L-aspartate-beta-decarboxylase mutant, genetically engineered host bacteria and application thereof

Abstract

The application belongs to the technical field of biological catalysis, and discloses an L-aspartate-beta-decarboxylase mutant and a genetically engineered host bacterium and application thereof. The amino acid sequence of the L-aspartate-beta-decarboxylase mutant is shown in SEQ ID NO. 4. The results of enzymatic property research show that the optimal pH of the mutant is changed from 6.0 of the wild type to 7.0, and the mutant shows stronger tolerance in thermal stability and pH stability. The enzyme activity of the mutant to L-aspartate is 208.9 U / mg, which is 4.17 times of the wild type. Under the optimal reaction condition, the mutant recombinant bacterium is used for whole-cell catalysis of DL-aspartate to generate optically pure D-aspartate and L-alanine, and the mutant recombinant bacterium shows excellent catalytic activity. The conversion rate of 2 mol / L DL-aspartate in a 10 L reaction system is 100% in 5 h, and the enantiomeric excess value (e.e. value) of the products D-aspartate and L-alanine is greater than 99.9%. The application not only obtains a green and efficient biological catalyst, but also lays a theoretical and technical foundation for green biological manufacturing of chiral amino acids.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically to an L-aspartate-β-decarboxylase mutant and its genetically engineered host bacteria and their applications. Background Technology

[0002] D-Aspartic acid (D-Asp) is an important non-natural chiral amino acid with wide applications in the pharmaceutical and food industries. In the pharmaceutical field, D-aspartic acid is a key chiral intermediate for synthesizing various active pharmaceutical ingredients, such as N-methyl-D-aspartic acid (NMDA) receptor-related drugs, side chains of certain lactam antibiotics, and antiviral drugs. In the food industry, D-aspartic acid can be used to improve food texture and enhance flavor. With the rapid development of chiral drugs and functional foods, the market demand for high-purity D-aspartic acid continues to grow, making the development of efficient, economical, and environmentally friendly D-aspartic acid production methods of significant industrial value.

[0003] Currently, the main methods for producing D-aspartic acid include chemical resolution and enzymatic methods. Chemical resolution typically uses chiral resolving agents, which suffers from lengthy steps, expensive resolving agents, difficulty in achieving the required optical purity of the product, and environmental pollution, significantly limiting its industrial application. Enzymatic methods, due to their advantages of mild reaction conditions, high stereoselectivity, and low production costs, have become the mainstream technology for D-aspartic acid production. Among these, the selective decarboxylation of DL-aspartic acid using L-aspartate-β-decarboxylase (EC 4.1.1.12) is one of the most mature enzymatic processes.

[0004] L-Aspartate β-decarboxylase is a lyase with pyridoxal 5′-phosphate (PLP) as a cofactor. It can highly specifically catalyze the removal of the β-carboxyl group from L-aspartate in a mixture of DL-aspartate and D-aspartate, producing L-alanine and carbon dioxide, while having no effect on D-aspartate. After the reaction, the remaining target product in the system is D-aspartate, avoiding environmental pollution at the source and representing a highly promising green preparation method.

[0005] Although enzymatic synthesis has significant advantages, naturally derived L-aspartate-β-decarboxylases often fall short of the requirements for industrial production in terms of catalytic efficiency, stability, and applicability for D-aspartate synthesis. Therefore, developing L-aspartate-β-decarboxylase mutants with high catalytic activity and stability has great application value. Summary of the Invention

[0006] The purpose of this invention is to provide an L-aspartate-β-decarboxylase mutant, a genetically engineered host bacterium, and their applications, thereby solving the problems of poor catalytic efficiency and stability of naturally derived L-aspartate-β-decarboxylases in the prior art. This mutant exhibits excellent catalytic activity towards L-aspartate and DL-aspartate substrates.

[0007] To achieve the above objectives, the present invention provides an L-aspartate-β-decarboxylase mutant, wherein the amino acid sequence of the mutant contains an L31A mutation site compared with the amino acid sequence of the wild-type L-aspartate-β-decarboxylase shown in SEQ ID NO.2; the mutant has enzymatic activity that catalyzes the decarboxylation of L-aspartate in DL-aspartate to produce L-alanine, and the enzyme activity is at least 2 times higher than that of the wild type. After reacting with DL-aspartate as a substrate for 5 hours, L-aspartate in DL-aspartate is completely converted, and the conversion rate of DL-aspartate is 3.85 times higher than that of the parent.

[0008] Furthermore, the amino acid sequence of the mutant is shown in SEQ ID NO. 4.

[0009] Furthermore, the mutant has at least 95% homology with the amino acid sequence shown in SEQ ID NO.2.

[0010] According to a preferred embodiment of the present invention, the mutated amino acid in the L-aspartate-β-decarboxylase mutant is the 31st amino acid site.

[0011] Specifically, the leucine L at position 31 can be mutated into alanine A, phenylalanine F, threonine T, serine S, aspartic acid D, and lysine K, with the preferred mutation being alanine A.

[0012] A second aspect of the present invention provides a polynucleotide sequence encoding the L-aspartate-β-decarboxylase mutant described herein.

[0013] Furthermore, the polynucleotide sequence is as shown in SEQ ID NO.3, or is a codon-optimized nucleotide sequence for the Escherichia coli host that encodes an amino acid sequence with at least 95% homology to the sequence shown in SEQ ID NO.4.

[0014] Furthermore, the polynucleotide sequence is as shown in SEQ ID NO.1, or is a codon-optimized nucleotide sequence for the Escherichia coli host that encodes an amino acid sequence with at least 95% homology to the sequence shown in SEQ ID NO.2.

[0015] A third aspect of the present invention provides a recombinant expression vector comprising the polynucleotide sequence described herein.

[0016] Furthermore, the recombinant expression vector includes a regulatory element cis-linked upstream and / or downstream of the polynucleotide sequence for regulating the expression of the polynucleotide sequence in prokaryotic host cells.

[0017] Furthermore, the recombinant expression vector is a pET series prokaryotic expression vector; the regulatory element includes at least one of the T7 promoter, lac operon, ribosome binding site, and transcription terminator.

[0018] A fourth aspect of the present invention provides a genetically engineered host bacterium transformed with the recombinant expression vector described in the present invention.

[0019] Furthermore, the host cell is Escherichia coli.

[0020] The fifth aspect of this invention provides a method for preparing an L-aspartate-β-decarboxylase mutant, comprising the following steps: (1) Activate the genetically engineered host bacteria described in this invention, inoculate them into a liquid culture medium containing antibiotic resistance that matches the selection marker of the recombinant expression vector, and culture them until OD500. 600 =0.6, and an inducer was added to induce the strain to heterologously express the L-aspartate-β-decarboxylase mutant; the inducer was isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM; the induction temperature was 20 ℃ and the induction time was 20 h.

[0021] (2) Collect the induced bacterial cells, and obtain a crude enzyme solution containing L-aspartate-β-decarboxylase mutant after ultrasonic disruption and centrifugation solid-liquid separation. Purify the crude enzyme solution to obtain purified L-aspartate-β-decarboxylase mutant protein.

[0022] The sixth aspect of this invention provides the application of the L-aspartate-β-decarboxylase mutant and / or the genetically engineered host bacteria described in this invention in enzyme-catalyzed transformation using L-aspartate or DL-aspartate as substrates.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects: 1. Compared with the original L-aspartate-β-decarboxylase, the L31A mutant L-aspartate-β-decarboxylase of this invention significantly improves the conversion rate of the substrate L-aspartate. Furthermore, its enzyme activity reaches 208.9 U / mg, a 3.17-fold increase compared to the wild-type enzyme's 50.1 U / mg. This increased enzyme activity means higher catalytic efficiency per unit enzyme protein, resulting in a higher reaction rate with the same enzyme dosage.

[0024] 2. The L-aspartic acid-β-decarboxylase mutant L31A of the present invention uses 2 mol / L DL-aspartic acid as a substrate to construct a reaction system. Under the conditions of 37 ℃, pH 7.0, and 600 rpm for 5 h, L-aspartic acid in DL-aspartic acid is completely converted. After separation and purification, the enantiomeric excess value (ee value) of D-aspartic acid is >99.9%, and the enantiomeric excess value (ee value) of L-alanine is >99.9%.

[0025] 3. Compared with the original L-aspartate-β-decarboxylase, the L-aspartate-β-decarboxylase mutant L31A of the present invention has improved both thermal stability and pH stability to varying degrees. Moreover, the optimal pH of mutant L31A changes from 6.0 of wild type to 7.0, and the optimal acid-base environment for enzymatic reaction shifts towards neutral.

[0026] 4. The preferred catalytic temperature for the L-aspartic acid-β-decarboxylase mutant is 37 °C, and the preferred pH is 7.0. The reaction conditions are mild. Compared with the high temperature, high pressure, and precious metal catalysts required by traditional chemical synthesis methods, it has the advantages of low cost and environmental safety. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only drawings of some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is an SDS-PAGE electrophoresis image of the L-aspartate-β-decarboxylase and its mutants of the present invention, where the marker is the protein molecular weight standard, Lane 1 is the supernatant of wild-type cell lysate; Lane 2 is the purified wild-type protein; Lane 3 is the supernatant of L31A cell lysate; and Lane 4 is the purified L31A protein. Figure 2 This is a figure showing the results of the study on the optimal temperature of L-aspartate-β-decarboxylase and its mutants in this invention; Figure 3 This is a figure showing the results of the study on the optimal pH of L-aspartate-β-decarboxylase and its mutants in this invention; Figure 4 This is a figure showing the results of the study on the thermal stability of L-aspartate-β-decarboxylase and its mutants in this invention; Figure 5 This is a figure showing the results of the study on the pH stability of L-aspartate-β-decarboxylase and its mutants in this invention; Figure 6 This is a figure showing the results of research on the whole-cell catalysis of DL-aspartic acid by the L-aspartate-β-decarboxylase and its mutants of the present invention. Detailed Implementation

[0029] 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.

[0030] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0031] In this invention, the expression regulatory element refers to a functional nucleic acid sequence capable of driving the transcription and translation of the target coding nucleic acid molecule in the host cell, including but not limited to promoters, operons, ribosome binding sites, and transcription terminators.

[0032] In this invention, the cis-linking refers to the sequential linking of the expression regulatory element and the target coding nucleic acid molecule in a manner that enables the expression regulatory function, i.e., the operative linking conventionally referred to in the art.

[0033] In this invention, the recombinant expression vector refers to all recombinant nucleic acid molecules that can carry foreign genes into host cells and achieve their expression, including various types such as recombinant plasmids, recombinant bacteriophages, recombinant viral vectors, and recombinant artificial chromosomes.

[0034] The relevant sequences used in this invention are as follows: SEQ ID NO.1: SEQ ID NO.2: MSKDYQSLAKLSPFELKDELIKIASSDGNRLMLNAGRGNPNFLATTPRRAFFRLGLFAAAESELSYSYMTTVGVGGLAKIDGIEGRFERYIAENRDQEGVRFLGKSLSYVRDQLGLDPAAFLHEMVDGILGCNYPVPPRMLNISEKIVRQYIIREMGADAIPSESVNLFAVEGGTAAMAYIFESLKLNGLLKAGDKVAIGMPVFTPYIEIPELAQYALEEVAINADPSLNWQYPDSELDKLKDPAIKIFFCVNPSNPPSVKMDQRSLERVRNIVAEHRPDLMILTDDVYGTFADDFQSLFAICPENTLLVYSFSKYFGATGWRLGVVAAHQQNVFDLALDKLQESEKVALDHRYRSLLPDVRSLKFIDRLVADSRAVALNHTAGLSTPQQVQMALFSLFALMDEADEYKHTLKQLIRRRETTLYRELGMPPLRDENAVDYYTLIDLQDVTAKLYGEAFSEWAVKQSSTGDMLFRIADETGIVLLPGRGFGSNRPSGRASLANLNEYEYAAIGRALRKMADELYAEYSGQAQNL SEQ ID NO.3: SEQ ID NO.4: MSKDYQSLAKLSPFELKDELIKIASSDGNRAMLNAGRGNPNFLATTPRRAFFRLLGLFAAAESELSYMTTVGVGGLAKIDGIEGRFERYIAENRDQEGVRFLGKSLSYVRDQLGLDPAAFLHEMVDGILGCN YPVPPRMLNISEKIVRQYIIREMGADAIPSESVNLFAVEGGTAAMAYIFESLKLNGLLKAGDKVAIGMPVFTPYIEIPELAQYALEEVAINADPSLNWQYPDSELDKLKDPAIKIFFCVNPSNPPSVKMDQRS LERVRNIVAEHRPDLMILTDDVYGTFADDFQSLFAICPENTLLVYSFSKYFGATGWRLGVVAAHQQNVFDLALDKLQESEKVALDHRYRSLLPDVRSLKFIDRLVADSRAVALNHTAGLSTPQQVQMALFSLF ALMDEADEYKHTLKQLIRRRETTLYRELGMPPLRDENAVDYYTLIDLQDVTAKLYGEAFSEWAVKQSSTGDMLFRIADETGIVLLPGRGFGSNRPSGRASLANLNEYEYAAIGRALRKMADELYAEYSGQAQNL The L-aspartate-β-decarboxylase of this invention belongs to type I PLP-dependent enzymes.

[0035] The embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the invention. For those skilled in the art, other equivalent embodiments based on the present invention can be obtained without creative effort, and all such embodiments should be considered to fall within the protection scope of the present invention.

[0036] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0037] Conversion rate = (Amount of reactants already reacted / Amount of initial reactants) × 100%.

[0038] Enzyme activity is defined as the amount of enzyme required to consume 1 μmol L-aspartic acid per minute under reaction conditions at 37 °C.

[0039] Method for determining the activity of L-aspartate-β-decarboxylase: Add approximately 3 µg of enzyme solution to a 1 mL centrifuge tube, then add 2 mol / L L-aspartate, 0.5 mmol / L PLP, and 20 mmol / L phosphate buffer at pH 7.0. React at 37 °C for 2 h at pH 7.0, and then detect the enzyme activity.

[0040] HPLC detection method: HPLC detection was performed at a wavelength of 200 nm (CROWNPAK CR(+), 4 x 150 mm x 5µm). The mobile phase was perchloric acid aqueous solution (pH 2.0), and isocratic elution was performed for 10 min. The injection volume was 5 μL, the flow rate was 0.4 mL / min, and the column temperature was 0 ℃.

[0041] Example 1: Alkaloid bacteria in feces ( Alcaligenes faecalis Cloning and functional verification of wild-type L-aspartate-β-decarboxylase (parental template of mutant) 1.1 Experimental Materials Alkaloid bacteria in feces ( Alcaligenes faecalis The target base sequence was obtained from GenBank: AF168368.1, and the target protein sequence was obtained from GenBank: AAK58507.1. The Gram-negative bacterial genomic DNA miniprep kit, PCR product purification kit, and agarose gel extraction kit were all purchased from Sangon Biotech (Shanghai) Co., Ltd. High-fidelity DNA polymerase, dNTPs, DNA Marker, pET-28a(+) prokaryotic expression vector, *E. coli* DH5α, and *E. coli* BL21(DE3) were all purchased from Sangon Biotech (Shanghai) Co., Ltd. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0042] 1.2 Cloning of wild-type L-aspartate-β-decarboxylase Strain activation and genome extraction: After activation, Alcaligenes faecalis was inoculated into LB liquid medium (NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L), and cultured at 37 ℃ and 180 rpm for 8 h with shaking. The cells were collected by centrifugation at 7000 rpm for 5 min. The DNA samples of Alcaligenes faecalis were purified strictly according to the instructions to obtain the whole genome DNA of the strain, which was stored at -20℃ for later use.

[0043] Primer design: Based on the conserved domain sequence of type I PLP-dependent enzymes and the candidate coding regions of the draft genome sequence of Alcaligenes faecalis, specific amplification primers were designed. The primer sequences are as follows: AfAsd-F:5'-ggaattc catatgagcaaggattatcagagtctgg-3' (SEQ ID NO. 5); AfAsd-R: 5'-ccc aagctt agagattctgggcttgtccg-3' (SEQ ID NO. 6).

[0044] PCR amplification: PCR amplification was performed using extracted whole-genome DNA as a template. PCR reaction system (50 μL): 25 μL 2×Taq Plus Master PCR Mix, 2 μL each of forward and reverse primers (10 μM), 2 μL genomic DNA template, and ddH2O to a final volume of 50 μL.

[0045] PCR reaction program: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 3 min, for a total of 30 cycles; 72 ℃ final extension for 5 min.

[0046] Product purification and sequencing: The PCR product was size-verified and purified by agarose gel electrophoresis. Specific steps were performed according to the FAVORGEN DNA Agarose Gel Extraction Kit instructions. The recovered gene fragment and pET-28a plasmid were double-digested at a molar ratio of 3:1 at 37 °C. The digested fragments were recovered and ligated overnight at 16 °C. The ligation product was transformed into *E. coli* DH5α competent cells, evenly spread on kanamycin-resistant plates, and incubated upside down at 37 °C for 16 h. After single colonies were picked to verify positive clones, the cells were cultured on LB medium, and the plasmid was extracted to obtain the recombinant plasmid pET-28a-AfAsd containing L-aspartate-β-decarboxylase and plasmid pET-28a.

[0047] 1.3 Sequence Analysis and Functional Validation Sequence analysis: The sample was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed that the amplified target gene was 1602 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1, named AfAsd. This gene encodes 533 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.

[0048] Stereoselectivity and basic enzyme activity verification: The recombinant plasmid pET-28a-AfAsd was transformed into Escherichia coli BL21(DE3) to obtain a wild-type engineered strain. After IPTG induction for expression and purification, the strain was subjected to a catalytic reaction using L-aspartic acid as a substrate.

[0049] Example 2: Construction of DH5α engineered strain 2.1 Experimental Materials Site-directed saturation mutagenesis kit and high-fidelity DNA polymerase were purchased from Sangon Biotech (Shanghai) Co., Ltd.; CROWNPAK CR(+) column was purchased from Daicel Chiral Pharmaceuticals (Shanghai) Co., Ltd.; other reagents and consumables were the same as in Example 1.

[0050] 2.2 Construction of DH5α engineered strains The recombinant plasmid pET-28a-AfAsd-L31A, the L-aspartate-β-decarboxylase mutant gene used in this invention, is obtained by using the recombinant plasmid pET-28a-AfAsd as a template and primer pair ① as primers.

[0051] ① Positive direction: 5'-ccgcattgagcatggcgcggtttccgtcgc-3' (SEQ ID NO.7); ①Reverse: 5'-gcgacggaaaccgcgccatgctcaatgcgg-3' (SEQ ID NO.8).

[0052] Using the wild-type recombinant plasmid pET-28a-AfAsd constructed in Example 1 as a template, full plasmid PCR amplification was performed: The PCR amplification system is as follows (25 μL): 12.5 μL of 2×Phanta® Max Buffer, 0.5 μL of dNTP Mix, 2 μL of primers, 1 μL of template, 0.5 μL of Phanta® Max Super-Fidelity DNA Polymerase, 1 μL of DMSO, and ddH2O to a final volume of 25 μL.

[0053] PCR amplification conditions: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 56 ℃ annealing for 20 s, 72 ℃ extension for 5 min 30 s, 15 cycles; 72 ℃ final extension for 10 min.

[0054] The PCR product was digested with Dpn I enzyme to extract the template plasmid, then transformed into *E. coli* DH5α competent cells. The entire bacterial culture was spread onto LB agar plates containing kanamycin (50 μg / mL) and incubated at 37°C for 16 h. Single colonies were picked and inoculated into 3 mL of LB liquid medium containing kanamycin (test tube), and cultured at 37°C and 180 rpm for 8 h. Full-length sequencing was performed, and the encoded nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4. All mutation sites were confirmed to be accurate, with no other random mutations, resulting in the mutant DH5α engineered strain DH5α-pET-28a-AfAsd-L31A.

[0055] Example 3: Construction of BL21(DE3) genetically engineered host strain 3.1 Experimental Materials The Plasmid Extraction Mini Kit was purchased from Shanghai Shuomeng Biotechnology Co., Ltd.; the other reagents were the same as in Example 1.

[0056] 3.2 Construction of genetically engineered host strains The mutant strain DH5α-pET-28a-AfAsd-L31A constructed in Example 2 was inoculated into LB liquid medium (test tube) containing 50 μg / mL kanamycin and cultured at 37 ℃ and 180 rpm for 8 h with shaking. The plasmid was extracted using a plasmid extraction kit to obtain the recombinant expression vector pET-28a-AfAsd-L31A. 1 μL of this vector was added to 100 μL of *E. coli* BL21(DE3) competent cells, incubated on ice for 30 min, heat-shocked at 42 ℃ for 90 s, immediately incubated on ice for 5 min, and then 900 μL of antibiotic-free LB liquid medium was added. The culture was then recovered at 37 ℃ and 180 rpm with shaking for 1 h. 100 μL of the recovered bacterial culture was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37 ℃ for 16 h. Single colonies were selected for colony PCR verification. Positive clones were identified as the successfully constructed genetically engineered host strain and named BL21(DE3)-pET-28a-AfAsd-L31A.

[0057] Example 4: Heterologous expression and purification of L-aspartate-β-decarboxylase mutant 4.1 Experimental Materials IPTG, Ni-NTA affinity chromatography column, and BCA protein quantification kit were all purchased from Shanghai Sangon Biotech Co., Ltd.; the other reagents were the same as in Example 1.

[0058] 4.2 Induction of expression and purification Induction of expression: The engineered strain BL21(DE3)-pET-28a-AfAsd-L31A constructed in Example 3 was inoculated into LB liquid medium (test tubes) containing 50 μg / mL kanamycin and cultured at 37 ℃ with shaking at 180 rpm for 8 h. Then, it was transferred to fresh LB liquid medium (containing 50 μg / mL kanamycin) at a 1% inoculation rate and cultured at 37 ℃ with shaking at 180 rpm until OD... 600 =0.6, add IPTG to a final concentration of 0.1 mM, adjust the temperature to 20 ℃, and induce expression for 20 h. A wild-type L-aspartate-β-decarboxylase engineered strain was set up as a control.

[0059] Cell collection and disruption: After induction, the cells were collected by centrifugation at 7000 rpm for 5 min at 4 ℃. The cells were resuspended in pre-cooled phosphate buffer (100 mM, pH 7.0) and washed twice. The cells were then resuspended again and disrupted by sonication under ice bath conditions (200 W power, 3 s sonication, 5 s interval, for a total of 99 cycles). The disruption solution was centrifuged at 7830 rpm for 10 min at 4 ℃, and the supernatant was collected as the crude enzyme solution of the mutant.

[0060] Protein purification: After the crude enzyme solution was filtered through a 0.22 μm filter membrane, it was loaded onto a pre-equilibrated Ni-NTA affinity chromatography column. First, impurities were eluted with PBS buffer containing 50 mM imidazole, and then the target protein was eluted with PBS buffer containing 200 mM imidazole. The elution peaks were collected, and the protein purity was detected by SDS-PAGE electrophoresis.

[0061] 4.3 Experimental Results SDS-PAGE electrophoresis results are as follows: Figure 1 The results showed that the engineered strain exhibited a distinct and specific protein band at approximately 50 kDa, consistent with the theoretical molecular weight of the mutant, and the protein purity after purification was >95%. It was comparable to the wild type, without the problem of excessive inclusion bodies, meeting the requirements for industrial-scale preparation.

[0062] Example 5: Determination of Optimal Temperature The purified enzyme solutions of both the mutant and wild-type enzymes from Example 4 were diluted to a final concentration of approximately 3 μg / mL. Using the wild-type enzyme as a control, and keeping other reaction conditions constant, the reactions were carried out at 30 ℃, 37 ℃, 45 ℃, 50 ℃, 55 ℃, and 60 ℃ for 2 h, after which inactivation was terminated. The optimal reaction temperature was then determined. The results are as follows: Figure 2 As shown, the optimal temperature for both the mutant and the wild-type enzyme is 45 °C, but the enzyme activity of the mutant is 3.23 times higher than that of the wild-type.

[0063] Example 6: Determination of Optimal pH The purified enzyme solutions of both the mutant and wild-type enzymes from Example 4 were diluted to a final concentration of approximately 3 μg / mL. Using the wild-type enzyme as a control, and keeping other reaction conditions constant, buffer solutions with pH values ​​of 3.0, 4.0, 5.0 (citrate-sodium citrate buffer), 6.0, 7.0, and 8.0 (phosphate buffer) were added to the reaction system. The reaction was terminated by inactivation after reacting at 37 °C for 2 h, and the optimal reaction pH was determined. The results are as follows: Figure 3 As shown, the optimal pH for the wild-type enzyme is 6.0, while the optimal pH for the mutant is 7.0. Furthermore, at pH 7.0, the specific enzyme activity of the wild-type is 50.1 U / mg, while that of the mutant is 208.9 U / mg, representing a 3.17-fold increase compared to the wild-type.

[0064] Example 7: Thermal Stability Determination The pure enzyme solutions of both the mutant and wild-type enzymes from Example 4 were diluted to a final concentration of approximately 3 μg / mL. Using the wild-type enzyme as a control, and keeping other reaction conditions unchanged, equal volumes of the pure enzyme solutions of the mutant and wild-type were incubated in water baths at 30 ℃, 37 ℃, 45 ℃, 50 ℃, 55 ℃, and 60 ℃ for 30 min, respectively. The reaction was then terminated by inactivation at 37 ℃ for 2 h, and the remaining enzyme activity was measured. The results are as follows: Figure 4 As shown, the residual enzyme activity of mutant enzyme L31A was higher than that of wild type.

[0065] Example 8: pH stability determination The purified enzyme solutions of both the mutant and wild-type enzymes from Example 4 were diluted to a final concentration of approximately 3 μg / mL. Using the wild-type enzyme as a control, and keeping other reaction conditions constant, equal volumes of the purified mutant and wild-type enzyme solutions were added to buffer solutions with pH values ​​of 3.0, 4.0, 5.0 (citrate-sodium citrate buffer), 6.0, 7.0, and 8.0 (phosphate buffer), respectively, to achieve the same concentration. The solutions were incubated on ice for 30 min, then reacted at 37 °C for 2 h to terminate the reaction. The remaining enzyme activity was then measured. The results are as follows: Figure 5 As shown, the residual enzyme activity of mutant enzyme L31A was higher than that of wild type.

[0066] Example 9: Whole-cell catalytic DL-aspartic acid reaction After induction, cells were collected by centrifugation and subjected to whole-cell catalytic reactions. Wild-type cells were reacted in a 10 L reaction system (containing bacterial cells, 20 mmol / L phosphate buffer at pH 7.0, and DL-aspartic acid substrate) at pH 6.0 and a reaction temperature of 37 °C, with a single addition of DL-aspartic acid to a final concentration of 2 mol / L. Mutants were reacted in a 10 L reaction system (containing bacterial cells, 80 mmol / L phosphate buffer at pH 7.0, and DL-aspartic acid substrate) at pH 7.0 and a reaction temperature of 37 °C, with a single addition of DL-aspartic acid to a final concentration of 2 mol / L. L-aspartic acid consumption was measured every hour. Results are as follows: Figure 6 As shown, the transformation rate of mutant L31A reached 100% after 5 h, while the transformation rate of wild type was 20.6%.

[0067] After the conversion reaction was completed, 2% diatomaceous earth was added to the system to adsorb bacterial proteins and various small molecule impurities. The mixture was then stirred in a 90 °C water bath for 30 min, followed by centrifugation at 5000 r / min for 20 min. The supernatant was collected and decolorized with 2% activated carbon, stirred in a 90 °C water bath for 30 min, and then filtered. The supernatant was collected and concentrated to 1 / 3 of its original volume using a rotary evaporator. After cooling to room temperature, 10 mol / L HCl was slowly added dropwise to adjust the pH to the isoelectric point of aspartic acid, 2.8. A large amount of crystals precipitated in the system. These crystals were dissolved in deionized water, mixed, and filtered to remove ammonium salt impurities. The resulting crystals were then thoroughly mixed with ethanol and filtered to remove Cl. - After drying, white D-aspartic acid crystals are obtained. The enantiomeric excess (ee value) of D-aspartic acid is >99.9%.

[0068] The mother liquor after separating D-aspartic acid was used to separate L-alanine using a 732# strong acid ion exchange resin. First, a low-concentration ammonia solution was used to completely elute the small amount of residual aspartic acid in the mother liquor, which had a weaker affinity for the resin. Then, the ammonia concentration was increased to elute L-alanine, which has a stronger affinity for the resin, thereby improving separation efficiency and shortening the process time. The pH and amino acid composition of each eluent were analyzed. The eluent containing only L-alanine was collected, concentrated by rotary evaporation until a white solid precipitated, mixed with ethanol, filtered, and finally dried to obtain a white L-alanine crystalline product. The enantiomeric excess (ee value) of L-alanine was >99.9%.

[0069] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. An L-aspartate-β-decarboxylase mutant, characterized in that, The amino acid sequence of the mutant contains the L31A mutation site compared with the amino acid sequence of the wild-type L-aspartate-β-decarboxylase shown in SEQ ID NO. 2; the mutant has enzyme activity that catalyzes the decarboxylation of L-aspartate to produce L-alanine, and the enzyme activity is at least 2 times higher than that of the wild type.

2. The L-aspartate-β-decarboxylase mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

4.

3. The L-aspartate-β-decarboxylase mutant according to claim 1, characterized in that, The mutant has at least 95% homology with the amino acid sequence shown in SEQ ID NO.

2.

4. A polynucleotide sequence encoding the L-aspartate-β-decarboxylase mutant according to any one of claims 1-3.

5. The polynucleotide sequence according to claim 4, characterized in that, The polynucleotide sequence is as shown in SEQ ID NO.3, or is a codon-optimized nucleotide sequence for the Escherichia coli host that encodes an amino acid sequence with at least 95% homology to the sequence shown in SEQ ID NO.

4.

6. A recombinant expression vector comprising the polynucleotide sequence of claim 4.

7. The recombinant expression vector according to claim 6, characterized in that, The recombinant expression vector is a pET series prokaryotic expression vector.

8. Transformation of a genetically engineered host bacterium with the recombinant expression vector of claim 6.

9. A method for preparing an L-aspartate-β-decarboxylase mutant, characterized in that, Includes the following steps: (1) Activate the genetically engineered host bacteria as described in claim 8, inoculate it into a liquid culture medium containing antibiotic resistance matching the selection marker of the recombinant expression vector, and culture it until OD. 600 =0.6, and an inducer was added to induce the strain to heterologously express the L-aspartate-β-decarboxylase mutant; the inducer was isopropyl-β-D-thiogalactoside, with a final concentration of 0.1 mM; the induction temperature was 20 ℃, and the induction time was 20 h; (2) Collect the induced bacterial cells, and obtain a crude enzyme solution containing L-aspartate-β-decarboxylase by ultrasonic disruption and centrifugation. Purify the crude enzyme solution to obtain purified L-aspartate-β-decarboxylase mutant protein.

10. The use of the L-aspartate-β-decarboxylase mutant according to any one of claims 1-3 and / or the genetically engineered host bacterium according to claim 8 in enzyme-catalyzed transformation using L-aspartate or DL-aspartate as substrates.

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