CN115851691A

CN115851691A - Aspartic acid ammonia lyase mutant and application thereof

Info

Publication number: CN115851691A
Application number: CN202211233368.3A
Authority: CN
Inventors: 汪钊; 曾茵; 章银军; 应向贤; 林行; 张连春; 陈梁; 刘学愚; 娄波; 何敏; 汪瑾; 汪军; 殷杭华; 白彦兵; 伊滨
Original assignee: Hangzhou Xinfu Science & Technology Co ltd; Zhejiang University of Technology ZJUT
Current assignee: Hangzhou Xinfu Science & Technology Co ltd; Zhejiang University of Technology ZJUT
Priority date: 2022-10-10
Filing date: 2022-10-10
Publication date: 2023-03-28

Abstract

The invention relates to the technical field of biology, in particular to an aspartate ammonia lyase mutant and an application thereof. The aspartate ammonia lyase mutant LmAspA-Mut has the capability of efficiently catalyzing high-concentration acrylic acid ammonification to generate beta-alanine. Wet thallus E.coliBL21 (DE 3)/pET 28a-LmAspA-Mut for expressing the mutant LmAspA-Mut is used as a biocatalyst, 315g/L of acrylic acid and 125g/L of ammonia water are used as substrates to construct a reaction system, the yield of the beta-alanine is up to 99%, and the product concentration is 385.5g/L.

Description

Aspartic acid ammonia lyase mutant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an aspartic acid ammonia lyase mutant and application thereof.

Background

Beta-alanine, also known as 3-aminopropionic acid, is a non-protein amino acid. Beta-alanine was discovered in uracil degradation products by Rose and Monroe, which were equal to 1972, is a precursor of various substances, and has been widely used in the fields of medicines, foods, chemicals, feeds, and the like. In the organism, beta-alanine can regulate metabolism in vivo and play a role of neurotransmitter or hormone regulator; in the field of animal breeding and production, the beta-alanine can improve the conversion capacity of animal feed, thereby improving the feed production performance; in the aspect of body movement, as the beta-alanine is a precursor required by in-vivo carnosine synthesis and is supplemented with a proper amount of beta-alanine, the content of the carnosine in-vivo muscles can be obviously improved; in the food field, the beta-alanine can be used as a flavoring agent and can effectively prevent food from being oxidized; in the environmental field, poly beta-alanine as high-efficiency purification coagulant can be used for water purification and clarification; in the pharmaceutical field, the main role of beta-alanine is the synthesis of pantothenic acid and calcium pantothenate.

The production methods of beta-alanine include chemical synthesis and biological synthesis, wherein the biological synthesis can be divided into microbial fermentation and biological catalysis. Currently, chemical synthesis is still the mainstream method in the production of beta-alanine, and the more representative synthesis methods are acrylic acid method and acrylonitrile method. The chemical method for synthesizing the beta-alanine has the advantages of low cost of production raw materials, high yield, mature technical process and the like, but the chemical method for synthesizing the beta-alanine usually needs to provide high-temperature and high-pressure environment or needs strong acid and strong alkali, has the defects of high energy consumption, harsh production conditions, more wastes and the like, can cause very serious influence on the environment, and does not meet the requirement of green development. Therefore, there is a need to establish an environmentally friendly method for producing beta-alanine. The biocatalysis method has the advantages of short production period, low production cost, less by-products, easy product separation, wide catalyst source and the like, and gradually becomes the hot research of the beta-alanine production at present. The production of beta-alanine by biological catalysis method mainly has two technological routes, one is taking beta-aminopropionitrile as substrate, and the other is taking L-aspartic acid or fumaric acid as substrate. The process route for producing beta-alanine by taking beta-aminopropionitrile as a substrate has low conversion rate, is limited by the self-synthesis way of microorganisms, can generate substrate inhibition, cannot improve the product concentration, has more byproducts, is difficult to separate and purify, and is not suitable for industrial production. A process route using L-aspartic acid or fumaric acid as a substrate mainly relates to L-aspartic acid-alpha-decarboxylase and aspartate ammonia lyase. L-aspartate-alpha-decarboxylase is responsible for decarboxylation of L-aspartate to beta-alanine in the biosynthetic pathway. Wild-type L-aspartic acid-alpha-decarboxylase has the problems of low activity, poor thermal stability, mechanism inactivation and the like, although a large number of targeted modification results are reported, the improvement range is not large, and the cost is high when the L-aspartic acid is used as a catalytic substrate.

Disclosure of Invention

In view of this, the invention provides an aspartate ammonia-lyase mutant and an application thereof. The mutant is LmAspA-Mut and has the capability of efficiently catalyzing high-concentration acrylic acid ammonification to generate beta-alanine

In order to achieve the above object, the present invention provides the following technical solutions:

an aspartate ammonia lyase mutant, which is obtained by mutating at least one site of T189V, M323I, K326M and N328A of wild type aspartate ammonia lyase.

The invention selects aspartate ammonia lyase LmAspA from Lysinibacillus mannius as a research object, and successfully obtains a mutant containing four-site mutation through homologous modeling, molecular docking and mutation introduction, wherein the four-site mutation comprises at least one of T189V, M323I, K326M and N328A.

Wherein, the mutant with the four mutations is marked as LmAspA-Mut, and the amino acid sequence of the mutant is shown as SEQ ID NO. 4.

The aspartate ammonia lyase LmAspA does not have the activity of catalyzing the ammonification of acrylic acid, and the aspartate ammonia lyase mutant LmAspA-Mut obtained by the transformation of the invention shows extremely high activity in the reaction of catalyzing the ammonification of acrylic acid to generate beta-alanine, thereby being a biocatalyst with extremely high application potential.

In the invention, the amino acid sequence of the wild type aspartate ammonia lyase is shown as SEQ ID NO. 1.

The invention also provides a nucleic acid for coding the aspartate ammonia lyase mutant.

In some embodiments, the nucleic acid encoding the mutant aspartate ammonia lyase has the nucleotide sequence shown in SEQ ID No. 5.

The invention also provides biological material containing the nucleic acid, wherein the biological material is an expression vector or a recombinant host.

In some embodiments, the expression vector containing the coding gene of the aspartate ammonia lyase mutant LmAspA-Mut is obtained by the following method:

the aspartate ammonia lyase LmAspA gene shown in SEQ ID NO.2 is subjected to codon optimization to obtain the optimized LmAspA gene, the nucleotide sequence is shown as SEQ ID NO.3, and the amino acid sequence corresponding to the aspartate ammonia lyase LmAspA is shown as SEQ ID NO. 1. The codon-optimized LmAspA gene (SEQ ID NO. 3) is artificially synthesized and inserted between EcoRI and HindIII of pET28a to obtain a recombinant plasmid pET28a-LmAspA. The recombinant plasmid pET28a-LmAspA is used as a template, a primer with mutant base is utilized to amplify the whole plasmid through reverse PCR, the obtained PCR product is digested and methylated by Dpn I enzyme, the enzyme digestion product is converted into Escherichia coli E.coli BL21 (DE 3), and the engineering bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut containing the aspartate ammonia lyase mutant LmAspA-Mut gene can be obtained, wherein the expression vector containing the recombinant aspartate ammonia lyase mutant coding gene is named as pET28a-LmAspA-Mut.

The invention also provides the application of the aspartate ammonia lyase mutant, the nucleic acid or the biological material in preparing beta-alanine.

Specifically, in the application, the aspartate ammonia lyase mutant, the nucleic acid or the biological material is used for catalyzing the preparation of beta-alanine by adding ammonia into acrylic acid.

The invention also provides a method for preparing beta-alanine, which comprises the following steps:

fermenting and culturing a recombinant host expressing the aspartate ammonia lyase mutant to obtain wet thalli;

the wet bacteria or the aspartate ammonia lyase mutant is used for catalyzing the reaction of acrylic acid and ammonia water, and the reaction liquid is separated and purified to obtain the beta-alanine.

In some embodiments, the method for preparing the wet biomass comprises: the recombinant host expressing the aspartate ammonia lyase mutant is used for preparing seed solution, then the seed solution is cultured and induced to express to obtain induced culture solution, the induced culture solution is centrifuged, supernatant liquid is discarded, and wet thalli are collected.

In some embodiments, the method for preparing wet cells comprises:

encoding aspartate-containing ammonia lyase mutantsInoculating genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut into LB liquid culture medium containing 100 mu g/mL kanamycin, culturing at 37 ℃ for 12h to obtain seed liquid, inoculating the seed liquid into fresh LB liquid culture medium containing 100 mu g/mL kanamycin in an inoculum size of 2% in volume concentration, and culturing at 37 ℃ to OD ₆₀₀ 0.5-0.7, adding IPTG with final concentration of 0.2mM, inducing at 24 deg.C for 10h to obtain inducing culture solution, centrifuging the inducing culture solution at 4 deg.C and 8000rpm for 10min, discarding supernatant, and collecting wet thallus.

In the method for preparing beta-alanine provided by the invention, the reaction is as follows: reacting for 5 hours at the temperature of 25-60 ℃ and the rpm of 50-400; in some embodiments, the reaction is: the reaction was carried out at 40 ℃ and 150rpm for 5h. In some embodiments, in the reaction system, the amount of the catalyst is 40g/L calculated by wet bacteria, the amount of the substrate acrylic acid is 315g/L, and the amount of the ammonia water is 125g/L.

In the present invention, the separation and purification of the reaction solution comprises: separating and purifying the product by an organic solvent precipitation method; in some embodiments, the reaction solution is purified by separation as follows: at V _{Anhydrous ethanol} ：V _{Reaction supernatant} Is 8:1, standing for 2 hours at room temperature, filtering and removing supernatant, and drying a crystallized product at 60 ℃ to obtain the beta-alanine.

Compared with the prior art, the invention has the following beneficial effects: the aspartate ammonia lyase LmAspA does not have the capability of catalyzing the ammonification of acrylic acid to generate beta-alanine, and the four-point mutation T189V/M323I/K326M/N328A is introduced, so that the aspartate ammonia lyase mutant LmAspA-Mut has the capability of efficiently catalyzing the ammonification of high-concentration acrylic acid to generate beta-alanine. Wet thalli E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut for expressing the mutant LmAspA-Mut is used as a biocatalyst, 315g/L of acrylic acid and 125g/L of ammonia water are used as substrates to construct a reaction system, the reaction is carried out for 5 hours at 40 ℃ and 150rpm, the yield of beta-alanine is up to 99%, and the product concentration is 385.5g/L.

Drawings

FIG. 1 is a schematic diagram of the aspartic acid ammonia lyase catalyzing the alanine plus ammonia synthesis of beta-alanine;

FIG. 2 is an agarose gel electrophoresis of aspartate ammonia lyase encoding genes LmAspA and pET-28a; lane M is marker; lane 1 is aspartate ammonia lyase encoding gene LmAspA; lane 2 is pET-28a;

FIG. 3 is an agarose gel electrophoresis of plasmid pET28a-LmAspA after reverse PCR amplification; lane M is marker;

lanes

1, 2, 3 are plasmid inverse PCR products;

FIG. 4 is an SDS-PAGE pattern of the aspartate ammonia lyase mutant LmAspA-Mut; lane M is marker; lane 1 is crude enzyme solution prepared from E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut without induction; lane 2 is the crude enzyme solution prepared from the induced wet thallus E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut, and lane 3 is the pure enzyme solution obtained by separating and purifying the aspartate ammonia lyase mutant LmAspA-Mut;

FIG. 5 is a HPLC detection profile of beta-alanine;

FIG. 6 is a HPLC detection standard curve for beta-alanine;

FIG. 7 shows the process of the aspartic acid ammonia lyase mutant LmAspA-Mut catalyzing the reaction of acrylic acid with ammonia;

FIG. 8 is an HPLC detection profile of a 99% pure beta-alanine standard;

FIG. 9 is a HPLC detection profile of beta-alanine separated and purified from the reaction solution;

FIG. 10 shows beta-alanine standards ¹ H NMR s (a) and ¹³ a C NMR (b) spectrum;

FIG. 11 shows the separation and purification of the resulting beta-alanine from the reaction mixture ¹ H NMR s (a) and ¹³ c NMR (b) spectrum.

Detailed Description

The invention provides an aspartate ammonia-lyase mutant and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1: construction of genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA

The coding gene (nucleotide sequence is shown as SEQ ID NO. 2) of the LmAspA of aspartate ammonia lyase derived from Lysinibacillus mannaus is subjected to codon optimization, the nucleotide sequence of the LmAspA gene after the codon optimization is shown as SEQ ID NO.3, the amino acid sequence corresponding to the LmAspA of the aspartate ammonia lyase is shown as SEQ ID NO.1, and the LmAspA is synthesized by Hangzhou Okagaku biological technology company. The codon-optimized LmAspA coding gene (SEQ ID NO. 3) is artificially synthesized and inserted between EcoRI and HindIII of pET28a to obtain a recombinant plasmid pET28a-LmAspA, and an agarose gel electrophoresis picture of the aspartate ammonia lyase coding genes LmAspA and pET-28a is shown in figure 2.

The synthesized recombinant expression plasmid pET28a-LmAspA is taken out by 5 muL and added into the competence of 50 muL E.coli BL21 (DE 3), the tube wall is flicked and mixed evenly, and the mixture is placed on ice for 30min. The mixture was heated in a water bath at 42 ℃ for 90s and immediately placed on ice for 5min. 900. Mu.L of LB liquid medium was added to the tube and shake-cultured at 37 ℃ for 1 hour. The culture broth was centrifuged at 4000rpm for 3min, and 900. Mu.L of the supernatant was taken out. The cells were suspended in the remaining medium, and 100. Mu.L of the suspension was applied to LB solid medium containing 100. Mu.g/mL kanamycin. Culturing in 37 deg.C incubator overnight for 14h to obtain genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA.

Composition of LB liquid medium: 5g/L of yeast extract, 10g/L of tryptone, 10g/L of NaCl, distilled water as solvent and 7.0-7.5 of pH.

Example 2: construction of recombinant expression plasmid pET28a-LmAspA-Mut and genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut

1. Recombinant expression plasmid pET28a-LmAspA-Mut

Primers were designed using the plasmid pET28a-LmAspA prepared in example 1 as a template, and single-point mutation, three-point combination mutation and four-point combination mutation of the amino acid at the critical position (T189/M323/K326/N328) were performed to find the optimal combination. The whole plasmid was cloned by inverse PCR and transformed into E.coli BL21 (DE 3). Extracting plasmid sequencing, and analyzing a sequencing result by using software, wherein the sequence contains an open reading frame with the length of 1410bp, and mutant plasmids are obtained, wherein the 189 th threonine is successfully changed into valine, the 323 rd methionine is mutated into isoleucine, the 326 th lysine is mutated into methionine, and the 328 th asparagine is mutated into alanine. And then transforming the mutant plasmid into E.coli BL21 (DE 3), extracting the plasmid and sequencing to successfully obtain the mutant plasmid pET28a-LmAspA-Mut, wherein the nucleotide sequence of the mutant LmAspA-Mut is shown as SEQ ID NO.5, and the amino acid sequence of the mutant LmAspA-Mut is shown as SEQ ID NO. 4. The primers are as follows:

M1-F 5’-GGTCGCACTCATCTGCAGGATGCAGTTCCG-3’(SEQ ID NO.6)；

M1-R 5’-CAGATGAGTGCGACCCATTTTAATGATGCC-3’(SEQ ID NO.7)；

M2-F 5’-AGCATTATGCCGGGTATGGTTGCACCGGTT-3’(SEQ ID NO.8)；

M2-R 5’-ACCCGGCATAATGCTGCTGCCAGGCTGACG-3’(SEQ ID NO.9)；

M3-F 5’-CCGGGTAAGGTTGCACCGGTTATGCCGGAA-3’(SEQ ID NO.10)；

M3-R 5’-TGCAACCTTACCCGGCATAATGCTGCTGCC-3’(SEQ ID NO.11)；

M4-F 5’-AAGGTTAATCCGGTTATGCCGGAAGTTATG-3’(SEQ ID NO.12)；

M4-R 5’-AACCGGATTAACCTTACCCGGCATAATGCT-3’(SEQ ID NO.13)；

M5-F 5’-GGACGTACACATCTTCAAGATGCTGTTCCA-3’(SEQ ID NO.14)；

M5-R 5’-AAGATGTGTACGTCCCATTTTAATAATGCC-3’(SEQ ID NO.15)；

M6-F 5’-TCAATTATGCCAGGTATGGTGGCCCCTGTT-3’(SEQ ID NO.16)；

M6-R 5’-ACCTGGCATAATTGATGAACCAGGCTGTCT-3’(SEQ ID NO.17)；

M7-F 5’-CCAGGTAAAGTGGCCCCTGTTATGCCAGAA-3’(SEQ ID NO.18)；

M7-R 5’-GGCCACTTTACCTGGCATAATTGATGAACC-3’(SEQ ID NO.19)；

M8-F 5’-AAAGTGAACCCTGTTATGCCAGAAGTTATG-3’(SEQ ID NO.20)；

M8-R 5’-AACAGGGTTCACTTTACCTGGCATAATTGA-3’(SEQ ID NO.21)；

M9-F 5’-GGACGTACACATCTTCAAGATGCTGTTCCA-3’(SEQ ID NO.22)；

M9-R 5’-AAGATGTGTACGTCCCATTTTAATAATGCC-3’(SEQ ID NO.23)。

the reverse PCR amplification system is shown in Table 1.

TABLE 1 PCR amplification reaction System

The PCR reaction process is as follows: pre-denaturation at 95 ℃ for 2min; thereafter, the resulting mixture was completely denatured at 95 ℃ for 15s, annealed at 62 ℃ for 15s, and elongated at 72 ℃ for 6min. Repeating the denaturation to extension step for 30 times, extending for 10min at 72 ℃, and cooling to 4 ℃. The PCR product was detected by 0.8% agarose gel electrophoresis, and a bright band was observed at about 6500bp in FIG. 3, which is consistent with the theoretical value of the plasmid.

2. Genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut

The PCR product was digested at 37 ℃ for 1h to remove methylated template, and the digestion system is shown in Table 2.

TABLE 2 digestion System for methylated template in PCR products

And (3) directly transforming and expressing a PCR product (the nucleotide sequence of the mutant gene LmAspA-Mut carried by the plasmid is shown in SEQ ID NO.5, and the amino acid sequence of the mutant gene LmAspA-Mut is shown in SEQ ID NO. 4) after the Dpn I enzyme digestion to obtain an E.coli genetic engineering bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut. The transformant is inoculated into LB liquid culture medium containing 100 mug/mL kanamycin after colony PCR verification, cultured for 12h at 37 ℃, centrifuged to collect thalli, extracted with plasmid and sent to sequencing. The sequencing result is analyzed by software, the threonine at the 189 th position is successfully changed into valine, the methionine at the 323 th position is mutated into isoleucine, the lysine at the 326 th position is mutated into methionine, and the asparagine at the 328 th position is mutated into alanine.

Example 3: inducible expression, separation and purification of aspartate ammonia lyase LmAspA and mutant LmAspA-Mut thereof

Obtaining wet thalli by shake flask culture: inoculating engineering bacteria E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut containing aspartate ammonia lyase mutant coding gene into LB liquid culture medium containing 100 mu g/mL kanamycin, culturing at 37 ℃ for 12h to obtain seed liquid, inoculating the seed liquid into fresh LB liquid culture medium containing 100 mu g/mL kanamycin in an inoculation amount with the volume concentration of 2%, and culturing at 37 ℃ until OD is achieved ₆₀₀ 0.5-0.7, adding IPTG with final concentration of 0.2mM, inducing at 24 deg.C for 10h to obtain inducing culture solution, centrifuging at 4 deg.C and 8000rpm for 10min, discarding supernatant, and collecting wet thallus.

And (3) culturing in a fermentation tank to obtain wet thalli: the genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut constructed in example 2 was inoculated into LB liquid medium containing 100. Mu.g/mL kanamycin and cultured at 37 ℃ for 12 hours to obtain a seed solution. Operating in a clean bench, 3mL Kana resistance was added to MgSO ₄ In solution, the inoculating loop is ignited, and MgSO is placed in the fire circle ₄ The Kana solution was poured into the medium, kanamycin was added to the fermentation medium to a final concentration of 100. Mu.g/mL, and the seed solution was inoculated from the fire ring to the fermentor at an inoculum size of 3%. Adjusting the rotation speed to 600rpm, adjusting the ventilation rate to 6L/min, and culturing at 37 deg.C to OD ₆₀₀ After 4-6 hours (about 3.5 hours), IPTG is added into 50mL LB liquid culture medium in a clean bench operation, the LB culture medium containing IPTG is added into a fermentation tank through an inoculation port under the protection of a fire ring, and the final concentration of the IPTG in the fermentation culture medium is 0.2mM. Reducing the temperature of the fermentation tank to 24 ℃ through a circulating water system, adjusting the rotating speed to 500rpm, continuously culturing for 10h, and inducing the thalli to express the protein. Pouring the fermentation liquid into a centrifuge cup, centrifuging at 8000rpm and 4 deg.C for 10min with a high-speed refrigerated centrifuge, and discarding the supernatantThe obtained cells were washed with 50mM Tris-HCl buffer (pH 8.0) in a resuspension manner, and the cell suspension was put into a 50mL centrifuge tube, centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was discarded to collect wet cells.

Adding appropriate amount of Tris-HCl (pH 8.0) buffer solution into 1g of wet thallus according to the proportion of adding 20ml of Tris-HCl buffer solution (pH 8.0), carrying out ultrasonic disruption for 15min (working 1s and intermittent 3 s) under 40% power, centrifuging the disruption solution for 10min at 4 ℃ and 8000rpm, and repeatedly centrifuging for three times to obtain a supernatant crude enzyme solution. According to Ni-NTA metal chelate affinity chromatography (from Bio-Rad, for Ni) ²⁺ Column, column inner diameter 1.6cm, column height 15 cm) instruction, taking 15mL of crude enzyme solution to load to pre-balance Ni ²⁺ And eluting the hybrid protein and the target protein in the column by using eluents (composed of corresponding concentration of imidazole and 300mM of sodium chloride, solvent is 50mM Tris-HCl buffer solution, pH 8.0) containing 5mM of imidazole, 50mM of imidazole, 100mM of imidazole, 200mM of imidazole and 500mM of imidazole in sequence, wherein the elution speed is 2.5mL/min, each concentration of eluent elutes 3 column volumes, collecting the effluent corresponding to the eluent containing 200mM of imidazole, centrifuging the eluent at 4 ℃ and 5000rpm by using an ultrafiltration tube with the molecular weight cutoff of 10kDa for 30min for desalination and concentration, and taking trapped fluid, namely LmAspA-Mut (T189V/M323I/K326M/N328A) enzyme solution to store at-20 ℃ for later use.

The purity of the pure enzyme solution of the aspartate ammonia lyase mutant LmAspA-Mut is verified by SDS-PAGE gel electrophoresis, and the result of the SDS-PAGE gel electrophoresis is shown in figure 4. The aspartic acid ammonia lyase mutant LmAspA-Mut is a single band after SDS-PAGE electrophoresis. The subunit theoretical size of the aspartate ammonia lyase mutant LmAspA-Mut is about 52kDa, and the apparent size of the mutant on SDS-PAGE electrophoresis accords with the theoretical molecular weight.

Similarly, wet cells of E.coli BL21 (DE 3)/pET 28a-LmAspA, which induced expression of LmAspA, were obtained according to the procedure of example 3 and used as a control for whole cell catalytic reaction.

Example 4: HPLC analysis of beta-alanine in process of catalyzing acrylic acid to generate beta-alanine by adding ammonia to generate beta-alanine through enzyme method

Preparing a mobile phase: phase A: 2.05g of anhydrous sodium acetate was weighed, 1.5mL of glacial acetic acid was transferred, 1L of ultrapure water was added thereto, and the mixture was dissolved by stirring and then filtered through a 0.22 μm aqueous membrane. Phase B: accurately measuring 1L of liquid-phase methanol, and filtering with 0.45 μm organic filter membrane. A. And uniformly mixing the phase B according to the formula (v: v) 1, and performing ultrasonic treatment in an ultrasonic cleaning instrument for 30min to obtain a mobile phase.

HPLC detection conditions: the chromatographic column is Welchrom

A (250X 4.6/5 μm) column; the flow rate of the mobile phase is 1mL/min; the detection wavelength is 360nm; the detection temperature was set at 40 ℃. According to the peak-out time of the beta-alanine standard sample with different concentrations, the retention time of the beta-alanine is determined to be 6.194min, and the retention time of the derivatization reagent is determined to be 4.515min (figure 5). The retention times of 2.713, 3.148, 3.293, 5.822, 6.589 and 9.668min are all the by-product peaks brought by derivatization reaction.

Preparing 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06M beta-alanine water solution, respectively taking 100 mu L into a 1.5mLEP tube, adding 100 mu L Na ₂ CO ₃ /NaHCO ₃ Buffer (pH 9.0), 100. Mu.L 1%2, 4-dinitrofluorobenzene-acetonitrile solution, reacted in a metal bath at 60 ℃ in the dark for 1 hour, and then 700. Mu.L NaH was added ₂ PO ₄ /Na ₂ HPO ₄ Buffer (pH 7.0). Centrifuging at 12000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, introducing into brown liquid phase bottle, detecting with HPLC, and determining beta-alanine peak time and peak area corresponding to each concentration. The β -alanine concentration was plotted against the corresponding peak area to obtain a standard curve of β -alanine concentration against peak area, as shown in fig. 6. The standard curve equation is y =7.0407 × 10 ⁸ X+3.3855×10 ⁶ Y is the peak area, x is the beta-alanine concentration (in M), R ² 0.9992, the linear relationship is good.

The catalytic reaction solution was centrifuged at 12000rpm for 10min. 100 mu L of supernatant was taken and added to 900 mu L of ultrapure water, and the dilution was repeated once for a total of 100 times. 100. Mu.L of the diluted reaction solution was put into a 1.5mL EP tube, and 100. Mu.L of Na was added ₂ CO ₃ /NaHCO ₃ Buffer (pH 9.0), 100. Mu.L 1%2, 4-dinitrofluorobenzene-acetonitrile solution, reacted in a metal bath at 60 ℃ in the dark for 1 hour, and then 700. Mu.L NaH was added ₂ PO ₄ /Na ₂ HPO ₄ Buffer (pH 7.0). Centrifuging at 12000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, and detecting the peak area of beta-alanine corresponding to each mutant catalytic reaction solution by HPLC to compare catalytic ability.

Example 5: aspartic acid ammonia lyase mutant LmAspA-Mut for catalyzing acrylic acid and adding ammonia to prepare beta-alanine

Wet thalli obtained by fermentation culture of engineering bacteria E.coli BL21 (DE 3)/pET 28a-LmAspA-Mut containing aspartate ammonia lyase mutant coding genes is used as a catalyst, the dosage of the catalyst is 40g/L, 315g/L of acrylic acid and 125g/L of ammonia water are added as substrates, and water is added to supplement the reaction system to 10mL. And (3) reacting the constructed 10mL catalytic reaction system in a water bath shaking table at 40 ℃ and 150rpm, taking 10 mu L of reaction liquid every 1h to dilute by 100 times, carrying out DNFB derivatization reaction, detecting the content of beta-alanine by HPLC, calculating the concentration of the beta-alanine according to a standard curve, and further calculating the product yield. The relationship between the product yield and the reaction time is shown in FIG. 7, in a 10mL reaction system, when the reaction temperature is 40 ℃, the yield of beta-alanine can reach 99% within 5h, and the corresponding product concentration is 385.5g/L. Under the same condition, wet thalli obtained by fermentation culture of an engineering bacterium E.coli BL21 (DE 3)/pET 28A-LmAspA containing an aspartate ammonia lyase coding gene is used as a reference, and the generation of beta-alanine is not detected in a reaction solution, which indicates that the introduced mutation T189V/M323I/K326M/N328A endows the aspartate ammonia lyase mutant AspA-Mut with the capability of efficiently catalyzing high-concentration acrylic acid to generate beta-alanine by adding ammonia.

Centrifuging the reaction solution to collect the supernatant according to V _Ethanol ：V _{Reaction supernatant} Adding absolute ethyl alcohol into the supernatant according to the proportion of 8. 3.56mg of a 99% pure beta-alanine standard sample and 3mg of purified beta-alanine powder were dissolved in 1mL of ultrapure water, 100. Mu.L of each was subjected to DNFB derivatization, and HPLC analysis was performed, and the detection patterns and results are shown in FIGS. 8 and 9. The retention time of the beta-alanine standard was 6.158min, corresponding to a peak area of 33744935 (FIG. 8), a retention time of purified beta-alanine of 6.166min, corresponding to a peak area of 28198334 (FIG. 9), and a purity of 98.17% of beta-alanine was calculated.

10mg of a 99% pure beta-alanine standard and purified beta-alanine powder were dissolved in 700. Mu.L of D ₂ In O, performing nuclear magnetic detection, and detecting the map as shown in figure 10 and figure 11, wherein the purified product and the beta-alanine standard sample ¹ H NMR spectrum and ¹³ the C NMR spectra are consistent, and the product is confirmed to be beta-alanine.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. An aspartate ammonia lyase mutant, which is obtained by mutating at least one site of T189V, M323I, K326M and N328A of wild type aspartate ammonia lyase;

the amino acid sequence of the wild aspartic acid ammonia lyase is shown in SEQ ID NO. 1.

2. The mutant aspartate ammonia lyase according to claim 1, wherein its amino acid sequence is represented by SEQ ID No. 4.

3. Nucleic acid encoding the aspartate ammonia-lyase mutant of claim 1 or 2.

4. The nucleic acid of claim 3, wherein the nucleotide sequence is set forth in SEQ ID No. 5.

5. A biological material comprising the nucleic acid of claim 3 or 4, said biological material being an expression vector or a recombinant host.

6. Use of an aspartate ammonia-lyase mutant according to claim 1 or 2, a nucleic acid according to claim 3 or 4, or a biomaterial according to claim 5 for the preparation of β -alanine.

7. The application according to claim 6, characterized in that it is: catalyzing acrylic acid and adding ammonia to prepare beta-alanine.

8. A method of producing beta-alanine, comprising:

fermenting and culturing a recombinant host expressing the aspartate ammonia lyase mutant of claim 1 or 2 to obtain wet thalli;

catalyzing the reaction of acrylic acid and ammonia water by using the wet thalli or the aspartate ammonia lyase mutant of claim 1 or 2, and separating and purifying reaction liquid to obtain the beta-alanine.

9. The method according to claim 8, wherein the method for preparing the wet cell mass comprises: preparing seed solution by using a recombinant host for expressing the aspartate ammonia lyase mutant of claim 1 or 2, culturing, performing induction expression to obtain an induction culture solution, centrifuging the induction culture solution, discarding supernatant, and collecting wet thalli.

10. The method of claim 8, wherein the reaction is: the reaction was carried out at 40 ℃ and 150rpm for 5h.