Aminopeptidase mutants, encoding genes, recombinant vectors, recombinant strains and their applications

Abstract

The application relates to the field of enzyme engineering, and discloses an aminopeptidase mutant, a coding gene of the aminopeptidase mutant, a recombination carrier, a recombination strain and application of the aminopeptidase mutant, wherein the aminopeptidase mutant is an enzyme with at least one mutation of S88L, Y150C and Q268F in an amino acid sequence shown in SEQ ID NO:1. The aminopeptidase mutant can catalyze synthesis of L-carnosine from L-histidine in the presence of beta-alanine methyl ester, has high selectivity, high L-carnosine yield and low impurity content, and has the value of industrial application.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, specifically to an aminopeptidase mutant, a gene encoding the aminopeptidase mutant, a recombinant vector, a recombinant strain, the application of at least one of the aminopeptidase mutant, gene, recombinant vector, and recombinant strain in the synthesis of L-carnosine, and a method for synthesizing L-carnosine. Background Technology

[0002] L-Carnosine, also known as β-alanyl-L-histidine, is a dipeptide formed by the condensation of β-alanine and L-histidine. It is a natural antioxidant found in living organisms. As a natural dipeptide, carnosine is abundant in the muscles and brains of mammals. In vivo, carnosine has pH buffering, antioxidant, free radical scavenging, and anti-aging effects. Clinically, it is used to relieve visual fatigue and treat cataracts; zinc-containing carnosine can be used to treat gastric ulcers. Compared with other antioxidants, L-carnosine has the advantages of strong antioxidant capacity, no toxic side effects, and various physiological activities, showing broad application prospects in medicine, health care, hygiene, and cosmetic fields.

[0003] There are several methods for producing L-carnosine. Because carnosine is abundant in animal muscle, it was initially extracted directly from animal tissues. However, this method resulted in low yields and poor purity and has long been phased out. Traditional chemical synthesis methods require the activation and protection of the active groups of L-histidine and β-alanine, followed by the removal of the protecting groups after a subsequent condensation reaction. While this method yields high results, the entire synthesis process involves numerous reaction steps and requires highly toxic agents such as hydrazine hydrate. Since hydrazine residues are unacceptable in L-carnosine products, the extraction and purification processes are extremely demanding. Several enzymatic synthesis methods have been reported, including the reverse hydrolysis of β-alanine and L-histidine catalyzed by dipeptidase, the acyl transfer reaction between β-alanine / alanine ester and L-histidine catalyzed by aminopeptidase, and the ATP-powered condensation reaction of β-alanine and L-histidine catalyzed by carnosine synthase. Dipeptide hydrolases are limited by reaction equilibrium and require the addition of high concentrations of β-alanine, with L-histidine conversion rates reaching only around 30%. Carnosine synthase-catalyzed reactions rely on ATP, posing cost issues. Compared to these two types of enzymatic reactions, aminopeptidase-catalyzed reactions do not require high concentrations of histidine, are ATP-independent, and involve fewer reaction steps, making them more suitable for industrial applications.

[0004] Nevertheless, the reported aminopeptidases currently available have low activity, produce many byproducts, and are complex to separate and purify, which severely limits the practical application of this method. Therefore, it is necessary to develop aminopeptidases with high activity and high selectivity. Summary of the Invention

[0005] The purpose of this invention is to provide an aminopeptidase with high activity and high selectivity. Specifically, it provides an aminopeptidase, its encoding gene, recombinant vector, recombinant strain, and their applications. This aminopeptidase can selectively catalyze the synthesis of L-carnosine from L-histidine in the presence of β-alanine methyl ester with high yield and has value for industrial application.

[0006] To achieve the above objectives, the first aspect of the present invention provides an aminopeptidase mutant, wherein the aminopeptidase mutant is an enzyme with an amino acid sequence as shown in SEQ ID NO: 1 that has been mutated by at least one of S88L, Y150C and Q268F.

[0007] Preferably, the aminopeptidase mutant is the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0008] A second aspect of the present invention provides a gene encoding an aminopeptidase mutant, said gene comprising a nucleotide sequence encoding an aminopeptidase mutant as described above.

[0009] Preferably, the gene encoding the aminopeptidase mutant is a gene with a nucleotide sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0010] A third aspect of the present invention provides a recombinant vector containing the genes described above.

[0011] A fourth aspect of the present invention provides a recombinant strain containing the gene described above or the recombinant vector described above.

[0012] The fifth aspect of the present invention provides the use of at least one of the aminopeptidase mutants, genes, recombinant vectors, and recombinant strains described above in the synthesis of L-carnosine.

[0013] The sixth aspect of the present invention provides a method for synthesizing L-carnosine, the method comprising catalyzing the synthesis of L-carnosine from L-histidine in the presence of β-alanine methyl ester or a salt thereof and an aminopeptidase mutant as described above.

[0014] The aminopeptidase mutant of the present invention can selectively catalyze the synthesis of L-carnosine from L-histidine in the presence of β-alanine methyl ester, and has a high L-carnosine yield and low impurity content, making it valuable for industrial application. Detailed Implementation

[0015] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0016] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0017] The first aspect of the present invention provides an aminopeptidase mutant, wherein the aminopeptidase mutant is an enzyme with an amino acid sequence as shown in SEQ ID NO: 1 that has been mutated by at least one of S88L, Y150C and Q268F.

[0018] The enzyme shown in SEQ ID NO: 1 is Gryganskiella cystogenkine The aminopeptidases from which this information originates. S88L means that the 88th amino acid of the enzyme shown in SEQ ID NO: 1 has been mutated from serine (S) to leucine (L); Y150C means that the 150th amino acid of the enzyme shown in SEQ ID NO: 1 has been mutated from tyrosine (Y) to cysteine ​​(C); Q268F means that the 268th amino acid of the enzyme shown in SEQ ID NO: 1 has been mutated from glutamine (Q) to phenylalanine (F).

[0019] A mutation in at least one of S88L, Y150C, and Q268F means that the mutant may contain a mutation at one of the sites of S88L, Y150C, and Q268F, or a mutation at two or three sites simultaneously.

[0020] In some embodiments, the aminopeptidase mutant comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The inventors have found that, under this preferred embodiment, the mutant enzyme exhibits higher activity and further improves the efficiency of catalyzing the synthesis of L-carnosine from L-histidine.

[0021] In this invention, the mutant can be formulated into a corresponding enzyme preparation, specifically, the enzyme preparation can exist in solid, semi-solid or liquid form.

[0022] A second aspect of the present invention provides a gene encoding an aminopeptidase mutant, said gene comprising a nucleotide sequence encoding an aminopeptidase mutant as described above.

[0023] The nucleotide sequences provided by this invention can generally be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombination. Typically, the obtained nucleotide sequence is cloned into a vector, then transformed into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the proliferated host cells using conventional methods. Alternatively, the relevant nucleotide sequence can also be synthesized using known artificial chemical synthesis methods.

[0024] Preferably, the gene encoding the aminopeptidase mutant comprises a gene with the nucleotide sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The terminal TAA in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 encodes a terminator.

[0025] A third aspect of the present invention provides a recombinant vector containing the genes described above.

[0026] The recombinant vector in this invention is constructed by inserting the nucleotide sequence into the multiple cloning site of the expression vector. The expression vector usually refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.

[0027] The expression vector can be a plasmid commonly used in the art, such as a pET series plasmid. In some embodiments, the expression vector includes, but is not limited to, pET22b plasmid, pET-29a plasmid, pET-21a plasmid, pET-50a plasmid, etc.

[0028] A fourth aspect of the present invention provides a recombinant strain containing the gene described above or the recombinant vector described above.

[0029] The recombinant strains in this invention contain the recombinant vector described above or have the genes described above integrated into their genome. The recombinant strains can be prokaryotic cells, such as bacterial cells (e.g., *Escherichia coli*). E.coli (DH5α); or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells.

[0030] In this invention, the recombinant vector can be transformed, transduced, or transfected into host cells (strains) using conventional methods in the art, such as chemical transformation by calcium chloride method or high-voltage electroporation transformation.

[0031] The fifth aspect of the present invention provides the use of at least one of the aminopeptidase mutants, genes, recombinant vectors, and recombinant strains described above in the synthesis of L-carnosine.

[0032] The sixth aspect of the present invention provides a method for synthesizing L-carnosine, the method comprising catalyzing the synthesis of L-carnosine from L-histidine in the presence of β-alanine methyl ester or a salt thereof (e.g., β-alanine methyl ester hydrochloride) and an aminopeptidase mutant as described above.

[0033] In some embodiments, the content of β-alanine methyl ester or its salt in the catalytic system is 50-200 g / L, for example, 50 g / L, 80 g / L, 120 g / L, 150 g / L, 180 g / L, 200 g / L, or any combination of two of the above values; the content of L-histidine is 40-80 mmol / L, for example, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, or any combination of two of the above values. The content is based on the total volume of the catalytic system. The inventors have found that at this preferred concentration, both the problem of limited catalytic reaction rate caused by insufficient histidine at low concentrations is avoided, and the potential inhibition of mutant enzyme activity by histidine at high concentrations is prevented, further ensuring the mutant's continuous and efficient catalysis of L-histidine to L-carnosine synthesis.

[0034] It is understood that the catalytic system comprises β-alanine methyl ester or its salt, L-histidine, aminopeptidase mutant, and water.

[0035] In some embodiments, the catalytic conditions include: a pH of 7-10, such as 7, 8, 9, 10, or any combination of two of the above values; and a temperature of 20-50°C, such as 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or any combination of two of the above values.

[0036] In some embodiments, β-alanine methyl ester or its salt and / or L-histidine are added to the catalytic system in batches. Those skilled in the art can adjust this according to the specific circumstances.

[0037] The present invention will be described in detail below through embodiments. Unless otherwise specified, all operations can be performed in the manner conventional in the art.

[0038] In the following examples, the pET-29a plasmid was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Escherichia coli E.coli DH5α and BL21 (DE3) were purchased from Titan Technologies Inc.

[0039] All other raw materials and reagents are commercially available products.

[0040] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH adjusted to 6.5, autoclaved for 21 min, ready for use.

[0041] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, pH adjusted to 7.0, autoclaved for 21 min, ready for use.

[0042] Example 1

[0043] This example illustrates the discovery and modification of aminopeptidase.

[0044] Through literature review, NCBI enzyme gene mining, and multiple sequence alignment, the inventors selected the aminopeptidase that synthesizes carnosine, derived from... Gryganskiella cystogenkine The wild-type aminopeptidase, named WT, may have problems such as low catalytic efficiency. Therefore, based on structural simulation and molecular docking, the inventors selected hot spots near the active pocket and some amino acids that are spatially close to the active site. Finally, they screened out three mutants that may improve its selectivity and catalytic activity: S88L, Y150C, and Q268F, which were named M1, M2, and M3, respectively.

[0045] The amino acid sequences of the wild type and the three mutants are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and their corresponding nucleotide sequences are SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, as shown in Table 1.

[0046] Table 1. Amino acid and nucleotide sequences of aminopeptidase

[0047]

[0048]

[0049]

[0050] Note: * indicates a terminator.

[0051] Example 2

[0052] This embodiment illustrates the construction of wild-type aminopeptidase and its mutant engineered bacteria.

[0053] 2.1 Construction of wild-type aminopeptidase and its mutant vector

[0054] The wild-type aminopeptidase sequence was synthesized by Jiangsu Saisofe Biotechnology Co., Ltd., and the synthesized aminopeptidase genes were ligated into pET-29a. Who I and Hind pET-29a, located at the cleavage site of enzyme III, is preserved in our laboratory. To construct the M1 / M2 / M3 expression vector, a plasmid containing the wild-type aminopeptidase sequence was used as a template. Primers were designed at the mutation site for inverse PCR. The amplified PCR product was digested overnight with Dpn I and then inactivated at 80℃ for 20 min before being transferred to… E.coli DH5α competent cells were cultured in LB liquid medium for 50 min, then spread onto LB solid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C with the medium inverted. Single clones were picked and sent to a sequencing company for sequencing verification. The sequencing results were analyzed using SnapGene software to confirm that the amino acid mutation at the mutation site was correct, thus obtaining the recombinant vector containing the aminopeptidase mutation site.

[0055] 2.2 Construction and expression of wild-type aminopeptidase and its mutant host bacteria

[0056] To obtain engineered bacteria expressing wild-type aminopeptidase and its mutants, four recombinant plasmids were transformed into BL21(DE3) competent cells, cultured in LB liquid medium for 50 min, and then plated onto LB solid medium containing 100 μg / mL kanamycin and incubated overnight at 37°C inverted. The resulting single colonies were the engineered bacteria expressing wild-type aminopeptidase and its mutants.

[0057] Each engineered bacterial strain was streaked onto LB agar plates and incubated at 37°C for 12 h. Single colonies were then inoculated into 5 ml of LB liquid medium containing 100 μg / mL kanamycin and incubated at 37°C with shaking for 12 h. Finally, a 1% (v / v) inoculum was transferred to 100 ml of fresh LB liquid medium also containing 100 μg / mL kanamycin and incubated at 37°C with shaking until OD (out of control) was reached.600 When the concentration reaches approximately 0.6, add IPTG to a final concentration of 0.1 mmol / L and incubate at 25°C for 20 h. After incubation, centrifuge the culture medium at 12000 rpm for 10 min, discard the supernatant, and collect the bacterial cells. These bacterial cells are those expressing wild-type aminopeptidase and mutant proteins.

[0058] Example 3: Catalytic reaction of wild type and its mutant

[0059] 3.1 Obtaining aminopeptidase protein

[0060] The bacterial cells collected after induced expression were washed twice with 0.1M, pH 8.5 Tris-HCl buffer to obtain resting cells. The obtained cells were then resuspended in 0.1M, pH 8.5 Tris-HCl buffer, sonicated on ice, and the supernatant was collected by high-speed centrifugation and filtered through a 0.22μm filter membrane to obtain crude enzyme solution containing aminopeptidase.

[0061] 3.2 Validation of Catalytic Reaction

[0062] Histidine was added to the crude enzyme solution containing wild-type and mutant enzymes to achieve a final concentration of 60 mmol / L. The solution was stirred to dissolve, and then 120 mmol / L β-alanine methyl ester hydrochloride was added, bringing the total reaction volume to 100 mL. After reacting at 30 °C for 12 h, 1 mL of the reaction solution was taken dropwise and 50 μL of stop solution (3M HCl) was added to terminate the reaction. The mixture was shaken to mix, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The selectivity and yield were analyzed by liquid chromatography, and the results are shown in Table 2.

[0063] Yield is calculated using the following formula: Actual yield / Theoretical yield × 100%, where the theoretical yield is calculated based on the reaction equation process.

[0064] Table 2. Response status of wild type and mutant

[0065]

[0066] Histidine conversion: Under histidine concentration of 60 mmol / L, both wild-type aminopeptidase and mutants catalyzed the conversion of histidine to carnosine. The carnosine yields for WT, M1, M2, and M3 were 61.5%, 75.9%, 77.5%, and 81.7%, respectively. Under histidine concentration of 60 mmol / L, the yield of mutant M3 was 1.3 times higher than that of wild-type aminopeptidase.

[0067] Example 4: Optimization of catalytic reaction conditions for mutant M3

[0068] 4.1 pH optimization

[0069] The inventors discovered that mutant M3 significantly enhances the catalytic activity of aminopeptidase. Therefore, under the conditions of mutant M3, the reaction conditions were optimized, and four pH values ​​(pH 7, pH 8, pH 9, and pH 10) were selected for the catalytic reaction. Specifically, after obtaining bacterial cells expressing M3 under the aforementioned conditions, the cells were resuspended in phosphate buffer, Tris-HCl buffer, and Gly-NaOH buffer at different pH values, and then subjected to the same catalytic conditions. The carnosine yield was analyzed, and the results are shown in Table 3.

[0070] Table 3. Reaction of M3 at different pH values

[0071]

[0072] Within the pH range of 7-10, the mutant M3 exhibited the highest carnosine yield of 85.6% at pH 8.0.

[0073] 4.2 Temperature Optimization

[0074] With histidine concentration kept constant (60 mmol / L) and pH 8.0, the prepared reaction system was subjected to catalytic reaction at 20, 30, 40 and 50 °C, and the yield was analyzed. The results are shown in Table 4.

[0075] Table 4. Reaction of M3 at different temperatures

[0076]

[0077] Between 20 and 50°C, the highest carnosine yield of mutant M3 was 89.3% at 40°C.

[0078] 4.3 Optimization of β-alanine methyl ester hydrochloride concentration

[0079] The enzyme-catalyzed reaction converts the product into impurities. While high concentrations of β-alanine methyl ester hydrochloride can promote the forward synthesis of the catalytic reaction, the continued reaction of the product in the later stages leads to a decrease in yield. Therefore, different concentrations of β-alanine methyl ester hydrochloride in the system were investigated to catalyze the reaction and analyze the carnosine yield. The results are shown in Table 5.

[0080] Table 5. Reaction of M3 with β-alanine methyl ester hydrochloride at different concentrations.

[0081]

[0082] With histidine concentration kept constant (60 mmol / L), the production and yield of carnosine showed a trend of first increasing and then decreasing with the increase of β-alanine methyl ester hydrochloride. When the concentration of β-alanine methyl ester hydrochloride was 90 mmol / L, the carnosine yield of mutant M3 was the highest, at 91.4%.

[0083] 4.4 Optimization of the addition method of β-alanine methyl ester hydrochloride

[0084] The inventors discovered that under optimal conditions, further increasing the concentration of β-alanine methyl ester hydrochloride led to a reaction between the product and histidine. This resulted in the product yield not increasing further with continued increases in the β-alanine methyl ester hydrochloride concentration, and even decreasing in some cases, while the impurity yield gradually increased. Therefore, the method of adding β-alanine methyl ester hydrochloride was investigated. While keeping the total amount of β-alanine methyl ester hydrochloride added constant (the concentration of β-alanine methyl ester hydrochloride was 90 mmol / L during a single addition), the catalytic reaction was carried out according to the methods shown in Table 6, and the carnosine yield was analyzed. The results are shown in Table 6.

[0085] Table 6. Reaction of M3 under different histidine addition methods

[0086]

[0087] With histidine concentration kept constant (60 mmol / L), the mutant M3 showed the highest carnosine yield of 93.6% after being dissolved and uniformly added for 4 hours under different β-alanine methyl ester hydrochloride addition methods.

[0088] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An aminopeptidase mutant, characterized in that, The aminopeptidase mutant is an enzyme with an amino acid sequence as shown in SEQ ID NO:1 that has been mutated by any one of S88L, Y150C and Q268F.

2. The aminopeptidase mutant according to claim 1, characterized in that, The aminopeptidase mutant is the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

3. A gene encoding an aminopeptidase mutant, characterized in that, The gene encoding the aminopeptidase mutant contains a nucleotide sequence encoding the aminopeptidase mutant of claim 1 or 2.

4. The gene according to claim 3, characterized in that, The gene encoding the aminopeptidase mutant has the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

5. A recombinant vector, characterized in that, The recombinant vector contains the gene as described in claim 3 or 4.

6. A recombinant bacterial strain, characterized in that, The recombinant strain contains the gene described in claim 3 or 4 or the recombinant vector described in claim 5.

7. The use of at least one of the aminopeptidase mutant of claim 1 or 2, the gene of claim 3 or 4, the recombinant vector of claim 5, and the recombinant strain of claim 6 in the synthesis of L-carnosine.

8. A method for synthesizing L-carnosine, characterized in that, The method comprises catalyzing the synthesis of L-carnosine from L-histidine in the presence of β-alanine methyl ester or a salt thereof and the aminopeptidase mutant of claim 1 or 2.

9. The method according to claim 8, characterized in that, In the catalytic system, the content of β-alanine methyl ester or its salt is 50-200 g / L, and the content of L-histidine is 40-80 mmol / L; The catalytic conditions include a pH of 7-10 and a temperature of 20-50°C.

10. The method according to claim 9, characterized in that, β-alanine methyl ester or its salt and / or L-histidine are added to the catalytic system in batches.

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