Aminoacyl-histidine dipeptidase mutants and uses thereof

Abstract

The present application relates to an aminoacyl histidine dipeptidase mutant and application thereof. The present application relates to the field of enzyme catalysis, and aims to provide an aminoacyl histidine dipeptidase mutant with improved enzyme activity. Specifically, the present application provides an aminoacyl histidine dipeptidase mutant, which is any one of the following (1) or (2): (1) an amino acid sequence as shown in SEQ ID NO: 4, 6 or 8; (2) an amino acid sequence with at least 98% homology to the amino acid sequence shown in (1) and having the activity of catalyzing β-alanine and L-histidine to generate L-carnosine.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis, and more specifically, to an aminoacylhistidine dipeptidase mutant with enhanced enzyme activity and its applications. Background Technology

[0002] L-Carnosine (β-alanyl-L-histidine) is a naturally occurring dipeptide composed of β-alanine and L-histidine linked by peptide bonds. It possesses various biological activities, including antioxidant, anti-aging, anti-fatigue, and immunomodulatory effects, and has broad application prospects in medicine, food, and cosmetics. It is widely present in vital tissues such as muscles, brain, and heart, playing a crucial role in maintaining normal physiological functions. In the pharmaceutical field, L-carnosine can be used to treat various diseases, such as hypertension, heart disease, age-related cataracts, and ulcers, and also has adjuvant therapeutic effects in anti-tumor treatment and wound healing. In the food industry, it can be used as a natural antioxidant and food additive to extend shelf life and improve food quality. In the cosmetics industry, it can delay skin aging and reduce wrinkle formation.

[0003] Currently, the main methods for producing L-carnosine are chemical synthesis and biosynthesis. Chemical synthesis has many drawbacks, such as complicated synthesis steps, the need for complex protection and deprotection of the active groups of the substrate, harsh reaction conditions, often requiring high temperature, high pressure, strong acid or strong alkali, resulting in low yield, high cost, high pollution, and difficulty in guaranteeing product purity.

[0004] Biosynthesis offers significant advantages, with mild reaction conditions typically conducted at room temperature and pressure in an aqueous phase, adhering to green chemistry principles and producing products with high optical purity. Enzymatic synthesis is a crucial biosynthetic route, but existing technologies face several challenges. For instance, the synthesis of L-carnosine catalyzed by aminopeptidase requires activation of the carboxyl group of β-alanine or the amino group of L-histidine, a complex process that introduces environmental pollution and increased costs. β-amino acid ester acyltransferases exhibit poor substrate selectivity, leading to numerous impurities and hindering industrialization. While the reverse hydrolysis reaction catalyzed by carnosine hydrolases does not require substrate activation, the reported types of carnosine hydrolases are limited and have low activity, resulting in long reaction times, low product concentrations, and low substrate conversion rates, thus limiting the efficient synthesis of L-carnosine and failing to meet industrial demands. The dipeptidase encoded by the pepD gene can catalyze the synthesis of L-carnosine from L-histidine and β-alanine; however, current technologies using PepD-catalyzed reverse hydrolysis yield low amounts of L-carnosine. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, the technical problem to be solved by the present invention is to provide an aminoacylhistidine dipeptidase mutant, which has high activity and can be used for efficient catalytic synthesis of L-carnosine, and has the advantages of low raw material cost, simple operation and environmental friendliness.

[0006] Furthermore, the present invention also provides the use of the aminoacylhistidine dipeptidase mutant in the production of L-carnosine or its derivatives.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] 1. An aminoacylhistidine dipeptidase mutant, characterized in that the mutant is any one of (1) or (2) below:

[0009] (1) An amino acid sequence as shown in SEQ ID NO:4, 6 or 8;

[0010] (2) An amino acid sequence that has at least 98% homology with the amino acid sequence shown in (1) and has aminoacylhistidine dipeptidase activity that catalyzes the formation of L-carnosine from β-alanine and L-histidine.

[0011] 2. The aminoacylhistidine dipeptidase mutant according to item 1, wherein the mutant is obtained by introducing an amino acid mutation into a wild-type aminoacylhistidine dipeptidase derived from *Histophilus somni*. 3. A nucleic acid molecule encoding the aminoacylhistidine dipeptidase mutant according to item 1 or 2.

[0012] 4. A recombinant expression vector comprising the nucleic acid molecules described in Project 3;

[0013] Optionally, the recombinant expression vector is an expression vector suitable for prokaryotic microorganisms.

[0014] 5. A host cell comprising the nucleic acid molecule described in item 3 or the recombinant expression vector described in item 4, and capable of expressing the aminoacylhistidine dipeptidase mutant;

[0015] Optionally, the host cell is Escherichia coli.

[0016] 6. The use of the aminoacylhistidine dipeptidase mutant described in Project 1 or 2, the nucleic acid molecule described in Project 3, the recombinant expression vector described in Project 4, or the host cell described in Project 5 in improving the enzyme activity of aminoacylhistidine dipeptidase or preparing L-carnosine or L-carnosine derivatives.

[0017] 7. A method for preparing L-carnosine, characterized in that the aminoacylhistidine dipeptidase mutant described in item 1 or 2 or the host cell expressing the aminoacylhistidine dipeptidase mutant described in item 5 is added to the reaction system.

[0018] 8. The preparation method according to Project 7, wherein the reaction system further includes substrates β-alanine and L-histidine.

[0019] Beneficial effects

[0020] To provide a one-step method for synthesizing L-carnosine, this invention, through structural studies of PepD, performs a series of site-directed mutagenesis on PepD, transfers the mutated pepD gene into host cells, and screens the cells to obtain a PepD mutant with enhanced enzyme activity. This mutant exhibits enhanced enzyme activity and has broad prospects for industrial development and application. Attached Figure Description

[0021] Figure 1 The results of molecular docking between aminoacylhistidine dipeptidase and L-carnosine were shown. Detailed Implementation

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] In this article, L-carnosine (β-alanyl-His), also known as β-alanyl-L-histidine, is a dipeptide composed of β-alanine and L-histidine. It possesses various biological activities, including antioxidant, anti-aging, anti-fatigue, and immunomodulatory effects. Its chemical formula is shown below:

[0024] .

[0025] In this paper, aminoacylhistidine dipeptidase (PepD) is a type of metallohydrolase widely found in bacteria, belonging to the M20 family of peptidases. Under physiological conditions, PepD can catalyze the hydrolysis of L-aminoacyl-L-histidine dipeptides containing α-peptide bonds, breaking them down into the corresponding free amino acids.

[0026] Carnosine (β-alanyl-L-histidine) is a dipeptide containing a β-peptide bond. PepD derived from wild-type *Histophilus somni* exhibits weak hydrolytic activity against it. Enzymatic reactions are thermodynamically reversible. Through the principle of "reverse hydrolysis," by significantly increasing the concentration of the substrates (β-alanine and L-histidine), the chemical equilibrium shifts towards the condensation and formation of the dipeptide. At this point, driven by the high substrate concentration, PepD catalyzes the dehydration condensation reaction, thereby achieving the synthesis of L-carnosine. This is a conventional and mature technique in enzyme engineering for peptide synthesis using hydrolases.

[0027] In this invention, by mutating PepD and optimizing reaction conditions, its hydrolytic enzyme activity is inhibited while its activity in catalyzing dipeptide synthesis is enhanced, so that the mutant enzyme exhibits higher catalytic efficiency and yield in L-carnosine synthesis.

[0028] In this paper, the wild-type aminoacylhistidine dipeptidase PepD is derived from Haemophilus somni, and its amino acid sequence is shown in SEQ ID NO: 2.

[0029] In this document, the aminoacylhistidine dipeptidase PepD mutant, relative to the aminoacylhistidine dipeptidase PepD shown in SEQ ID NO: 2, has the following amino acid substitutions: E145D and / or E171A.

[0030] Specifically, when the amino acid is replaced with E145D, the amino acid sequence of the mutant is shown in SEQ ID NO: 4; when the amino acid is replaced with E171A, the amino acid sequence of the mutant is shown in SEQ ID NO: 6; and when the amino acid is replaced with both E145D and E171A, the amino acid sequence of the mutant is shown in SEQ ID NO: 8.

[0031] In this document, the aminoacylhistidine dipeptidase PepD mutant has an amino acid sequence as shown in SEQ ID NO:4, 6 or 8.

[0032] In this document, the aminoacylhistidine dipeptidase PepD mutant may also be an amino acid sequence having at least 98% homology (e.g., 98% or 99% homology) with SEQ ID NO:4, 6 or 8, and having aminoacylhistidine dipeptidase activity that catalyzes the production of L-carnosine from β-alanine and L-histidine. Preferably, the mutant is obtained by introducing amino acid mutations into wild-type aminoacylhistidine dipeptidase derived from Haemophilus somnius, and the amino acid mutations include E145D and / or E171A.

[0033] The presence of aminoacylhistidine dipeptidase activity refers to the ability to catalyze the synthesis of the dipeptide L-carnosine from the substrates β-alanine and L-histidine.

[0034] The aminoacylhistidine dipeptidase PepD mutant can be prepared by DNA recombination technology, specifically including: (1) gene cloning, cloning the gene (cDNA) encoding the enzyme from the target organism, or directly obtaining it through commercial synthesis. (2) vector construction: inserting the target gene into a suitable expression vector (plasmid) to construct a recombinant expression vector; wherein, to facilitate the expression of the target gene, the vector may preferably carry a strong promoter (such as T7, lac, AOX1), a selection marker (such as an antibiotic resistance gene) and a protein tag (such as His-tag, GST-tag, for subsequent purification). (3) construction of recombinant engineered bacteria / cells: transforming or transfecting the recombinant expression vector into prokaryotic or eukaryotic cells. (4) expression: fermenting and culturing the recombinant engineered bacteria / cells under appropriate conditions and inducing expression to obtain recombinant cells expressing the aminoacylhistidine dipeptidase PepD mutant. To obtain the enzyme preparation further, the method may further include: (5) cleaving the recombinant cells to obtain a crude enzyme solution containing the aminoacylhistidine dipeptidase PepD mutant, which can be directly used for the preparation of L-carnosine without further purification. To obtain an enzyme preparation with higher purity to optimize the reaction effect, the method may further include: (6) separating and purifying the crude enzyme solution to obtain the purified aminoacylhistidine dipeptidase PepD mutant.

[0035] In this paper, the recombinant engineered bacteria / cells may be selected from prokaryotic or eukaryotic systems. Prokaryotic systems include Escherichia coli, Bacillus subtilis, and Lactococcus lactis; eukaryotic systems include yeasts (such as Pichia pastoris and Saccharomyces cerevisiae) or insect / mammalian cells.

[0036] This paper provides the application of the above-mentioned aminoacylhistidine dipeptidase pepD mutant or host cells expressing the above mutant in improving the enzyme activity of aminoacylhistidine dipeptidase pepD or in the preparation of L-carnosine or L-carnosine derivatives.

[0037] In this invention, the aminoacylhistidine dipeptidase PepD mutant is obtained by introducing specific amino acid substitutions on the basis of the wild-type aminoacylhistidine dipeptidase pepD shown in SEQ ID NO: 2, wherein the amino acid substitutions are: E145D and / or E171A.

[0038] The host cell described in this invention refers to a microbial cell used to express the above-mentioned aminoacylhistidine dipeptidase PepD mutant, which is obtained by introducing a nucleic acid molecule encoding the mutant (or a recombinant expression vector containing the nucleic acid molecule).

[0039] In this invention, the host cell is preferably a prokaryotic host cell, such as Escherichia coli strains like BL21 (DE3) and Rosetta (DE3), which have advantages such as rapid growth, high expression levels, and ease of operation. Other prokaryotic hosts may also be used, including but not limited to Bacillus subtilis, Lactococcus lactis, and Corynebacterium glutamicum.

[0040] When used to prepare L-carnosine, the aminoacylhistidine dipeptidase pepD mutant or host cell of the present invention can be used in any of the following forms:

[0041] 1) Whole-cell catalyst: After fermentation culture, the cell bodies are collected and directly added to the reaction system as a catalyst without the need for crushing and purification, making the operation simple.

[0042] 2) Crude enzyme solution: After the bacterial cells are broken and centrifuged, the supernatant is discarded and the precipitate is resuspended to obtain the crude enzyme solution (or enzyme solution) containing the aminoacylhistidine dipeptidase pepD mutant.

[0043] 3) Purified enzyme: The crude enzyme solution is purified by chromatography to obtain high-purity enzyme protein, which is suitable for scenarios where high product purity is required or enzyme immobilization is required.

[0044] 4) Immobilized enzymes / cells: Purified enzymes or cells are immobilized on a carrier (such as sodium alginate, chitosan, resin, etc.) to enable reuse and continuous production.

[0045] This invention provides a method for preparing L-carnosine, which involves adding the aforementioned aminoacylhistidine dipeptidase pepD mutant or host cells expressing this mutant to a reaction system to catalyze the reaction of β-alanine with L-histidine to generate L-carnosine. Specific steps include:

[0046] (1) Preparation of substrate solution: Dissolve β-alanine and L-histidine in buffer solution or water, and adjust the pH to a suitable range. The concentration of β-alanine is 5.0-7.5 mol / L, for example 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 mol / L, preferably 6.5 mol / L; the concentration of L-histidine is 0.10-0.25 mol / L, for example 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.25 mol / L, preferably 0.18 mol / L; the pH is 7-10, preferably pH 8.

[0047] (2) Preparation of enzyme solution: After measuring the OD600 of the bacterial culture obtained by fermenting and inducing expression of PepD protein from host cells, take 1 mL of bacterial culture and centrifuge at 10000 rpm for 1 min, discard the supernatant and add 1 mL of PBS buffer to resuspend, thus obtaining the enzyme solution; all experimental groups used bacterial cultures with the same OD600 to prepare enzyme solutions to ensure the consistency of enzyme amount in the reaction system.

[0048] (3) Catalytic reaction: Take the substrate solution obtained in step (1), add the whole-cell enzyme solution obtained in step (2), and react on a constant temperature shaker (e.g., 220 rpm). Set up a blank control (without enzyme in the substrate solution) and treat it under the same conditions.

[0049] The volume ratio of substrate solution to enzyme solution is (8-12):1, for example, 10:1. For example, 1 mL of substrate solution is added to 0.1 mL of enzyme solution. The enzyme reaction conditions are a temperature of 35-40℃, for example, 35, 36, 37, 38, 39, or 40℃, preferably 37℃; and a reaction time of 15-25 minutes, preferably 20 minutes.

[0050] (4) Ending the reaction: The reaction is terminated by heating, acid or alkali treatment (e.g., inactivating the sample by placing it in a boiling water bath to stop the enzyme reaction).

[0051] After the reaction is complete, L-carnosine is isolated and purified or the yield of L-carnosine is measured.

[0052] The determination of the yield of the product L-carnosine involved centrifugation of the reaction solution (12000 rpm, 5 min), followed by HPLC analysis of the supernatant. Specific HPLC conditions were as follows:

[0053] Chromatographic column: Yuexu Ultimate HILIC Amphion II ion column;

[0054] Mobile phase: Acetonitrile : (0.05 mol / L potassium dihydrogen phosphate aqueous solution, pH adjusted to 3.0 with phosphoric acid) = 75 : 25 (volume ratio)

[0055] The mobile phase preparation steps are as follows: (1) Prepare a 0.05 mol / L potassium dihydrogen phosphate aqueous solution and adjust the pH to 3.0 ± 0.1 with phosphoric acid; (2) Mix the solution prepared in (1) with acetonitrile at a volume ratio of 25:75, and then filter the mixture with a 0.45 μm organic phase filter membrane. The filtrate is degassed by ultrasonication.

[0056] (3) Liquid phase conditions: column temperature: 35℃; wavelength: 206nm; flow rate: 1.5mL / min; injection volume: 10uL.

[0057] In this paper, the composition of LB liquid medium is as follows: per 1L of medium, it includes: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, deionized water to a final volume of 1L, and pH adjusted to 7.0-7.5 with NaOH.

[0058] In this study, the composition of the antibiotic-free LB medium was: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and pH adjusted to 7.0-7.4.

[0059] Example 1: Obtaining aminoacylhistidine dipeptidase and aminoacylhistidine dipeptidase mutants

[0060] 1. Bioinformatics analysis of pepD gene structure - AutoDock Vina molecular docking

[0061] The aminoacylhistidine dipeptidase pepD (NCBI: WP_011608162.1, nucleotide sequence shown in SEQ ID NO: 1, amino acid sequence shown in SEQ ID NO: 2) from Haemophilus neonatorum was used as a substrate for molecular docking in AutoDock Vina to observe its interaction. AutoDock Vina is a conventional molecular simulation software commonly used to study the interaction of complexes between biomacromolecules (e.g., proteins) and small molecules (e.g., ligands). The results are as follows: Figure 1As shown, L-carnosine forms hydrogen bonds with residues at positions E145 and E171 of pepD, and these residues are key residues in the active pocket. Therefore, to obtain aminoacylhistidine dipeptidase pepD with higher activity, the inventors performed point mutations on the above key residues and obtained aminoacylhistidine dipeptidase pepD mutants with potentially improved activity through a series of screenings, namely pepD-E145D, pepD-E171A, and pepD-E145D-E171A. Based on this, the required gene coding sequences (pepD original, pepD-E145D, pepD-E171A, pepD-E145D-E171A) were designed. The nucleotide sequence of the mutant pepD-E145D is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; the nucleotide sequence of the mutant pepD-E171A is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; the nucleotide sequence of the combined mutant pepD-E145D-E171A is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. As shown in Figure 8, the gene sequences of the wild-type pepD and its mutants designed above were given to Hongxun Biosynthesis. The synthesized gene was linked with the pet28a vector (purchased from Nanjing Genscript Biotech Co., Ltd.) to form an expression vector. The recombinant expression vectors were named pET28a-pepD original, pET28a-pepD-E145D, pET28a-pepD-E171A, and pET28a-pepD-E145D-E171A, respectively.

[0062] 2. Construction of recombinant bacteria

[0063] (1) Preparation of competent Escherichia coli BL21 cells: Freshly activated single colonies of E. coli BL21 (purchased from Nanjing Genscript Biotech Co., Ltd.) were picked from LB plates and inoculated into 5 ml of LB liquid medium. The cells were cultured at 37°C with shaking for about 12 h until the late logarithmic growth phase. The bacterial suspension was then inoculated into 100 ml of LB liquid medium at a ratio of 1:100 and cultured at 37°C with shaking for 2-3 h until the OD600 = 0.5. The culture was transferred to centrifuge tubes, placed on ice for 10 min, and then centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was discarded, and the cells were gently resuspended in 10 ml of pre-cooled 0.05 mol / L CaCl2 solution. The cells were placed on ice for 15-30 min and then centrifuged at 3000 rpm for 10 min at 4°C. Discard the supernatant, add 4 ml of pre-cooled 0.05 mol / L CaCl2 solution containing 15% glycerol, gently suspend the cells, and place on ice for a few minutes to obtain a competent cell suspension. Aliquot the competent cells into 200 μl portions and store at -80°C.

[0064] (2) Plasmid transformation

[0065] Remove competent cells from the -80℃ freezer and thaw them on ice. Add the original, pET28a-pepD, pET28a-pepD-E145D, pET28a-pepD-E171A, and pET28a-pepD-E145D-E171A expression vectors obtained in step 1, respectively, and incubate on ice for 30 min to allow DNA molecules to fully adsorb onto the surface of the competent cells. Simultaneously, turn on the water bath and adjust it to 42℃. After the ice bath, remove the competent cells with adsorbed DNA from the ice and heat shock them at 42℃ for 90 s. Then quickly incubate on ice for about 1 min. Add about 800 μL of antibiotic-free LB (which can be pre-prepared at 37℃), and then incubate at 37℃ and 220–250 rpm for 40 min. After resuscitation, cells were collected by centrifugation at 4000 rpm for 5 min, then resuspended in about 100 μl of antibiotic-free LB agar, plated onto the corresponding selective LB plates, cultured overnight, and screened to obtain BL21 strains that successfully introduced the expression vector.

[0066] Among them, the BL21 strain that successfully introduced the original plasmid pET28a-pepD was named CAR001, the BL21 strain that successfully introduced the plasmid pET28a-pepD-E145D was named CAR002, the BL21 strain that successfully introduced the plasmid pET28a-pepD-E171A was named CAR003, and the BL21 strain that successfully introduced the plasmid pET28a-pepD-E145D-E171A was named CAR004.

[0067] 3. Fermentation culture for the production of aminoacylhistidine dipeptidase

[0068] Seed culture: CAR001, CAR002, CAR003 and CAR004 strains were inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C to prepare seed culture.

[0069] Expanded culture and induced expression: The seed culture was transferred at an inoculum of 5% to a 1L shake flask containing 100mL LB medium, and IPTG was added to make the final concentration of IPTG 0.2mmol / L. The culture was induced at 30℃ for 16h to obtain the fermentation broth.

[0070] Example 2: Aminoacylhistidine dipeptidase and aminoacylhistidine dipeptidase mutants for enzymatic production of carnosine

[0071] 1. Enzyme activity assay of aminoacylhistidine dipeptidase:

[0072] (1) Preparation of substrate solution: Weigh 11.57 g β-alanine and 0.558 g L-histidine, put them into a 50 mL centrifuge tube, add about 15 mL of water to dissolve them, and adjust the pH to 8 using 10% NaOH. Make up the volume to 20 mL.

[0073] (2) Prepare enzyme solution: Take the above fermentation broth, measure the OD600 value, adjust the bacterial broth expressing different PepD mutants to the same OD600 value, take 1 mL, centrifuge at 10000 rpm for 1 min, add 1 mL PBS to resuspend, and obtain enzyme solution.

[0074] (3) Perform the enzyme reaction: Take 1 mL of substrate solution, add 0.1 mL of enzyme solution, and react at 220 rpm for 30 min on a constant temperature shaker at 37℃. Set up a blank control (without enzyme in the substrate solution) and treat it under the same conditions.

[0075] (4) End the reaction: After the reaction is complete, immediately put the sample into a boiling water bath for 5 minutes to inactivate it and stop the enzyme reaction.

[0076] (5) Determination of the concentration of L-carnosine: The carnosine yield of wild-type strain CAR001 was 1.55 g / L, the carnosine yield of mutant strain CAR002 reached 1.97 g / L, the carnosine yield of mutant strain CAR003 reached 2.05 g / L, and the carnosine yield of mutant strain CAR004 reached 3.83 g / L. Under the same culture conditions, the yield of CAR004 was 2.47 times that of wild-type strain CAR001, which laid a good foundation for the large-scale industrial production of carnosine by green enzymatic method.

[0077] SEQ ID NO: 1, nucleotide sequence of wild-type aminoacylhistidine dipeptidase PepD

[0078]

[0079] SEQ ID NO: 2, Amino acid sequence of wild-type aminoacylhistidine dipeptidase PepD

[0080] MSDLQSLQPKLLWQWFDQICAIPHPSYKEEQLAQFIINWAKTKGFFAERDEVGNVLIRKPATVGMENRKPVVLQAHLDMVPQANEGTNHNFDQDPILPYIDGDWVKAKGTTLGADNGIGMA SALAVLESNDIAHPELEVLLTMTEERGMEGAIGLRPNWLRSEILINTDTEENGEIYIGCAGGENADLELPIEYQVNNFEHCYQVVLKGLRGGHSGVDIHTGRANAIKVLLRFLAELQQNQPH FDFTLANIRGGSIRNAIPRESVATLVFNGDITVLQSAVQKFADVIKAELALTEPNLIFTLEKVEKPQQVFSSQCTKNIIHCLNVLPNGVVRNSDVIENVVETSLSIGVLKTEDNFVRSTML VRSLIESGKSYVASLLKSLASLAQGNINLSGDYPGWEPQSHSDILDLTKTIYAQVLGTDPEIKVIHAGLECGLLKKIYPTIDMVSIGPTIRNAHSPDEKVHIPAVETYWKVLTGILAHIPSR

[0081] SEQ ID NO: 3, nucleotide sequence of mutant pepD-E145D

[0082]

[0083] SEQ ID NO: 4, Amino acid sequence of mutant pepD-E145D

[0084] MSDLQSLQPKLLWQWFDQICAIPHPSYKEEQLAQFIINWAKTKGFFAERDEVGNVLIRKPATVGMENRKPVVLQAHLDMVPQANEGTNHNFDQDPILPYIDGDWVKAKGTTLGADNGIGMASALAVLESNDIAHPELEVLLTMTDERGMEGAIGLRPNWLRSEILINTDTEENGEIYIGCAGGENADLELPIEYQVNNFEHCYQVVLKGLRGGHSGVDIHTGRANAIKVLLRFLAELQQNQPHFDFTLANIRGGSIRNAIPRESVATLVFNGDITVLQSAVQKFADVIKAELALTEPNLIFTLEKVEKPQQVFSSQCTKNIIHCLNVLPNGVVRNSDVIENVVETSLSIGVLKTEDNFVRSTMLVRSLIESGKSYVASLLKSLASLAQGNINLSGDYPGWEPQSHSDILDLTKTIYAQVLGTDPEIKVIHAGLECGLLKKIYPTIDMVSIGPTIRNAHSPDEKVHIPAVETYWKVLTGILAHIPSR

[0085] SEQ ID NO: 5, Nucleotide sequence of mutant pepD-E171A

[0086]

[0087] SEQ ID NO: 6, Amino acid sequence of mutant pepD-E171A

[0088] MSDLQSLQPKLLWQWFDQICAIPHPSYKEEQLAQFIINWAKTKGFFAERDEVGNVLIRKPATVGMENRKPVVLQAHLDMVPQANEGTNHNFDQDPILPYIDGDWVKAKGTTLGADNGIGMASALAVLESNDIAHPELEVLLTMTEERGMEGAIGLRPNWLRSEILINTDTAENGEIYIGCAGGENADLELPIEYQVNNFEHCYQVVLKGLRGGHSGVDIHTGRANAIKVLLRFLAELQQNQPHFDFTLANIRGGSIRNAIPRESVATLVFNGDITVLQSAVQKFADVIKAELALTEPNLIFTLEKVEKPQQVFSSQCTKNIIHCLNVLPNGVVRNSDVIENVVETSLSIGVLKTEDNFVRSTMLVRSLIESGKSYVASLLKSLASLAQGNINLSGDYPGWEPQSHSDILDLTKTIYAQVLGTDPEIKVIHAGLECGLLKKIYPTIDMVSIGPTIRNAHSPDEKVHIPAVETYWKVLTGILAHIPSR

[0089] SEQ ID NO: 7, Nucleotide sequence of mutant pepD-E145D-E171A

[0090]

[0091] SEQ ID NO: 8, Amino acid sequence of mutant pepD-E145D-E171A

[0092] MSDLQSLQPKLLWQWFDQICAIPHPSYKEEQLAQFIINWAKTKGFFAERDEVGNVLIRKPATVGMENRKPVVLQAHLDMVPQANEGTNHNFDQDPILPYIDGDWVKAKGTTLGADNGIGMASALAVLESNDIAHPELEVLLTMTDERGMEGAIGLRPNWLRSEILINTDTAENGEIYIGCAGGENADLELPIEYQVNNFEHCYQVVLKGLRGGHSGVDIHTGRANAIKVLLRFLAELQQNQPHFDFTLANIRGGSIRNAIPRESVATLVFNGDITVLQSAVQKFADVIKAELALTEPNLIFTLEKVEKPQQVFSSQCTKNIIHCLNVLPNGVVRNSDVIENVVETSLSIGVLKTEDNFVRSTMLVRSLIESGKSYVASLLKSLASLAQGNINLSGDYPGWEPQSHSDILDLTKTIYAQVLGTDPEIKVIHAGLECGLLKKIYPTIDMVSIGPTIRNAHSPDEKVHIPAVETYWKVLTGILAHIPSR

Claims

1. An aminoacylhistidine dipeptidase mutant, characterized in that, The mutant is any one of (1) or (2) below: (1) An amino acid sequence as shown in SEQ ID NO:4, 6 or 8; (2) An amino acid sequence that has at least 98% homology with the amino acid sequence shown in (1) and has aminoacylhistidine dipeptidase activity that catalyzes the formation of L-carnosine from β-alanine and L-histidine.

2. The aminoacylhistidine dipeptidase mutant according to claim 1, wherein, The mutant was obtained by introducing an amino acid mutation into a wild-type aminoacylhistidine dipeptidase derived from *Histophilus somni*.

3. A nucleic acid molecule encoding the aminoacylhistidine dipeptidase mutant of claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid molecule of claim 3; Optionally, the recombinant expression vector is an expression vector suitable for prokaryotic microorganisms.

5. A host cell comprising the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4, and capable of expressing the aminoacylhistidine dipeptidase mutant; Optionally, the host cell is Escherichia coli.

6. The use of the aminoacylhistidine dipeptidase mutant of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, or the host cell of claim 5 in improving the enzyme activity of aminoacylhistidine dipeptidase or in preparing L-carnosine or L-carnosine derivatives.

7. A method for preparing L-carnosine, characterized in that, Add the aminoacylhistidine dipeptidase mutant of claim 1 or 2 or the host cell expressing the aminoacylhistidine dipeptidase mutant of claim 5 to the reaction system.

8. The preparation method according to claim 7, wherein, The reaction system also includes the substrates β-alanine and L-histidine.

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