Aminopeptidase mutant, preparation method thereof and application of aminopeptidase mutant in preparation of L-carnosine

Abstract

The invention relates to the technical field of enzyme engineering, in particular to an aminopeptidase mutant, a preparation method of the aminopeptidase mutant and application of the aminopeptidase mutant to preparation of L-carnosine. The aminopeptidase mutant is obtained by mutating wild type aminopeptidase and can be used for preparing the L-carnosine through reverse hydrolysis of beta-alanine methyl ester and L-histidine. The L-carnosine produced by using the aminopeptidase mutant disclosed by the invention has the following advantages: 1, the conversion rate is greater than or equal to 95%, the conversion time is short, and relatively cheap alanine methyl ester can be used as a raw material to catalytically prepare the L-carnosine; 2, the production process is simple, the reaction conditions are mild, and the green chemistry concept is met;

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to an aminopeptidase mutant, a preparation method thereof and use of the aminopeptidase mutant in preparing L-carnosine. Background Art

[0002] L-carnosine, also known as β-alanyl-L-histidine, is a dipeptide obtained by condensing two amino acids, β-alanine and L-histidine. It is a white crystalline solid and a natural antioxidant in living organisms. As a natural dipeptide, L-carnosine is present in large quantities in the muscles, brain tissues, and lens tissues of animals, with concentrations as high as 20 mM. In vivo, carnosine has pH buffering, antioxidant, anti-free radical, and anti-aging effects. Clinically, it is used to reduce visual fatigue and treat cataracts; zinc-containing carnosine can be used to treat gastric ulcers. In addition, carnosine and its derivatives also have broad application prospects in the fields of food additives, cosmetics, and health products.

[0003] There are many methods for the production of L-carnosine. Since carnosine has a high content in animal muscle, carnosine was directly extracted from animal tissue in the early stage. This method has low yield and poor purity and has been eliminated long ago. The method commonly used at present is chemical synthesis, which uses phthalic anhydride to protect the amino group of alanine, uses dichlorothionyl to activate the carboxyl group, and then reacts with trimethylsilane-protected L-histidine to obtain a group-protected carnosine derivative, and then deprotects to obtain free L-carnosine. This method has a high yield, but there are many reaction steps and harsh reaction conditions. In the reaction process, it is necessary to meter and consume and generate a large amount of environmentally harmful reagents, and the deprotection of phthalyl needs to use the highly toxic reagent hydrazine, and the product L-carnosine does not allow the residue of hydrazine, so the extraction and purification of the product have high requirements. In general, although the chemical synthesis method has developed earlier, it generally requires complex protection-deprotection steps, and there are problems such as cumbersome synthesis steps, harsh reaction conditions, low product yield, large pollution, and residual toxic reagents.

[0004] In comparison, the biosynthesis method has the advantages of short synthesis route, mild reaction conditions, environmental friendliness, high product optical purity, etc. It is more suitable for industrial application and will gradually replace the traditional chemical synthesis method. Aminopeptidase can catalyze the condensation of β-alanine or β-alanine methyl ester and L-histidine to generate L-carnosine. The reaction process has few reaction steps, simple process, high efficiency, mild conditions, and the whole process is green and environmentally friendly, so it has a good prospect for industrial application. However, the existing aminopeptidases have low catalytic activity for L-histidine, resulting in low carnosine production, so it is necessary to develop aminopeptidases with high catalytic activity. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an aminopeptidase mutant and a preparation method thereof and its use in preparing L-carnosine, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned object and other related objects, the present invention provides an aminopeptidase mutant, which is a protein obtained by mutating the wild-type aminopeptidase having an amino acid sequence as shown in SEQ ID No. 2, wherein the mutation site is selected from any one or more of the following: position 82, position 90, position 98, position 105, position 117, position 132, position 137, position 144, position 145, position 150, position 183, position 186, position 223, position 278, position 283, position 319 or position 325.

[0007] The present invention also provides an isolated polynucleotide encoding the aforementioned aminopeptidase mutant.

[0008] The present invention also provides a nucleic acid construct, which comprises the isolated polynucleotide.

[0009] The present invention also provides an engineered cell, wherein the engineered cell contains the nucleic acid construct or the exogenous isolated polynucleotide is integrated into the genome.

[0010] The present invention also provides a method for preparing the aminopeptidase, comprising the following steps: culturing the engineered cells, inducing the expression of the aminopeptidase and continuing the culturing, and after the culturing is completed, collecting the bacterial cells and separating them to obtain the enzyme solution of the aminopeptidase.

[0011] The present invention also provides use of the aminopeptidase, the isolated polynucleotide, the nucleic acid construct or the engineered cell in preparing L-carnosine.

[0012] The present invention also provides a method for synthesizing L-carnosine, which comprises the following steps: mixing the aminopeptidase with L-histidine and beta-alanine methyl ester for reaction, and collecting the supernatant after the reaction to obtain L-carnosine.

[0013] As described above, the aminopeptidase mutant of the present invention, the preparation method thereof and the use thereof for preparing L-carnosine have the following beneficial effects: compared with the wild-type aminopeptidase, the substrate conversion rate is increased from 41% to 95%. The catalytic efficiency of the mutant is significantly improved, and the efficient synthesis of carnosine is achieved. Relatively cheap β-alanine methyl ester is used as a raw material to reduce the cost of industrial application, provide a supplementary solution for the synthesis of carnosine, and have great significance for the green catalytic synthesis and industrial application of carnosine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Shown is a schematic diagram of the route for synthesizing L-carnosine by the aminopeptidase of the present invention.

[0015] Figure 2 Shown is a protein gel image of the mutant W90H / N105Y / D132N / E278D / I325Q.

[0016] Figure 3 Shown is the result of UPLC detection of carnosine product of mutant W90H / N105Y / D132N / E278D / I325Q. DETAILED DESCRIPTION

[0017] The invention provides an aminopeptidase mutant, which is a protein obtained by mutating a wild-type aminopeptidase having an amino acid sequence as shown in SEQ ID No. 2, wherein the mutation site is selected from any one or more of the following: the 82nd, 90th, 98th, 105th, 117th, 132nd, 137th, 144th, 145th, 150th, 183rd, 186th, 223rd, 278th, 283rd, 319th or 325th position.

[0018] In certain embodiments of the present invention, the mutation site is selected from any 1, 2, 3, 4, 5 or more of the following: position 82, position 90, position 98, position 105, position 117, position 132, position 137, position 144, position 145, position 150, position 183, position 186, position 223, position 278, position 283, position 319 or position 325.

[0019] In certain embodiments of the present invention, the amino acid at position 82 of the aminopeptidase mutant is any one of V, W, R, P or K; preferably, the amino acid at position 82 is R or K. That is, the C at position 82 of the amino acid sequence shown in SEQ ID No. 2 is mutated to R or K.

[0020] In certain embodiments of the present invention, the amino acid at position 90 of the aminopeptidase mutant is A, H, L, V, P, M or K; preferably, the amino acid residue at position 90 is A, H or P. That is, the amino acid residue at position 90 of the amino acid sequence shown in SEQ ID No. 2 is mutated from W to A, H or P.

[0021] In certain embodiments of the present invention, the amino acid at position 98 of the aminopeptidase mutant is L, W, T or Y; preferably, the amino acid at position 98 is L or Y. That is, the amino acid residue at position 98 of the amino acid sequence shown in SEQ ID No. 2 is mutated from F to L or Y.

[0022] In certain embodiments of the present invention, the amino acid at position 105 of the aminopeptidase mutant is E, F, T, Y or C; preferably, the amino acid at position 105 is F, Y or C. That is, the amino acid residue at position 105 of the amino acid sequence shown in SEQ ID No. 2 is mutated from N to F, Y or C.

[0023] In certain embodiments of the present invention, the amino acid at position 117 of the aminopeptidase mutant is T, R, D, C or S; preferably, the amino acid at position 117 is T, D or C. That is, the amino acid residue at position 117 of the amino acid sequence shown in SEQ ID No. 2 is mutated from F to T, D or C.

[0024] In certain embodiments of the present invention, the amino acid at position 132 of the aminopeptidase mutant is N, V, G, C or A; preferably, the amino acid at position 132 is N or V. That is, the amino acid residue at position 132 of the amino acid sequence shown in SEQ ID No. 2 is mutated from D to N or V.

[0025] In certain embodiments of the present invention, the amino acid at position 137 of the aminopeptidase mutant is I, F, W, R or Q; preferably, the amino acid residue at position 137 is W or Q. That is, the amino acid residue at position 137 of the amino acid sequence shown in SEQ ID No. 2 is mutated from P to W or Q.

[0026] In certain embodiments of the present invention, the amino acid at position 144 of the aminopeptidase mutant is C, M, G, K, F or P; preferably, the amino acid at position 144 is C, M or P. That is, the amino acid residue at position 144 of the amino acid sequence shown in SEQ ID No. 2 is mutated from D to C, M or P.

[0027] In certain embodiments of the present invention, the 145th amino acid of the aminopeptidase mutant is R, V, F, Y or H; preferably, the 145th amino acid is H or R. That is, the 145th amino acid residue of the amino acid sequence shown in SEQ ID No. 2 is mutated from G to H or R.

[0028] In certain embodiments of the present invention, the amino acid at position 150 of the aminopeptidase mutant is A, Y, S, H or K; preferably, the amino acid at position 150 is A or H. That is, the amino acid residue at position 150 of the amino acid sequence shown in SEQ ID No. 2 is mutated from I to A or H.

[0029] In certain embodiments of the present invention, the amino acid at position 183 of the aminopeptidase mutant is P, W, F, L or A; preferably, the amino acid at position 183 is F or L. That is, the amino acid residue at position 183 of the amino acid sequence shown in SEQ ID No. 2 is mutated from M to F or L.

[0030] In certain embodiments of the present invention, the amino acid at position 186 of the aminopeptidase mutant is M, L, I, C or R; preferably, the amino acid at position 186 is L or R. That is, the amino acid residue at position 186 of the amino acid sequence shown in SEQ ID No. 2 is mutated from H to L or R.

[0031] In certain embodiments of the present invention, the amino acid at position 223 of the aminopeptidase mutant is H, Q, L, E, N or S; preferably, the amino acid at position 223 is E or S. That is, the amino acid residue at position 223 of the amino acid sequence shown in SEQ ID No. 2 is mutated from A to E or S.

[0032] In certain embodiments of the present invention, the amino acid at position 278 of the aminopeptidase mutant is A, C, D, G or V; preferably, the amino acid residue at position 278 is A, D or V. That is, the amino acid residue at position 278 of the amino acid sequence shown in SEQ ID No. 2 is mutated from E to A, D or V.

[0033] In certain embodiments of the present invention, the amino acid at position 283 of the aminopeptidase mutant is C, Q, T, S, F or W; preferably, the amino acid at position 283 is S or W. That is, the amino acid residue at position 283 of the amino acid sequence shown in SEQ ID No. 2 is mutated from G to S or W.

[0034] In certain embodiments of the present invention, the amino acid at position 319 of the aminopeptidase mutant is W, T, C, A or N; preferably, the amino acid at position 319 is W or C. That is, the amino acid residue at position 319 of the amino acid sequence shown in SEQ ID No. 2 is mutated from S to W or C.

[0035] In certain embodiments of the present invention, the amino acid at position 325 of the aminopeptidase mutant is M, N, C, V, K or Q; preferably, the amino acid at position 325 is M, C or Q. That is, the amino acid residue at position 325 of the amino acid sequence shown in SEQ ID No. 2 is mutated from I to M, C or Q.

[0036] In certain embodiments of the invention, the aminopeptidase mutant comprises any of the following mutations: W90H, F98L, F117T, F117C, D132N, P137Q, D144M, D144P, I150H, M183F, H186R, A223S, I325Q, C82K / F117C, W90H / N105Y, W90H / I325Q, F98L / I150H, F117T / G145H, D132N / E27 8D, D132N / G283S, D132V / S319C, M183F / I325Q, W90H / D132N / E278D, W90H / D132N / I325 Q, W90H / N105Y / I325Q, W90H / N105Y / D132N / I325Q, W90H / N105Y / D132N / E278D / I325Q.

[0037] The present invention also provides an isolated polynucleotide encoding the aforementioned aminopeptidase mutant.

[0038] The polynucleotide may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded.

[0039] The polynucleotide encoding the aminopeptidase of the present invention can be prepared by any appropriate technology well known to those skilled in the art, such as but not limited to recombinant DNA technology, chemical synthesis and the like.

[0040] Based on the disclosed amino acid sequence of the aminopeptidase, due to the degeneracy of codons, those skilled in the art can obtain the nucleotide sequence of the isolated polynucleotide while keeping the encoded amino acid sequence unchanged, which belongs to the routine technology in the field.

[0041] In certain embodiments of the present invention, the isolated polynucleotide comprises the nucleotide sequence shown in SEQ ID No.13.

[0042] W90H / N105Y / D132N / E278D / I325Q (SEQ ID No.13) The present invention also provides a nucleic acid construct, which comprises the isolated polynucleotide.

[0043] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, which include a vector backbone, i.e., an empty vector, and an expression framework.

[0044] The type of expression vector is not specifically limited. An expression vector refers to a nucleic acid molecule that allows the insertion of exogenous nucleotides without destroying the ability of the vector to replicate and / or integrate in a host cell. An expression vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. An expression vector may also include one or more selective marker genes and other genetic factors. An expression vector is a vector that contains necessary regulatory sequences to allow the transcription and translation of an inserted gene or genes. The expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0045] The prokaryotic expression vector is selected from an Escherichia coli expression vector, a Bacillus subtilis expression vector or a Streptomyces expression vector. In a preferred embodiment, the prokaryotic expression vector is selected from an Escherichia coli expression vector. The Escherichia coli expression vector is, for example, pET-29a(+).

[0046] The eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector or a mammalian expression vector.

[0047] The host cell is selected from eukaryotic host cells or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, C.elegans or nematodes or mammalian host cells. Examples of yeast host cells are Saccharomyces cerevisiae, Kluyveromyces lactis, K.lactis or Yarrowia lipolytica, Pichia pastoris. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, Vero or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells, such as Escherichia coli, Streptomyces, Bacillus subtilis, Salmonella typhi or mycobacteria.

[0048] Those skilled in the art can transfect the expression vector into host cells according to methods well known in the art to obtain cells containing the aminopeptidase encoding gene.

[0049] The present invention also provides an engineered cell, wherein the engineered cell contains the nucleic acid construct or the exogenous isolated polynucleotide is integrated into the genome.

[0050] In certain embodiments of the present invention, the engineered cell is Escherichia coli.

[0051] In certain embodiments of the present invention, the engineered cell is obtained by transforming a host cell with the nucleic acid construct. The host cell is Escherichia coli Escherichia coli BL21 (DE3).

[0052] The present invention also provides a method for preparing the aminopeptidase, comprising the following steps: culturing the engineered cells, inducing the expression of the aminopeptidase and continuing the culturing, and after the culturing is completed, collecting the bacterial cells and separating them to obtain the enzyme solution of the aminopeptidase.

[0053] In certain embodiments of the present invention, the culture temperature is 35-37.5°C.

[0054] In certain embodiments of the present invention, IPTG is used to induce the expression of aminopeptidase. Based on the total volume of the reaction system, the concentration of IPTG is 0.05-0.2 mM. The induction temperature is 18-22° C. and the induction time is 6-14 hours.

[0055] In certain embodiments of the present invention, the method for collecting and isolating bacterial cells is as follows: the culture system is centrifuged to collect bacterial cells, the bacterial cells are resuspended with potassium phosphate buffer, and the enzyme solution of the aminopeptidase is obtained after homogenization and disruption.

[0056] The aminopeptidase may be in the form of fermentation broth of the engineered cells, pure enzyme or lyophilized powder, for example, in the form of fermentation broth.

[0057] The present invention also provides use of the aminopeptidase, the isolated polynucleotide, the nucleic acid construct or the engineered cell in preparing L-carnosine.

[0058] In certain embodiments of the present invention, the use is for catalyzing the preparation of L-carnosine from L-histidine and / or β-alanine methyl ester.

[0059] The present invention also provides a method for synthesizing L-carnosine, which comprises the following steps: mixing the aminopeptidase with L-histidine and beta-alanine methyl ester for reaction, and collecting the supernatant after the reaction to obtain L-carnosine.

[0060] The aminopeptidase can participate in the reaction in the form of fermentation liquid of the engineered cells or enzyme liquid or lyophilized powder obtained after the fermentation liquid or lyophilized powder is broken.

[0061] In certain embodiments of the invention, the concentration of aminopeptidase in the reaction system is 5-100 U / mL, for example, 5-10 U / mL, 10-15 U / mL, 15-20 U / mL, 20-30 U / mL, 30-40 U / mL, 40-50 U / mL, 50-60 U / mL, 60-70 U / mL, 70-80 U / mL, 80-90 U / mL, 90-100 U / mL.

[0062] In the reaction system, L-histidine and β-alanine methyl ester are in excess relative to aminopeptidase.

[0063] In certain embodiments of the present invention, at the beginning of the reaction, the concentration of L-histidine is 150-250 mM.

[0064] In certain embodiments of the present invention, at the beginning of the reaction, the concentration of the β-alanine methyl ester is 300-350 mM.

[0065] In certain embodiments of the present invention, the reaction temperature is 10-30° C. The reaction temperature is, for example, 10-15° C., 15-20° C., 20-25° C., or 25-30° C.

[0066] In certain embodiments of the present invention, the reaction pH is 7.3-7.7, for example, the pH is 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7.

[0067] Those skilled in the art can flexibly adjust the reaction time according to the reaction progress. In certain embodiments of the present invention, the reaction time is 2.5 to 5.5 hours.

[0068] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0069] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0070] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0071] The specific formula of LB medium is: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L.

[0072] The specific formula of LB solid medium is: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar 15 g / L. Example 1: Preparation of aminopeptidase mutant gene sequence

[0073] The existing aminopeptidase library in the laboratory was screened, and the protease with the highest reaction activity was used as the initial protein for subsequent research. The selected aminopeptidase amino acid coding sequence (as shown in SEQ ID No.2) was codon optimized. This part of the work and subsequent gene synthesis were completed by Saisuofei Biotechnology Co., Ltd., and the nucleotide sequence is shown in SEQ ID No.1. The target gene was connected between NdeI and HindIII in the vector pET-29a(+), and the constructed plasmid was transferred into the Escherichia coli BL21(DE3) strain to obtain a recombinant bacterium.

[0074] MTSHPSPSQRSRIRDVVPSLIIGRHSPGRLNCLTDVPGVLVHTESLRRTTPSIVNTGVTTILPRKEWFHQGCHAAIYAFNGCGEMTGSHWLNESGILFSPIVLTNSFAVGACHTGIFEYAVSKYRNKQGLADWGLLPVVAETYDGYLSDIGAMAIKSDMTVRGIENASHGPVPEGNTGGGTGMMCHRFKGGTGSSSRVLPALNNRENGQ PKDINYTVAALVQANYGSKRDLKIGGIPIGRLIMREEEEEQQQQQQAQQDEKESGRNENTPPTSTSTERKKDGSIIVVLATDAPLHPTQLKRLATRATVGLARV GGWGSNSSGDIFLAFSTAAHIPIKSTSDRWIPTTNDSLDLLDTSTINSLFEAVADCVEEAIYNALTSAEDTQGPGGVTVKALPLDRLRELMKEHYVSVPFV (SEQ ID No.2) The glycerol tube containing the aminopeptidase recombinant bacteria was streaked onto a plate containing LB solid medium (50 μg / mL kanamycin) and cultured at 37°C overnight. A single colony was picked from the plate and inoculated into 5 mL LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm overnight. After obtaining the culture solution, the plasmid was extracted according to the instructions of the plasmid extraction kit.

[0075] Degenerate primers (primers containing NNK codons) of the sites to be mutated were synthesized at a gene synthesis company, and mutations were introduced into the aminopeptidase gene by PCR using the extracted pET-29a(+)-aminopeptidase plasmid as a template. The primers and PCR systems used are shown in Tables 1 and 2, respectively: Table 1 PCR primer design Table 2. PCR amplification system After PCR amplification, the amplified product was detected by 1.0% agarose gel electrophoresis, and the target band was bright. Figure 2The mutant W90H / N105Y / D132N / E278D / I325Q protein gel image is shown, and the band above 43kDa is in line with expectations. It was digested with DpnI, and the digestion product was purified by a purification kit after digestion, and then transformed into BL21 (DE3) competent cells, and then spread on the plate and placed in a 37°C constant temperature incubator for overnight culture. The library can be constructed when more than 200 single colonies grow on the plate the next day. Example 2: Preparation of aminopeptidase mutant library Example 2: Preparation of aminopeptidase mutant library

[0076] Add 200 μL LB liquid culture medium (50 μg / mL Kan) to each well of a 96-well plate (seed plate), pick a single well to the 96-well plate with a sterilized toothpick, and culture overnight at 37°C and 450 rpm to obtain seed solution. Add 140 μL LB liquid culture medium (50 μg / mL Kan) to each well of a new 96-well plate (expression plate), and then transfer the seed solution in the seed plate to another 96-well plate according to the corresponding position (inoculation volume is 10 μL), culture at 37°C and 450 rpm for 3 hours, add 50 μL LB culture medium (containing 0.4 mM IPTG, IPTG final concentration is 0.1 mM), and induce expression at 20°C for 12 hours. Add 50 μL of 50% glycerol to each well of the transferred seed plate, mix well, and store at -80°C. The above-mentioned expression plate fermentation liquid after induced expression was centrifuged (4°C, 4000 rpm, 10 min), the supernatant was discarded, and stored at -80°C. Example 3: Screening of aminopeptidase mutant library

[0077] Step 1: A 1 g / L lysozyme solution was prepared with Tris-HCl buffer (100 mM, pH 8.5), and 120 μL of lysozyme solution was added to each well of the expression plate. The cells were shaken at 37°C, 800 rpm for 1 h, and the supernatant after centrifugation (4°C, 4000 rpm, 10 min) was the crude enzyme solution of the active mutant to be tested. Add 120 μL Tris-HCl buffer (100 mM, pH 8.5) to each well of a new 96-well plate (plate A), take 20 μL of the mutant crude enzyme solution from the expression plate and add it to the corresponding position of plate A, add 60 μL of substrate mother solution (containing 200 mM L-histidine and 260 mM β-alanine methyl ester), and react for 1 h (20°C, 800 rpm); add 60 μL Tris-HCl buffer (100 mM, pH 8.5) to each well of a new 96-well plate (plate B), take 80 μL of the mutant crude enzyme solution from the expression plate and add it to plate B according to the corresponding position, add 60 μL of substrate mother solution (containing 200 mM carnosine and 260 mM β-alanine methyl ester), and react for 1 h (20°C, 800 rpm). After the reaction, 50 μL of 3M HCl was added and shaken at 20°C and 800 rpm for 10 min to quench the reaction. The reaction solution at the corresponding position was analyzed by UPLC, and mutants with product peak areas larger than the wild type or impurity peak areas smaller than the wild type were selected for sequencing and rescreening (a large product peak indicates that the activity of synthesizing carnosine is improved).

[0078] Step 2: The highly active mutants in step 1 were inoculated into a 96-well plate (seed plate), induced for expression according to the operation in Example 2, and rescreened according to the operation in step 1 in Example 3. The performance of the mutants was detected by UPLC, and the mutants whose product peak area was greater than that of the wild type or whose impurity peak area was less than that of the wild type were selected for enzyme activity determination and amplification verification. Example 4: Determination of enzyme activity of aminopeptidase mutants

[0079] The glycerol bacteria of the aminopeptidase mutant screened out in step 2 of Example 3 above were streaked and activated, and a single colony was picked from the plate and inoculated into 5 mL LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 220 rpm, and then transferred to 20 mL LB liquid medium (containing 50 μg / mL Kan) at a 1%-2% (V / V) inoculum, and cultured until OD 600The concentration of isopropyl-β-D-thiogalactoside (IPTG) was 0.6-0.8, and the final concentration of 0.1 mM was added. The expression was induced for 12 hours at 20°C and 220 rpm. The induced bacterial solution was centrifuged at 4°C and 12000 rpm to obtain wet bacteria, which were washed twice with Tris-HCl buffer (100 mM, pH 8.5), and then resuspended with an appropriate volume of Tris-HCl buffer. The resuspended solution was placed in an ice water bath and ultrasonically disrupted (set power 400W, stop for 3 seconds for each ultrasonic disruption for 1s, a total of 3min) to obtain a cell disruption solution, and the supernatant obtained by centrifugation at 4°C and 12000 rpm was the crude enzyme solution of the mutant to be tested.

[0080] The activity test reaction was carried out in a 2 mL ep tube. The substrate mother solution (L-histidine final concentration 60 mM, β-alanine methyl ester final concentration 78 mM) and aminopeptidase were added, and Tris-HCl buffer (100 mM, pH 8.5) was added to make up to a total volume of 1 mL. The reaction was carried out at 20°C and 220 rpm for 30 minutes. After the reaction, 100 μL of 3M HCl was added to terminate the reaction. The sample to be tested was obtained after centrifugation and filtration. The enzyme activity was determined by measuring the amount of carnosine produced in the reaction mixture. One unit of enzyme activity (1U) is defined as the amount of enzyme required to produce 1 μmol of carnosine per minute under the above standard conditions. All determinations were repeated three times. The substrate and product of the reaction were detected by UPLC using a ZORBAX SB-Aq (4.6*150 mm 5 μm) column at a wavelength of 210 nm. The mobile phase was 50 mM NaClO4, the flow rate was 1 mL / min, and the temperature was 25°C.

[0081] Screening, verification, and activity testing were performed according to the above method, and finally 27 mutants with significantly higher conversion rates than the wild type were obtained. The results are shown in Table 4, among which W90H / N105Y / D132N / E278D / I325Q had the highest conversion rate, and the enzyme activity per unit volume of fermentation broth was 12.5U / mL, which was 8.3 times that of the wild type aminopeptidase. The conversion rate was calculated as follows:

[0082] Table 4. Statistical results of the conversion rate of carnosine synthesis catalyzed by aminopeptidase mutants Aminopeptidase L-histidine conversion rate wild type 41% W90H 60% F98L 55% F117T 48% F117C 52% D132N 51% P137Q 49% D144M 55% D144P 52% I150H 51% M183F 53% H186R 58% A223S 66% I325Q 49% C82K / F117C 54% W90H / N105Y 74% W90H / I325Q 69% F98L / I150H 60% F117T / G145H 63% D132N / E278D 65% D132N / G283S 60% D132V / S319C 61% M183F / I325Q 68% W90H / D132N / E278D 77% W90H / D132N / I325Q 78% W90H / N105Y / I325Q 85% W90H / N105Y / D132N / I325Q 89% W90H / N105Y / D132N / E278D / I325Q 95% Example 5: Preparation of L-carnosine by wild-type aminopeptidase

[0083] Culture of bacteria and preparation of crude enzyme solution: After streaking the glycerol tube containing the engineered bacteria of wild-type aminopeptidase, a single colony was inoculated into 20 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C overnight with shaking. Transfer 1% of the inoculum to 1 L of fresh LB liquid medium containing 50 μg / ml kanamycin, and culture at 37°C with shaking until OD 600 When the concentration reached about 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the expression was induced overnight at 20°C. After the culture was completed, the culture solution was centrifuged at 4°C and 6500 rpm for 10 min, the bacteria were collected, and the bacterial sludge was resuspended in 100 mM pH 7.5 potassium phosphate buffer at a ratio of 1:10 (m / V), and the crude enzyme solution was prepared by high-pressure homogenization.

[0084] The reaction system for preparing L-carnosine was 150 mL, containing 200 mM L-histidine, 330 mM β-alanine methyl ester, and 5 mL of 300U / mL aminopeptidase crude enzyme solution. The reaction temperature was controlled at 10-30°C by a condensing cycler, and the pH was controlled at 7.5 by adding ammonia water during the reaction. After 3 hours of reaction, the residual concentration of L-histidine and the generated concentration of carnosine were detected by UPLC.

[0085] The final reaction results were as follows: conversion rate 42%, and carnosine production concentration 15.75 g / L. Example 6: Preparation of L-carnosine by aminopeptidase mutants

[0086] Culture of bacteria and preparation of crude enzyme solution: After activating the glycerol tubes of engineered bacteria containing aminopeptidase mutants W90H / N105Y, W90H / D132N / E278D, W90H / D132N / I325Q, W90H / N105Y / I325Q, W90H / N105Y / D132N / I325Q, and W90H / N105Y / D132N / E278D / I325Q by streaking on plates, pick a single colony and inoculate it into 20 mL LB liquid culture medium containing 50 μg / mL kanamycin, and shake culture at 37°C overnight. Transfer the inoculation volume to 1 L of fresh LB liquid culture medium containing 50 μg / ml kanamycin at a rate of 1%, and shake culture at 37°C until OD 600 When the concentration reached about 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the expression was induced overnight at 20°C. After the culture was completed, the culture solution was centrifuged at 4°C and 6500 rpm for 10 min, the bacteria were collected, and the bacterial sludge was resuspended in 100 mM pH7.5 potassium phosphate buffer at a ratio of 1:10 (m / V), and the crude enzyme solution was prepared by high-pressure homogenization.

[0087] The reaction system for preparing L-carnosine is 150 mL, containing 200 mM L-histidine, 330 mM β-alanine methyl ester, and 5 mL 2500U / mL crude aminopeptidase enzyme solution. The reaction temperature is controlled at 10-30°C by a condensing cycler, and the pH is controlled at 7.5 by adding ammonia water during the reaction. After 3 hours of reaction, the residual concentration of L-histidine and the concentration of carnosine generated are detected by UPLC. The schematic diagram of the route for synthesizing L-carnosine by aminopeptidase mutants is shown in the figure. Figure 1 shown.

[0088] The final reaction results are shown in the following table: Table 5. Statistical results of the conversion rate of carnosine synthesis catalyzed by aminopeptidase mutants Aminopeptidase mutant Conversion rate Carnosine production concentration (g / L) W90H / N105Y 76% 31.24 W90H / D132N / E278D 79% 33.78 W90H / D132N / I325Q 77% 33.17 W90H / N105Y / I325Q 87% 37.97 W90H / N105Y / D132N / I325Q 89% 39.45 W90H / N105Y / D132N / E278D / I325Q 95% 42.62 UPLC detection of carnosine produced by the W90H / N105Y / D132N / E278D / I325Q mutant. The results are as follows Figure 3 As shown, the left side of the figure is the substrate L-histidine peak, the middle is the product L-carnosine peak, and the right side is the impurity peak. The reaction conversion rate is high and the impurity yield is low.

[0089] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. An aminopeptidase mutant, characterized in that: The aminopeptidase mutant is a protein obtained by performing mutations on the wild-type aminopeptidase with the amino acid sequence shown in SEQ ID No. 2, and the aminopeptidase mutant includes W90H and N105Y mutations.

2. The aminopeptidase mutant according to claim 1, characterized in that The aminopeptidase mutant includes any one of the following mutations: W90H / N105Y / I325Q, W90H / N105Y / D132N / I325Q or W90H / N105Y / D132N / E278D / I325Q.

3. An isolated polynucleotide, characterized in that The polynucleotide encodes the aminopeptidase mutant according to any one of claims 1 to 2.

4. The isolated polynucleotide according to claim 3, characterized in that The polynucleotide comprises the nucleotide sequence shown as SEQ ID No.

13.

5. A nucleic acid construct, characterized in that The nucleic acid construct comprises the isolated polynucleotide of claim 3 or 4.

6. The nucleic acid construct according to claim 5, characterized in that The nucleic acid construct is a eukaryotic expression vector or a prokaryotic expression vector.

7. The nucleic acid construct according to claim 6, characterized in that The prokaryotic expression vector is selected from an Escherichia coli expression vector, a Bacillus subtilis expression vector or a Streptomyces expression vector.

8. An engineered cell, characterized in that: The engineered cell contains the nucleic acid construct according to any one of claims 5 to 7 or the exogenous isolated polynucleotide according to claim 3 integrated into its genome.

9. The method for preparing an aminopeptidase mutant according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: culturing the engineered cells according to claim 8, inducing the expression of aminopeptidase and continuing the culturing, and after the culturing is completed, collecting and separating the bacterial cells to obtain the enzyme solution of the aminopeptidase.

10. Use of the aminopeptidase mutant according to any one of claims 1 to 2, the isolated polynucleotide according to claim 3 or 4, the nucleic acid construct according to any one of claims 5 to 7 or the engineered cell according to claim 8 in the preparation of L-carnosine.

11. A method for synthesizing L-carnosine, characterized in that: The method comprises the following steps: mixing the aminopeptidase mutant according to any one of claims 1 to 2 or the aminopeptidase obtained by the preparation method according to claim 9 with L-histidine and β-alanine methyl ester for reaction, and collecting the supernatant after the reaction to obtain L-carnosine.

12. The method according to claim 11, characterized in that Also includes any one or more of the following features: 1) The concentration of aminopeptidase in the reaction system is 5~100U / mL; 2) At the beginning of the reaction, the concentration of L-histidine is 150-250 mM; 3) At the beginning of the reaction, the concentration of the β-alanine methyl ester is 300-350 mM; 4) The reaction temperature is 10~30℃; 5) The reaction pH is 7.3~7.7; 6) The reaction time is 2.5~3.5 hours.

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