Buffer A: 50 mM NaH₂PO₄ (pH 8.0), containing 300 mM NaCl and 20 mM imidazole; Buffer B: 50 mM NaH₂PO₄ (pH 8.0), containing 300 mM NaCl and 500 mM imidazole. The nickel column is washed with five column volumes of deionized water. The crude enzyme solution is loaded onto the nickel column, followed by washing with ten column volumes of Buffer A, and then eluting with fifteen to twenty column volumes of Buffer B. The eluent is collected. SDS-PAGE protein electrophoresis is used to verify the purification of the protein. Afterwards, the imidazole is removed by centrifugation through ultrafiltration, and the protein concentration is determined using Nanodrop. The purified protein is then aliquoted and stored at -80°C for later use.

[0052] The seventh technical solution adopted in this invention:

[0053] This invention provides the application of the dipeptidase or dipeptidase mutant described in this invention in the condensation of β-alanine and L-histidine to prepare L-carnosine. The application involves adding the dipeptidase or dipeptidase mutant described in this invention to a buffer solution containing β-alanine and L-histidine to catalyze the reverse hydrolysis of β-alanine and L-histidine to synthesize L-carnosine.

[0054] The buffer salt system of the buffer solution is not limited, as long as its pH range is between 6.0 and 9.0; the preferred buffer salt system is Tris-HCl with a pH of 8.0. The reaction temperature is 20-65℃, preferably 40℃. An appropriate amount of water-soluble or water-insoluble solvent may be added to the buffer solution. Other reaction conditions, such as substrate concentration and enzyme dosage, can be selected according to conventional conditions for this type of reaction in the art.

[0055] When the dipeptidase or dipeptidase mutant described in this invention is used as a catalyst, the crude enzyme solution containing the dipeptidase or dipeptidase mutant, or the pure dipeptidase or dipeptidase mutant, is immobilized onto a carrier to obtain an immobilized enzyme, which is then used as a catalyst.

[0056] Intermittent sampling was performed during the reaction, and analysis was conducted using liquid chromatography. A chiral crown ether column was used. A CR(+) 4.0 mm × 150 mm column was used as the liquid chromatography column, with a packing size of 5 μm. A perchloric acid (HClO4) aqueous solution with a pH of 1.0 was selected as the mobile phase, and the flow rate was 0.3 mL / min. -1 The detection was performed at a wavelength of 210 nm.

[0057] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0058] The dipeptidase or dipeptidase mutant described in this invention exhibits high activity, good thermal stability, and strong substrate tolerance. This dipeptidase or dipeptidase mutant can directly catalyze the condensation of β-alanine and L-histidine to synthesize L-carnosine, avoiding the substrate protection and deprotection steps required in chemical synthesis methods, and is more environmentally friendly. Compared to other reported L-carnosine synthesis methods, the method of this invention for preparing L-carnosine offers advantages such as mild reaction conditions, no need for substrate pretreatment, environmentally friendly process, and ease of industrial scale-up, demonstrating excellent prospects for industrial application development. Detailed Implementation

[0059] The present invention will now be described in detail with reference to specific embodiments.

[0060] Example 1: Screening of dipeptidase.

[0061] Using the dipeptidase SmPepD reported in the literature as a probe, BLAST was performed in the NCBI database. Sequences with 20-80% similarity to the probe were screened, and phylogenetic trees were constructed after multiple sequence alignment. Twenty candidate enzymes were selected, and corresponding recombinant plasmids of the dipeptidases were obtained by PCR or artificial synthesis. These plasmids were then transformed into *E. coli* to obtain the corresponding recombinant strains. The strains were inoculated into test tubes containing 4 mL of culture medium and cultured at 37°C with shaking at 200 rpm for 12 h. 1 mL of the bacterial culture was then transferred to a shake flask containing 100 mL of LB medium for induction culture. After 24 h, the bacterial cells were collected by centrifugation. The bacterial cells were resuspended in 10 mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated in an ice-water bath at 30% power for 15 min to obtain the crude enzyme solution.

[0062] The activity of the crude enzyme solution of the novel enzyme against L-carnosine was verified by a biocatalytic reaction. The total reaction volume was 0.2 mL, containing a final concentration of 100 mM L-carnosine, 50 mM Tris-HCl buffer (pH 8.0), and an appropriate concentration of crude enzyme. The reaction was carried out at 30 °C and 1000 rpm for 2 h with shaking. After the reaction was completed, 10 μL of the reaction solution was mixed with 990 μL of perchloric acid to terminate the reaction. After centrifugation at 13000 × g for 2 min, the mixture was filtered through a mixed cellulose membrane and analyzed by high performance liquid chromatography (HPLC). The activity of the screened novel enzyme against L-carnosine was determined based on the reaction results, which are shown in Table 1.

[0063] Table 1 Comparison of carnosine hydrolysis activities of different strains

[0064]

[0065]

[0066] Note: "SmPepD" is the probe sequence; "++" indicates that the hydrolysis activity is higher than that of the probe; "+" indicates that the hydrolysis activity is lower than that of the probe, but there is hydrolysis activity for L-carnosine; "-" indicates that no hydrolysis activity for L-carnosine was detected.

[0067] Example 2: Preparation of recombinant dipeptidase BmPepD

[0068] With forward primer 5'-CCG GAATTC ATGGTGCAAACAGTA-3', reverse primer 5'-CCG CTCGAG The dipeptidase BmPepD encoding gene screened in Example 1 was amplified using polymerase chain reaction (PCR). The amplified encoding DNA fragment was then digested with restriction endonucleases EcoRI and XhoI, and ligated with the pET-28a plasmid that had also been digested with EcoRI and XhoI to obtain the recombinant plasmid pET-28a-BmPepD.

[0069] The obtained recombinant plasmid was transformed into *E. coli* BL21. The constructed recombinant strain was inoculated into a test tube containing 4 mL of medium and cultured with shaking at 37°C for 12 h. 1 mL of the bacterial culture was then transferred to a shake flask containing 100 mL of LB medium and cultured at 37°C for 3 h. IPTG was added, and the culture was induced at 16°C for 24 h. The bacterial cells were collected by centrifugation. The bacterial cells were resuspended in 10 mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated at 30% power for 15 min in an ice-water bath to obtain crude enzyme solution. The nickel column was washed with five column volumes of deionized water. The crude enzyme solution was loaded onto the nickel column, followed by washing with ten column volumes of solution A, and then eluting with fifteen to twenty column volumes of solution B. The eluent was collected. Imidazole was removed by ultrafiltration, aliquoted, and glycerol was added before storage at -80°C. The synthetic activity of the purified BmPepD enzyme was 16.7 U mg. -1 .

[0070] Example 3: Molecular modification of recombinant dipeptidase BmPepD

[0071] A structural model of BmPepD was constructed using AlphaFold, and it was docked with substrate molecules and metal ions at substrate-binding or metal-binding sites. Five amino acid residues were selected as targets for site-directed saturation mutagenesis. Site-directed saturation mutagenesis was performed on the target sites using designed NNK degenerate codons. The mutants were cultured in deep-well plates, incubated overnight at 37°C with shaking, and then 50 μL was transferred to a secondary deep-well plate containing 600 μL of the mutant. After incubation at 37°C with shaking for 3 h, IPTG was added to a final concentration of 0.2 mM, and the plate was incubated at 16°C for 24 h. The cells were then centrifuged, lysozyme was added for disruption, and 100 μL of the enzyme solution was mixed with 100 μL of a substrate solution containing β-alanine (2 M) and L-histidine (200 mM) for the synthesis reaction. The reaction solution was analyzed by liquid chromatography for preliminary screening, revealing that twelve mutants showed increased activity compared to the parent BmPepD. The L-carnosine synthesis activity of the purified enzymes was measured after purification of these twelve mutants, and the results are shown in Table 2. The sequence numbers in Table 2 correspond to a series of sequences following Table 2. In the activity column, compared with the parent BmPepD, one plus sign "+" indicates that the mutant activity is increased by 1.2-5 times; two plus signs "++" indicate that the mutant activity is increased by 5-10 times; and three plus signs "+++" indicate that the mutant activity is increased by more than 10 times.

[0072] Table 2. List of dipeptidase BmPepD mutant sequences and corresponding improvements in L-carnosine synthesis activity.

[0073]

[0074]

[0075] The amino acid sequences of the BmPepD mutants corresponding to the labels in the table are as follows:

[0076] (1) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with serine;

[0077] (2) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with glutamine;

[0078] (3) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with glycine;

[0079] (4) Replace the valine at position 457 of the amino acid sequence shown in SEQ ID No. 2 with glycine;

[0080] (5) Replace the valine at position 457 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0081] (6) Replace valine at position 457 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0082] (7) Replace the valine at position 81 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0083] (8) Replace valine at position 81 of the amino acid sequence shown in SEQ ID No. 2 with tryptophan;

[0084] (9) Replace threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with serine and valine at position 457 with glycine;

[0085] (10) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with glycine and the valine at position 457 with isoleucine;

[0086] (11) Replace the valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with cysteine, and replace the valine at position 81 with cysteine;

[0087] (12) Replace valine at position 457 of the amino acid sequence shown in SEQ ID No.2 with isoleucine and valine at position 81 with tryptophan;

[0088] (13) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No.2 with serine, the valine at position 457 with glycine, and the valine at position 81 with cysteine;

[0089] Example 4: Dipeptidase mutant BmPepD M13 Preparation

[0090] Extract the recombinant plasmid pET-28a-BmPepD obtained as in Example 3. M13 The recombinant strain was transformed into *E. coli* BL21. The constructed recombinant strain was inoculated into a test tube containing 4 mL of culture medium and cultured with shaking at 37°C for 12 h. 1 mL of the bacterial culture was then transferred to a shake flask containing 100 mL of LB medium and cultured at 37°C for 3 h. IPTG was then added, and the culture was induced at 16°C for 24 h. The cells were then collected by centrifugation. The cells were resuspended in 10 mL of Tris-HCl buffer (100 mM, pH 8.0) and sonicated at 30% power for 15 min in an ice-water bath to obtain a crude enzyme solution. The nickel column was washed with five column volumes of deionized water. The crude enzyme solution was loaded onto the nickel column, followed by washing with ten column volumes of solution A, and then eluting with fifteen to twenty column volumes of solution B. The eluent was collected. Imidazole was removed by centrifugation through an ultrafiltration tube. The eluent was aliquoted, added to glycerol, and stored at -80°C for later use.

[0091] Example 5: Effect of temperature on dipeptidase BmPepD activity

[0092] At different temperatures (20-65℃), 100 μL of BmPepD was taken. M13 The purified enzyme (enzyme protein concentration 0.05 mg / mL) was added to 100 μL of Tris-HCl substrate solution (pH 8.0) containing β-alanine (4 M) and L-histidine (200 mM) for the synthesis reaction. The reaction was carried out with shaking for 15 min, and the results were analyzed by HPLC. The results are shown in Table 3. The enzyme exhibited the highest activity at 50 °C, and the enzyme activity at this temperature was defined as 100%. The relative activities at other temperatures were calculated. Before 50 °C, the enzyme activity increased with increasing temperature; above 50 °C, the enzyme activity decreased rapidly.

[0093] Table 3. Activity of dipeptidase BmPepD at different temperatures

[0094]

[0095] Example 6: Effect of pH on the activity of dipeptidase BmPepD

[0096] At an activity assay temperature of 40℃, 100 μL of pure enzyme (enzyme protein concentration 0.05 mg / mL) was added to 100 μL of substrate solution containing β-alanine (4 M), L-histidine (200 mM), PBS buffer (pH 6.0-7.5), or Tris-HCl buffer (pH 7.5-9.0) for the synthesis reaction. The reaction was carried out with shaking for 15 min, and the activity was analyzed by HPLC. The results are shown in Table 4. The enzyme exhibited the highest relative activity in Tris-HCl buffer at pH 8.0, defined as 100%. The relative activities at other pH values ​​were calculated.

[0097] Table 4. Activity of dipeptidase BmPepD at different pH values.

[0098]

[0099]

[0100] Example 7: 10 mL scale-up reaction of L-carnosine synthesis catalyzed by dipeptidase BmPepD and its mutant M13.

[0101] The synthesis of carnosine was carried out in a 50 mL Erlenmeyer flask with a total volume of 10 mL, containing saturated β-Ala, 0.2 M L-His, and 50 mM Tris-HCl buffer (pH 8.0), and 0.05 mg / mL BmPepD or its mutant purified enzyme. The reaction was conducted at 40 °C and 200 rpm, with intermittent sampling and HPLC analysis. After 8 h of reaction, using BmPepD as a catalyst, 48.2 mM L-carnosine was produced, with a specific yield of 436 g of enzyme. Car g catalyst -1 When using mutant M13, 61.3 mM of L-carnosine was produced, and the specific yield of the enzyme reached 554 g. Car g catalyst -1 .

[0102] The sequences involved in this invention are as follows:

[0103] SEQ ID No.1

[0104]

[0105] SEQ ID No.2

[0106] MVQTVNELIKHPVFYFFNEISAIPRESGNEKEISNYLVSFAKERSLEVIQDEALNVIIKKPATKGYEHAPAIILQGHMDMVCELNKGTVHDFEKDPLQLRIVEDMLYANGTTLGADNGIAV AYALALLDAQNIAHPSLEVVITTEEETTMGGAIAVNPAYFEGKIFINLDTEEDGKLLVSSAGGVKGVLRIPINWESSSNNSETYSLSIGGLRGGHSGMEIDKERGNANKLLGRVLYDLQQEL PYSLSSISGGLKSNAIPRESEAILSVEPSEVGKLENKIREWNEIVKNELQAADPSVYVKINKFSSVEKCFTRETTERIVQAIMLTPNGVQSMSMNIEGLVESSTNLGVITTTESEVVFQNE IRSSVKSLKEKIVSQVRILAQVVGGRVETKGNYPEWAYNGDSKIRELCKKVYKEKYGEEAEIIAIHAGIECGIFLEKIPGLDAISLGPDMYDVHTPDEHLSIPSTLKTWEYLLAVLKEANKI

[0107] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A dipeptidase mutant, characterized in that, The amino acid sequence of the dipeptidase mutant is shown below: (1) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with serine; (2) Replace threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with serine and valine at position 457 with glycine; (3) Replace the threonine at position 171 of the amino acid sequence shown in SEQ ID No. 2 with serine, the valine at position 457 with glycine, and the valine at position 81 with cysteine.

2. An isolated nucleic acid, characterized in that, The nucleic acid encodes the dipeptidase mutant as described in claim 1.

3. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 2.

4. A recombinant expression transformant, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. A method for preparing a dipeptidase mutant as described in claim 1, characterized in that, The method includes the following steps: culturing the recombinant expression transformant as described in claim 4, and isolating and obtaining the dipeptidase mutant.

6. The use of the dipeptidase mutant as described in claim 1 in the synthesis of L-carnosine.

7. The application according to claim 6, characterized in that, Includes the following steps: Using the dipeptidase mutant as described in claim 1, the reverse hydrolysis of β-alanine and L-histidine is catalyzed to generate L-carnosine, and then the product L-carnosine is separated and extracted from the reaction mixture.

8. The application according to claim 7, characterized in that, The reaction temperature was 20-65°C, the reaction pH was 6.0-9.0, the concentration of the substrate β-alanine was 200 mM to saturation, and the concentration of the substrate L-histidine was 100-200 mM.

9. The application according to claim 6, characterized in that, When the dipeptidase mutant is used as a catalyst, the crude enzyme solution containing the dipeptidase mutant or the pure dipeptidase mutant is immobilized on a carrier to obtain an immobilized enzyme, which is then used as a catalyst.

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