An asparagine synthetase a mutant

Abstract

The application discloses an asparagine synthetase A mutant and belongs to the technical field of bioengineering. The application uses L-aspartic acid and ATP as raw materials, uses the asparagine synthetase A (EcAsnA) mutant with improved activity to carry out biosynthesis of L-asparagine, and the overall catalytic process is simple and efficient. The enzyme is used to carry out an ammonia connection reaction, compared with the parent capable of catalyzing L-asparagine from L-aspartic acid, the EcAsnA L109A / T44I / Q66L has higher catalytic efficiency, the catalytic activity is increased to 3222.51 U / mg, which is 5.48 times of the parent (497.15 U / mg), and the catalytic activity is increased by 23.77% compared with EcAsnA L109K / K58R . The application provides a new synthesis method for rapidly synthesizing L-asparagine and provides a new idea for industrialized production of L-asparagine.

Description

Technical Field

[0001] This invention relates to an asparagine synthase A mutant, belonging to the field of bioengineering technology. Background Technology

[0002] Asparagine, one of the 20 most common amino acids, is widely used in medicine, food, and other fields. Recent studies have shown that compounds derived from asparagine have high medicinal value, exhibiting good biological activity in anti-inflammatory, antihypertensive, and hemostatic effects. Examples include captopril (ACEI) antihypertensive drugs, endorphin-2 (EM2) analgesics, and the anti-inflammatory drug N-[β-(p-substituted benzoyl)ethyl]asparagine. The main methods for synthesizing L-asparagine are chemical synthesis, plant extraction, and biosynthesis. Biosynthesis offers advantages such as simple processes, low equipment requirements, high production efficiency, low energy consumption, and low pollution, but it still suffers from problems such as low activity of key enzymes. Given the importance of L-asparagine, there is an urgent need to develop efficient methods for its synthesis to promote the production and application of L-Asparagine.

[0003] In existing biosynthetic methods, L-aspartic acid is used as a substrate, and asparagine synthase A is used to ammoniate the carboxyl group of its side chain to synthesize L-asparagine. Asparagine synthase A is a multifunctional enzyme that uses ATP as a cofactor to functionalize the CO bond of amino acids and is considered one of the important catalysts for amino acid amidation. Although mutants of this enzyme have been disclosed in existing technologies, the catalytic efficiency and specific activity of the currently disclosed enzymes still need further improvement. To address the above issues, this invention uses protein engineering techniques to modify the asparagine synthase A mutant EcAsnA. L109A / T44I Further modifications were made to obtain an asparagine synthase A with enhanced activity, enabling the low-cost industrial synthesis of L-asparagine. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the construction and property study of an asparagine synthase A mutant for the production of L-asparagine. The aim is to achieve efficient synthesis of L-asparagine from genetically engineered bacteria using inexpensive substrate L-aspartic acid and cofactor ATP, thereby solving the technical problem of low activity of asparagine synthase A in existing biological synthesis methods, which leads to low yield of L-asparagine and requires further improvement in production intensity.

[0005] This invention provides an asparagine synthase A mutant, wherein the mutant is a mutation of leucine at position 109, threonine at position 44, and glutamine at position 66 of the parent asparagine synthase A, and the amino acid sequence of the asparagine synthase A (EcAsnA) is shown in SEQ ID NO.1.

[0006] MKTAYIAKQRQISFVKSHFSRQLEERLGLIEVQAPILSRVGDGTQDNLSGCEKAVQVKVKALPDAQFEVVHSLAKWKRQTLGQHDFSAGEGLYTHMKALRPDEDRLSPLHSVYVDQWDWERVMGDGERQFSTLKSTVEAIWAGIKATEAAVSEEFGLAPFLPDQIHFVHSQELLSRYPDLDAKGRERAIAKDLGAVFLVGIGGKLSDGHRHDVRAPDYDDWSTPSELGHAGLNGDILVWNPVLEDAFELSSMGIRVDADTLKHQLALTGDEDRLELEWHQALLRGEMPQTIGGGIGQSRLTMLLLQLPHIGQVQCGVWPAAVRESVPSLL

[0007] In one embodiment, the nucleic acid sequence of the gene encoding the EcAsnA is as shown in SEQ ID NO.2.

[0008] SEQ ID NO.2:

[0009] atgaaaaccg cttacattgc caaacaacgt caaattagct tcgtgaaatc tcacttttctcgtcaactgg

[0010] aagaacgtct ggggctgatc gaagtccagg cgccgattct tagccgtgtg ggggatggcacgcaggataa

[0011] cttgtcgggc tgtgaaaaag cggtgcaggt aaaagtgaaa gctctgcctg atgcccagttcgaagtggtt

[0012] cattcactgg cgaagtggaa acgtcagacc ttagggcaac acgacttcag cgcgggcgaagggctgtaca

[0013] cgcacatgaa agcccttcgc cccgatgaag accgtctttc tccgttgcac tcggtctatgttgaccagtg

[0014] ggactgggaa cgcgtaatgg gcgacggtga gcgtcaattc tcgactctga aaagcacggtagaggcgatc

[0015] tgggcgggaa ttaaagcaac cgaagctgcg gttagcgaag agtttggcct ggcaccgttcctgccggatc

[0016] agatccactt cgtacacagc caggagttac tgtctcgtta tccggatctt gatgccaaagggcgtgagcg

[0017] ggcgatagcg aaagatcttg gcgcggtatt ccttgtcggg attggcggca agctgagcgatggtcatcgc

[0018] cacgacgtgc gcgcaccgga ttatgatgac tggagcaccc cgtcagagct gggccatgcgggtctgaacg

[0019] gcgatattct ggtgtggaac ccggtactgg aagatgcgtt tgagctttcc tccatggggatccgtgtaga

[0020] tgccgacacg ctgaagcatc aactggcgct gaccggtgac gaagatcgcc tggagctggagtggcatcag

[0021] gcgctgctgc gcggtgaaat gccgcagacc atcggcggcg gtatcggcca gtctcgtttgactatgctgc

[0022] tgctgcaact gccgcatatc ggccaggttc agtgtggagt atggccagct gctgttcgcgagagcgtccc

[0023] ttctctgctg taa

[0024] In one embodiment, the mutant is a mutation performed on the parental line as follows: leucine L at position 109 is mutated to alanine A, threonine T at position 44 is mutated to isoleucine I, and glutamine Q at position 66 is mutated to leucine L.

[0025] In one embodiment, the mutant is obtained by mutating amino acids at positions 109 and 44 and glutamine at position 66, relative to the parent EcAsnA, to obtain the mutant EcAsnA. L109A / T44I / Q66L .

[0026] In one embodiment, the mutant EcAsnA L109A / T44I / Q66L The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0027] SEQ ID NO.3:

[0028] MKTAYIAKQRQISFVKSHFSRQLEERLGLIEVQAPILSRVGDGIQDNLSGCEKAVQVKVKALPDALFEVVHSLAKWKRQTLGQHDFSAGEGLYTHMKALRPDEDRLSPAHSVYVDQWDWERVMGDGERQFSTLKSTVEAIWAGIKATEAAVSEEFGLAPFLPDQI HFVHSQELLSRYPDLDAKGRERAIAKDLGAVFLVGIGGKLSDGHRHDVRAPDYDDWSTPSELGHAGLNGDILVWNPVLEDAFELSSMGIRVDADTLKHQLALTGDEDRLELEWHQALLRGEMPQTIGGGIGQSRLTMLLLQLPHIGQVQCGVWPAAVRESVPSLL

[0029] SEQ ID NO.4:

[0030] atgaaaaccg cttacattgc caaacaacgt caaattagct tcgtgaaatc tcacttttctcgtcaactggaagaacgtct ggggctgatc gaagtccagg cgccgattct tagccgtgtg ggggatggcattcaggataacttgtcgggc tgtgaaaaag cggtgcaggt aaaggtgaaa gctctgcctg atgccctattcgaagtggttcattcactgg cgaagtggaa acgtcagacc ttagggcaac acgacttcag cgcgggcgaagggctgtacacgcacatgaa agcccttcgc cccgatgaag accgtctttc tccggcacac tcggtctatgttgaccagtgggactgggaa cgcgtaatgg gcgacggtga gcgtcaattc tcgactctga aaagcacggtagaggcgatctgggcgggaa ttaaagcaac cgaagctgcg gttagcgaag agtttggcct ggcaccgttcctgccggatcagatccactt cgtacacagc caggagttac tgtctcgtta tccggatctt gatgccaaagggcgtgagcgggcgatagcg aaagatcttg gcgcggtatt ccttgtcggg attggcggca agctgagcgatggtcatcgccacgacgtgc gcgcaccgga ttatgatgac tggagcaccc cgtcagagct gggccatgcgggtctgaacggcgatattct ggtgtggaac ccggtactgg aagatgcgtt tgagctttcc tccatggggatccgtgtagatgccgacacg ctgaagcatc aactggcgct gaccggtgac gaagatcgcc tggagctggagtggcatcaggcgctgctgc gcggtgaaat gccgcagacc atcggcggcg gtatcggcca gtctcgtttgactatgctgctgctgcaact gccgcatatcggccaggttc agtgtggagt atggccagct gctgttcgcgagagcgtcccttctctgctg taa

[0031] In one embodiment, the method for obtaining the EcAsnA mutant includes the following steps:

[0032] (1) The mutant of asparagine synthase A in Escherichia coli (EcAsnA) L109A / T44I Based on the amino acid sequence, the mutation site was determined; saturation mutation primers were designed to carry the code for EcAsnA. L109A / T44I Gene vectors are used as templates for saturation mutation; plasmid vectors containing mutants are constructed;

[0033] (2) Transform the mutant plasmid into the host cell;

[0034] (3) Select positive clones for fermentation culture and purify EcAsnA.

[0035] This invention also provides an encoding of the asparagine synthase A mutant, EcAsnA. L109A / T44I / Q66L The gene or a recombinant vector carrying the gene.

[0036] In one implementation, pET-28a(+) is used as the expression vector.

[0037] The present invention also provides expression of the above mutant EcAsnA L109A / T44I / Q66L Or recombinant cells carrying the aforementioned genes or the recombinant vector.

[0038] In one embodiment, the recombinant cells are bacteria or fungi as host cells.

[0039] The present invention also provides a mutant EcAsnA containing the above-mentioned asparagine synthase A. L109A / T44I / Q66L The recombinase catalyst is any of the following forms:

[0040] (1) Cultivate EcAsnA containing the asparagine synthase A mutant. L109A / T44I / Q66L The recombinant expression transformant was used to isolate transformant cells containing the recombinant asparagine synthase A mutant enzyme;

[0041] (2) Cultivate EcAsnA containing the aforementioned asparagine synthase A mutant. L109A / T44I / Q66L The recombinant expression transformant was obtained, and the transformant cells containing the recombinant asparagine synthase A mutant enzyme were isolated. The transformant cells containing the recombinant asparagine synthase A mutant enzyme were lysed to obtain the cell lysate.

[0042] (3) Cultivate EcAsnA containing the aforementioned asparagine synthase A mutant. L109A / T44I / Q66L The recombinant expression transformant was obtained, and the transformant cells containing the recombinant asparagine synthase A mutant enzyme were isolated. The transformant cells containing the recombinant asparagine synthase A mutant enzyme were lysed to obtain cell lysate. The cell lysate containing the recombinant asparagine synthase A mutant enzyme was freeze-dried to obtain lyophilized enzyme powder.

[0043] The present invention also provides a method for improving the catalytic activity of asparagine synthase A on substrates and increasing specific enzyme activity. The method involves mutating leucine at position 109 of asparagine synthase A, as shown in SEQ ID NO.1, to alanine, threonine at position 44, to isoleucine, and glutamine at position 66, to leucine.

[0044] This invention also provides a method for increasing the yield of L-asparagine, wherein the method comprises using L-aspartic acid as a substrate and employing the above-mentioned EcAsnA L109A / T44I / Q66L L-asparagine was prepared by reacting mutants, or the above-mentioned recombinant cells, or the above-mentioned recombinase catalyst.

[0045] In one embodiment, the system further contains MgCl2 and ATP;

[0046] In one embodiment, the concentration of NH4Cl is 150–250 mM; the concentration of MgCl2 is 150–250 mM; the concentration of ATP is 150–250 mM; and the concentration of L-aspartic acid is 150–250 mM.

[0047] In one embodiment, the system has a pH of 7.5-8.5, a temperature of 30-40°C, and a rotation speed of 200-300 rpm.

[0048] The present invention also provides a genetically engineered bacterium, wherein the bacterium uses *E. coli* as the substrate cell and expresses the above-mentioned asparagine synthase A mutant EcAsnA. L109A / T44I / Q66L .

[0049] In one embodiment, the genetically engineered bacteria are derived from Escherichia coli strains.

[0050] In one implementation, Escherichia coli BL21(DE3) is used as the expression host.

[0051] In one implementation, a carrier code EcAsnA is constructed. L109A / T44I / Q66L The gene expression vector EcAsnA L109A / T44I / Q66L-pET-28a, EcAsnA L109A / T44I / Q66L -pET-28a is introduced into E. coli BL21(DE3) to form EcAsnA L109A / T44I / Q66L -pET-28a-BL21(DE3) genetically engineered bacteria.

[0052] In one implementation, a carrier code EcAsnA is constructed. L109A / T44I / Q66L The gene expression vector EcAsnA L109A / T44I / Q66L -pET-28a, EcAsnA L109A / T44I / Q66L -pET-28a is introduced into E. coli BL21(DE3) to form EcAsnA L109A / T44I / Q66L -pET-28a-BL21(DE3) genetically engineered bacteria.

[0053] This invention also provides a method for preparing L-asparagine, wherein the method comprises: [the method involves] preparing the asparagine synthase A mutant EcAsnA... L109A / T44I / Q66L The recombinant asparagine synthase A mutant enzyme or the recombinant enzyme catalyst prepared by the above-mentioned recombinant cells or genetically engineered bacteria is added to a system containing L-aspartic acid and NH4Cl for degradation.

[0054] In one embodiment, the system further contains MgCl2 and ATP;

[0055] In one embodiment, the concentration of NH4Cl is 150–250 mM; the concentration of MgCl2 is 150–250 mM; the concentration of ATP is 150–250 mM; and the concentration of L-aspartic acid is 150–250 mM.

[0056] In one embodiment, the system has a pH of 7.5-8.5, a temperature of 30-40°C, a reaction time of 30-90 min, a rotation speed of 200-300 rpm, and a catalyst dosage of 20-30 g / L of genetically engineered bacteria.

[0057] In one embodiment, the catalytic system is 10 mL, wherein L-aspartic acid is 200 mM, NH4Cl is 200 mM, ATP200 is 200 mM, MgCl2 is 200 mM, and the amount of catalyst added is 20 g / L of genetically engineered bacteria.

[0058] In one embodiment, the buffer solution of the catalytic system is 200 mM Tris-HCl.

[0059] In one embodiment, the catalytic system has a pH of 8.0, a catalytic reaction temperature of 37°C, and a catalytic reaction time of 0.5 h.

[0060] The present invention also provides the above-mentioned asparagine synthase A mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned use in the preparation of L-asparagine or products containing L-asparagine.

[0061] Beneficial effects

[0062] This invention designs a whole-cell biosynthesis of L-asparagine using L-aspartic acid and ATP as raw materials and an enhanced-activity asparagine synthase A (EcAsnA) mutant. The overall catalytic process is simple and efficient. Compared to existing EcAsnA enzymes capable of catalyzing the conversion of L-aspartic acid to L-asparagine, the EcAsnA in this invention... L109A / T44I / Q66L The catalytic efficiency was further improved, reaching [a higher level]. 3222.51U / mg Compared with wild-type enzymes and currently reported EcAsnA L109K / K58R Compared to the previous methods, the yields were improved by 5.48 times and 23.77%, respectively. This invention provides a new synthetic method for the rapid synthesis of L-asparagine and offers new ideas for the industrial production of L-asparagine. Attached Figure Description

[0063] Figure 1 The results of the initial screening for half-saturation mutations of enzyme EcAsnA at amino acid (Q) at position 66.

[0064] Figure 2 The results of the initial screening for half-saturation mutations of enzyme EcAsnA at amino acid (K) at position 58.

[0065] Figure 3 The results of the initial screening for half-saturation mutations of enzyme EcAsnA at amino acid (R) position 105.

[0066] Figure 4 The results of the initial screening for half-saturation mutation of enzyme EcAsnA at amino acid (D) at position 104.

[0067] Figure 5 The results of the initial screening for half-saturation mutations of enzyme EcAsnA at amino acid (K) position 53 are shown.

[0068] Figure 6 The results of the initial screening for half-saturation mutations of enzyme EcAsnA at amino acid (R) position 210.

[0069] Figure 7 EcAsnA wild-type, mutant EcAsnA L109A EcAsnA L109K EcAsnA L109A / T44I EcAsnA L109K / K58R and, EcAsnA L109A / T44I / Q66L EcAsnAL109A / T44I / D104K EcAsnA L109A / T44I / K58R EcAsnA L109A / T44I / R105P EcAsnA L109A / T44I / K53N and EcAsnA L109A / T44I / R210P The yield of L-asparagine produced by catalysis for 0.5 h at a 10 mL preparation scale is shown in the figure.

[0070] Figure 8 EcAsnA wild-type, mutant EcAsnA L109A EcAsnA L109K EcAsnA L109A / T44I EcAsnA L109K / K58R and, EcAsnA L109A / T44I / Q66L EcAsnA L109A / T44I / D104K EcAsnA L109A / T44I / K58R EcAsnA L109A / T44I / R105P EcAsnA L109A / T44I / K53N and EcAsnA L109A / T44I / R210P The enzyme activity.

[0071] Figure 9 The mutant EcAsnA L109A / T44I / Q66L Thermal stability diagram.

[0072] Figure 10 The mutant EcAsnA L109A / T44I / Q66L pH stability graph. Detailed Implementation

[0073] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0074] Gene source: The EcAsnA gene involved in this invention is derived from Escherichia coli. The pET-28a(+) plasmid was purchased from Novagen (Madison, WI, USA). The ClonExpress II One Step Cloning Kit, Primer Star Max, DpnI, etc., were purchased from TaKaRa (Dalian, China). All EcAsnA mutants were obtained through molecular modification.

[0075] The host used was E. coli BL21(DE3), the vector was pET-28a(+), and the resistance was kanamycin. The following procedures involved mutagenesis of the parental genetically engineered strain E. coli-EcAsnA-pET-28a to screen for high-yielding strains.

[0076] The culture media involved in the following examples are as follows:

[0077] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, sterilized at 121°C for 20 min.

[0078] TB fermentation medium: tryptone 12 g / L, Angel yeast powder FM 802 24 g / L, glycerol 4 g / L, KH2PO4 2.31 g / L and K2HPO4 12.31 g / L.

[0079] The detection methods involved in the following embodiments are as follows:

[0080] Determination of L-aspartic acid and L-asparagine by HPLC: For specific steps, please refer to the literature Anal. Biochem., 1984, 136(1):195-201 (A high-performance liquid chromatography assay for asparagine synthetase).

[0081] L-Asparagine content determination: The reaction product was determined by high-performance liquid chromatography (HPLC). The HPLC detection conditions are the same as described above for the HPLC method for the determination of L-aspartic acid and L-asparagine.

[0082] Methods for detecting the activity of asparagine synthase A:

[0083] After mixing 200 mM L-aspartic acid, 200 mM NH4Cl, 200 mM MgCl2, and 200 mM ATP, the pH was adjusted to 8.0 with ammonia. EcAsnA pure enzyme with a final concentration of 0.5 mg / ml was added, and the reaction was carried out at 37℃ for 30 min at a rotation speed of 220 rpm. The reaction was terminated with 50 mM HCl after completion. The L-asparagine content was then detected using the OPA derivatization method. HPLC determination of L-aspartic acid and L-asparagine: For specific steps, please refer to the literature Anal. Biochem., 1984, 136(1):195-201 (A high-performance liquid chromatography assay for asparagine synthetase).

[0084] Asparagine synthase A activity is defined as the production of 1 μmol·L⁻¹ per minute. -1 The amount of enzyme in L-asparagine.

[0085] The following embodiments involve the transformation of competent states:

[0086] Transformation: The digestion products were introduced into E. coli BL21(DE3) competent cells via heat shock. Specific transformation steps:

[0087] (1) 10 μL of PCR product was introduced into 100 μL of E.coli BL21(DE3) competent cells;

[0088] (2) Ice bath for 30 minutes;

[0089] (3) Heat shock in a 42℃ water bath for 60 seconds, then remove and quickly place in ice for 3-5 minutes;

[0090] (4) Add 600 μL of antibiotic-free LB medium and mix well. Incubate at 37°C and 220 rpm for 1 h.

[0091] (5) Centrifuge at 4000 rpm for 2 min;

[0092] (6) Discard the supernatant, use the remaining 100-200 μL LB medium to mix the bacterial cells by blowing and spreading them onto a plate containing 0.05 mg / mL kanamycin resistance, and incubate at 37°C for about 12 hours.

[0093] Example 1: Construction of mutants

[0094] 1. Construction of recombinant vectors containing wild type

[0095] (1) Obtaining the recombinant vector

[0096] The target protein sequence AsnA was amplified from the genome of Escherichia coli (shown as SEQ ID NO.2) using primers AsnA-F (ATGGGTCGCGGATCCATGAAAACCGCTTACATTGCCA) and AsnA-R (ACGGAGCTCGAATTCTTACAGCAGAGAAGGGACGCT) and ligated into the pET-28a(+) vector.

[0097] SEQ ID NO.2:

[0098] atgaaaaccg cttacattgc caaacaacgt caaattagct tcgtgaaatc tcacttttctcgtcaactgg

[0099] aagaacgtct ggggctgatc gaagtccagg cgccgattct tagccgtgtg ggggatggcacgcaggataa

[0100] cttgtcgggc tgtgaaaaag cggtgcaggt aaaagtgaaa gctctgcctg atgcccagttcgaagtggtt

[0101] cattcactgg cgaagtggaa acgtcagacc ttagggcaac acgacttcag cgcgggcgaagggctgtaca

[0102] cgcacatgaa agcccttcgc cccgatgaag accgtctttc tccgttgcac tcggtctatgttgaccagtg

[0103] ggactgggaa cgcgtaatgg gcgacggtga gcgtcaattc tcgactctga aaagcacggtagaggcgatc

[0104] tgggcgggaa ttaaagcaac cgaagctgcg gttagcgaag agtttggcct ggcaccgttcctgccggatc

[0105] agatccactt cgtacacagc caggagttac tgtctcgtta tccggatctt gatgccaaagggcgtgagcg

[0106] ggcgatagcg aaagatcttg gcgcggtatt ccttgtcggg attggcggca agctgagcgatggtcatcgc

[0107] cacgacgtgc gcgcaccgga ttatgatgac tggagcaccc cgtcagagct gggccatgcgggtctgaacg

[0108] gcgatattct ggtgtggaac ccggtactgg aagatgcgtt tgagctttcc tccatggggatccgtgtaga

[0109] tgccgacacg ctgaagcatc aactggcgct gaccggtgac gaagatcgcc tggagctggagtggcatcag

[0110] gcgctgctgc gcggtgaaat gccgcagacc atcggcggcg gtatcggcca gtctcgtttgactatgctgc

[0111] tgctgcaact gccgcatatc ggccaggttc agtgtggagt atggccagct gctgttcgcgagagcgtccc

[0112] ttctctgctg taa

[0113] EcAsnA-pET-28a was prepared.

[0114] (2) Obtaining recombinant bacteria

[0115] After obtaining the recombinant expression plasmid EcAsnA-pET-28a, it was transformed into E.coli BL21(DE3), and the resulting positive engineered bacteria was named EcAsnA-pET-28a-BL21(DE3).

[0116] Using the recombinant expression plasmid EcAsnA-pET-28a as a template, PCR amplification was performed using primers L109A-F (gtctttctccgGCAcactcggtct at) and L109A-R (caacatagaccgagtgTGCcggagaa). The PCR product was then transformed into E. col i BL21(DE3), and the resulting positive engineered bacteria was named EcAsnA. L109A -pET-28a-BL21(DE3). Using plasmid EcAsn A L109A Using pET-28a-BL21(DE3) as a template, PCR amplification was performed using primers T44I-F (gtgtgggggatggcATTcaggataact) and T44I-R (cgacaagttatcctgAATgccatccccc). The PCR product was then transformed into E. coli BL21(DE3), and the resulting engineered bacterium was named EcAsnA. L109A / T44I -pET-28a-BL21(DE3).

[0117] Recombinant vectors EcAsnA-pET-28a and EcAsnA were prepared separately. L109A / T44I -pET-28a.

[0118] 2. Construction of mutants:

[0119] Primers for single and triple mutation sites of EcAsnA were designed as shown in Table 1. Mutants were constructed by whole plasmid PCR using the recombinant vector EcAsnA-pET-28a obtained in step 1 as a template.

[0120] Table 1: Mutant Primer Sequences

[0121]

[0122] The Prime Star Max system was used to construct the PCR amplification system. The recombinant plasmid EcAsnA-pET-28a was used as the template, and PCR was performed using the primers shown in Table 1. The Prime Star Max system is shown in Table 2. The PCR reaction conditions were: ① 98℃ for 30s; ② 98℃ for 10s; ③ 55℃ for 30s; ④ 72℃ for 1min 20s; ⑤ Repeat steps ② to ④ 34 times; ⑥ 72℃ for 10min; ⑦ Incubate at 12℃.

[0123] Table 2: Prime Star Max System Table

[0124]

[0125] The above PCR reaction system was incubated in a metal bath at 37°C for 30 min to digest the plasmid template (the digestion system consisted of: 0.3 μL of DpnI quick, 8.7 μL of the above PCR product, and 1 μL of 10×T Buffer). The digestion product was obtained after digestion.

[0126] Transformation: The digestion products were introduced into E. coli BL21(DE3) competent cells via heat shock.

[0127] Recombinant strains containing different mutants were prepared separately.

[0128] Example 2: Screening for single mutation sites

[0129] The strains expressing mutants at different sites obtained in Example 1 were screened.

[0130] 1. Screening of Q66 mutant strains

[0131] Sequentially sequenced single clones were selected and placed in LB liquid medium containing 0.05 mg / mL kanamycin resistance. After incubation at 220 rpm and 37 °C for 12 h, seed solutions were prepared.

[0132] The prepared seed culture was transferred into TB liquid medium at an inoculation rate of 2% and cultured at 220 rpm and 37°C. After culturing for 2-3 hours, IPTG was added to a final concentration of 0.2 mM and induced at 25°C for 16 hours. Then, the cells were collected by centrifugation at 10,000 rpm for 30 minutes.

[0133] A 10 ml reaction mixture (pH 8.0) was prepared, containing 200 mM L-aspartic acid, 200 mM NH4Cl, 200 mM MgCl2, and 200 mM ATP. Bacterial cells were added to a final concentration of 20 g / L, and the mixture was reacted at 37 °C for 30 min at 220 rpm. After the reaction, the mixture was centrifuged at 10,000 rpm for 60 min. The supernatant was diluted 50-fold and filtered through a 0.22 μm microfiltration membrane. The L-aspartic acid yield was determined by HPLC. The results are shown below. Figure 1 As shown.

[0134] The results showed that L-asparagine was successfully prepared by whole-cell catalysis using recombinant strains expressing AsnA mutants (Q66 mutated to Ile, Asn, Ala, Phe, Thr, Ser, Gly, Pro, Val, Arg, Leu, Lys, Tyr, Cys, and His, respectively). The yields of L-asparagine were 6.16 g / L, 6.47 g / L, 6.86 g / L, 6.11 g / L, 6.78 g / L, 6.12 g / L, 7.31 g / L, 7.41 g / L, 7.70 g / L, 7.73 g / L, 8.14 g / L, 6.74 g / L, 7.10 g / L, 7.29 g / L, and 7.12 g / L, respectively.

[0135] L-Asparagine was prepared using a recombinant strain expressing wild-type AsnA, with a yield of 4.98 g / L.

[0136] 2. Screening of K58 mutant strains

[0137] Following the method in Example 1, Lys at position 58 was mutated to another amino acid, and whole-cell catalysis was performed according to step 1. The results are as follows: Figure 2 As shown, L-asparagine was prepared by whole-cell catalysis using recombinant strains expressing AsnA mutants of Lys mutated to Phe, Ile, His, Tyr, Cys, Asn, Gln, Arg, Ser, Ala, and Leu, respectively. The yields of L-asparagine were 10.31 g / L, 9.59 g / L, 9.34 g / L, 9.25 g / L, 10.38 g / L, 9.24 g / L, 12.31 g / L, 9.46 g / L, 9.76 g / L, 9.80 g / L, 8.79 g / L, and 9.25 g / L, respectively.

[0138] 3. Screening of R105 mutant strains

[0139] Following the method in Example 1, Arg at position 105 was mutated to another amino acid, and whole-cell catalysis was performed according to step 1. The results are as follows: Figure 3 As shown, L-asparagine was prepared by whole-cell catalysis using recombinant strains expressing AsnA mutants mutated to Leu, Val, Tyr, Pro, Glu, Cys, Gly, Gln, Phe, and Ile, respectively. The yields of L-asparagine were 9.06 g / L, 7.93 g / L, 7.70 g / L, 8.86 g / L, 7.07 g / L, 6.96 g / L, 7.66 g / L, 8.46 g / L, 8.53 g / L, and 11.33 g / L, respectively.

[0140] 4. Screening of D104 mutant strains

[0141] Following the method in Example 1, Asp at position 104 was mutated to other amino acids, and whole-cell catalysis was performed according to step 1. The results are as follows: Figure 4 As shown, L-asparagine was prepared by whole-cell catalysis using recombinant strains expressing AsnA mutants mutated to Thr, Ile, Val, Ala, Pro, Glu, Leu, Lys, and Arg, respectively. The yields of L-asparagine were 7.76 g / L, 7.48 g / L, 7.23 g / L, 7.52 g / L, 7.71 g / L, 7.93 g / L, 7.49 g / L, 7.50 g / L, and 7.47 g / L, respectively.

[0142] 5. Screening of K53 mutant strains

[0143] Following the method in Example 1, Lys at position 53 was mutated to another amino acid, and whole-cell catalysis was performed according to step 1. The results are as follows: Figure 5 As shown, L-asparagine was prepared by whole-cell catalysis using recombinant strains expressing AsnA mutants of Lys mutated to Phe, Ile, His, Asn, Gln, Arg, and Ser, respectively. The yields of L-asparagine were 10.31 g / L, 9.59 g / L, 9.34 g / L, 12.31 g / L, 9.46 g / L, 9.76 g / L, 9.80 g / L, and 9.76 g / L, respectively.

[0144] 6. Screening of R210 mutant strains

[0145] Following the method in Example 1, Arg at position 210 was mutated to another amino acid, and whole-cell catalysis was performed according to step 1. The results are as follows: Figure 6As shown, L-asparagine was prepared by whole-cell catalysis using recombinant strains expressing AsnA mutants mutated to Ala and Pro, respectively. The yields of L-asparagine were 6.39 g / L and 7.95 g / L, respectively. When Arg was mutated to other amino acids, the yield of L-asparagine decreased to varying degrees.

[0146] Example 3: Screening of combined mutant strains

[0147] Primers for EcAsnA were designed, as shown in Table 1. The recombinant vector EcAsnA constructed in Example 1 was obtained by whole-plasmid PCR. L109A / T44I Using -pET-28a as a template, mutants were constructed.

[0148] Recombinant strains containing the triple mutant were prepared according to the method in Example 1:

[0149] EcAsnA L109A / T44I / Q66L -pET-28a-BL21(DE3),EcAsnA L109A / T44I / R105I -pET-28a-BL21(DE3),Ec AsnA L109A / T44I / R105P -pET-28a-BL21(DE3),EcAsnA L109A / T44I / D104K -pET-28a-BL21(DE3),EcAsnA L 109A / T44I / K53N -pET-28a-BL21(DE3),EcAsnA L109A / T44I / R210P -pET-28a-BL21(DE3),EcAsnA L109A / T44I / K58R -pET-28a-BL21(DE3).

[0150] Meanwhile, a strain expressing the L109K / K58R mutant, EcAsnA, was prepared according to the method published in CN118460485A. L109K / K58R -pET-28a-BL21(DE3),EcAsnA L109K -pET-28a-BL21(DE3), as a control.

[0151] Example 4: Study on the enzymatic properties of mutants

[0152] 1. Enzyme activity detection of mutant and wild-type enzymes

[0153] (1) Preparation of pure enzyme solution:

[0154] EcAsnA-pET28a-BL21(DE3) and EcAsnA were respectively... L109A -pET28a-BL21(DE3),EcAsnA L109K-pE T28a-BL21(DE3),EcAsnA L109A / T44I -pET28a-BL21(DE3),EcAsnA L109K / K58R -pET28a-BL21(DE3),EcAsnA L109A / T44I / Q66L -pET28a-BL21(DE3),EcAsnA L109A / T44I / D104K -pET28a-BL21(DE3),EcAsnA L 109A / T44I / K58R -pET28a-BL21(DE3),EcAsnA L109A / T44I / R105P -pET28a-BL21(DE3),EcAsnA L109A / T44I / K53N -pET28a-BL21(DE3) and EcAsnA L109A / T44I / R210P -pET28a-BL21(DE3) strains were inoculated into 3 mL of LB liquid medium and cultured overnight at 37°C.

[0155] Subsequently, 2% inoculum was transferred to 150 mL of TB liquid medium and incubated at 220 rpm and 37°C. When OD 600 When the pH value is between 0.4 and 0.8, add IPTG to a final concentration of 0.4 mM, induce culture at 25°C for 16 h, and collect the bacterial cells after centrifugation.

[0156] Add 10 mL of binding solution A (25 mM Tris-HCl, 250 mM NaCl, 20 mM imidazole, pH adjusted to 8.0 with HCl) to fully resuspend the bacterial cells. Then, place the centrifuge tube in an ice bath and put it into an ultrasonic cell disruptor. The ultrasonic disruption conditions are: working time 3.5 s, interval time 2.5 s, for a total of 10 min. Centrifuge the obtained disruption solution at low temperature and high speed at 4℃ and 10000 rpm for 30 min. Then, add it to a nickel column (5 mL) equilibrated with 25 mL of binding solution A. After the cell disruption solution has drained, add 50 mL of binding solution A to wash the nickel column. Then, add 10 mL of binding solution B (25 mM Tris-HCl, 250 mM NaCl, 500 mM imidazole, pH adjusted to 8.0 with HCl) to elute the target protein EcAsnA from the nickel column. Finally, the protein eluent was concentrated using a 30 kDa ultrafiltration tube and replaced with 0.2 M Tris-HCl (pH 8.0) 3-5 times to remove the high concentration of imidazole.

[0157] The following pure enzymes were prepared: wild-type EcAsnA and mutant EcAsnA. L109A EcAsnA L109K EcAsnA L 109A / T44I EcAsnAL109K / K58R and, EcAsnA L109A / T44I / Q66L EcAsnA L109A / T44I / D104K EcAsnA L109A / T44I / K58R EcAsnA L109A / T44I / R105P EcAsnA L109A / T44I / K53N and EcAsnA L109A / T44I / R210P .

[0158] (2) Detection of enzyme activity

[0159] After mixing 200 mM L-aspartic acid, 200 mM NH4Cl, 200 mM MgCl2, and 200 mM ATP, the pH was adjusted to 8.0 with ammonia. The amount of pure enzyme added was 0.5 mg / ml. The reaction was carried out at 37℃ for 30 min at a speed of 220 rpm. After the reaction was completed, the reaction was terminated with 50 mM HCl. The L-asparagine content was then detected by OPA derivatization. L-aspartic acid and L-asparagine were determined by HPLC. For specific steps, please refer to the literature Anal. Biochem., 1984, 136(1):195-201 (A high-performance liquid chromatography assay for asparagine synthetase). The enzyme activity of asparagine synthase A is defined as the production of 1 μmol·L⁻¹ per minute. -1 The amount of enzyme in L-asparagine.

[0160] The results are as follows Figure 8 As shown, EcAsnA wild type Mutant EcAsnA L109A EcAsnA L109K EcAsnA L109A / T44I EcAsnA L109K / K58R , EcAsnA L109A / T44I / Q66L EcAsnA L109A / T44I / D104K EcAsnA L109A / T44I / K58R EcAsnA L109A / T44I / R105P EcAsnA L109A / T44I / K53N and EcAsnA L109A / T44I / R210P The specific enzyme activities were respectively 497.15 U / mg, 1785.14U / mg, 2079.51U / mg, 1969.88U / mg, 2603.64U / mg, 3222.51U / mg 1469.98U / mg, 2447.38U / mg, 1383.21U / mg, 2069.13U / mg and 1738.55U / mg.

[0161] Optimal mutant EcAsnA L109A / T44I / Q66L Comparing EcAsnA and EcAsnAL109K / K58R These figures represent increases of 5.48 times and 23.77%, respectively.

[0162] 2. Thermal stability study

[0163] The mutant EcAsnA L109A / T44I / Q66L The purified enzyme, following the method in step 1, was incubated at 20, 25, 30, 37, and 45°C for 30 min, 60 min, 120 min, 300 min, and 600 min, respectively, and its activity was measured. The results are as follows: Figure 9 As shown, the mutant EcAsnA L109A / T44I / Q66L It is rapidly inactivated after incubation at 45℃ for 120 min. When the incubation temperature does not exceed 37℃, the activity remains essentially unchanged within 300 min, and decreases by 10-20% after 600 min.

[0164] Therefore, it can be concluded that when the reaction temperature does not exceed 37℃, the mutant EcAsnA L109A / T44I / Q66L Its reactivity is relatively stable.

[0165] 3. pH stability study

[0166] The mutant EcAsnA L109A / T44I / Q66L The purified enzyme, following the method in step 1, was incubated in buffer solutions with pH values ​​of 4, 5, 6, 7.4, 8, 9, 10, and 11 for 60 min, 120 min, 180 min, 300 min, and 600 min, respectively, and the enzyme activity was measured. The results are as follows: Figure 10 As shown, the mutant EcAsnA L109A / T44I / Q66L The mutant EcAsnA rapidly inactivated after incubation for 60 min in buffers with pH 4 and 5; when the buffer pH was 11, the activity decreased by approximately 10% after 600 min; and the mutant EcAsnA showed increased activity at pH 6-10. L109A / T44I / Q66L The activity remains essentially unchanged. Therefore, it can be concluded that the mutant EcAsnA... L109A / T44I / Q66L It exhibits good reaction stability at pH values ​​of 6-10.

[0167] Example 5: Whole-cell catalytic preparation of L-asparagine

[0168] The specific steps are as follows:

[0169] EcAsnA-pET28a-BL21(DE3) and EcAsnA were respectively... L109A -pET28a-BL21(DE3),EcAsnA L109K -pE T28a-BL21(DE3),EcAsnA L109A / T44I -pET28a-BL21(DE3),EcAsnA L109K / K58R-pET28a-BL21(DE3),EcAsnA L109A / T44I / Q66L -pET28a-BL21(DE3),EcAsnA L109A / T44I / D104K -pET28a-BL21(DE3),EcAsnA L 109A / T44I / K58R -pET28a-BL21(DE3),EcAsnA L109A / T44I / R105P -pET28a-BL21(DE3),EcAsnA L109A / T44I / K53N -pET28a-BL21(DE3) and EcAsnA L109A / T44I / R210P -pET28a-BL21(DE3) strain was inoculated into LB liquid medium containing 0.05 mg / mL kanamycin resistance, and after incubation at 220 rpm and 37℃ for 12 h, seed liquids were prepared.

[0170] The prepared seed culture was transferred into TB liquid medium at an inoculation rate of 2% (v / v) and cultured at 220 rpm and 37℃. After 2-3 h of culture, IPTG was added to a final concentration of 0.2 mM and induced at 25℃ for 16 h. Then, the cells were collected by centrifugation at 10000 rpm for 30 min.

[0171] A 10 ml reaction mixture (pH 8.0) containing 200 mM L-aspartic acid, 200 mM NH4Cl, 200 mM MgCl2, and 200 mM ATP was prepared. Bacterial cells were added to a final concentration of 20 g / L, and the mixture was reacted at 37 °C for 30 min at 220 rpm. After the reaction, the mixture was centrifuged at 10,000 rpm for 60 min. The supernatant was diluted 50-fold, filtered through a 0.22 μm microfiltration membrane, and the L-aspartic acid yield was determined by HPLC.

[0172] The results showed that EcAsnA L109A / T44I / Q66L The production intensity of -pET28a-BL21(DE3) is significantly higher than that of EcAsnA. L109K / K58R The production intensity and output of -pET28a-BL21(DE3) have also been significantly improved. Figure 7 ).

[0173] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An asparagine synthase A mutant, characterized in that, The asparagine synthase A mutant is obtained by mutating leucine at position 109 of asparagine synthase A (as shown in SEQ ID NO.1) to alanine, threonine at position 44 to isoleucine, and glutamine at position 66 to leucine.

2. The gene encoding the asparagine synthase A mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the asparagine synthase A mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector.

4. The recombinant cell according to claim 3, characterized in that, The recombinant cells are bacteria or fungi as host cells.

5. A recombinant enzyme catalyst containing the asparagine synthase A mutant of claim 1, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing the asparagine synthase A mutant and isolate transformant cells containing the recombinant asparagine synthase A mutant; (2) Culture recombinant expression transformants containing the asparagine synthase A mutant, isolate transformant cells containing the recombinant asparagine synthase A mutant, and break the transformant cells containing the recombinant asparagine synthase A mutant to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the asparagine synthase A mutant, isolate transformant cells containing the recombinant asparagine synthase A mutant, break the transformant cells containing the recombinant asparagine synthase A mutant, obtain cell lysate, and freeze-dry the cell lysate of the recombinant asparagine synthase A mutant to obtain lyophilized enzyme powder.

6. A method for improving the catalytic activity of asparagine synthase A on substrates and increasing its specific enzyme activity, characterized in that, The method involves mutating leucine at position 109 of asparagine synthase A, as shown in SEQ ID NO.1, to alanine, threonine at position 44 to isoleucine, and glutamine at position 66 to leucine.

7. A method for increasing the yield of L-asparagine, characterized in that, The method involves using L-aspartic acid and NH4Cl as substrates, and employing the asparagine synthase A mutant as described in claim 1, or the recombinant cells as described in claim 3 or 4, or the recombinant enzyme catalyst as described in claim 5, to prepare L-asparagine through reaction.

8. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria used Escherichia coli as the substrate cell. The asparagine synthase A mutant of claim 1 was expressed.

9. A method for preparing L-asparagine, characterized in that, The method involves adding the asparagine synthase A mutant of claim 1, or the recombinant cells of claim 3 or 4, or the genetically engineered bacteria of claim 8, or the recombinant asparagine synthase A mutant enzyme prepared using the genetically engineered bacteria of claim 8, or the recombinant enzyme catalyst of claim 5, to a system containing L-aspartic acid and NH4Cl for reaction, thereby preparing L-asparagine.

10. The method according to claim 9, characterized in that, The system also contains MgCl2 and ATP.

11. The method according to claim 10, characterized in that, In the system, the concentration of NH4Cl is 150~250 mM; the concentration of MgCl2 is 150~250 mM; the concentration of ATP is 150~250 mM; and the concentration of L-aspartic acid is 150~250 mM.

12. The method according to claim 11, characterized in that, The reaction conditions in the system are: pH 7.5-8.5, temperature 30-40℃, reaction time 30-90 min, and rotation speed 200-300 rpm.

13. The use of the asparagine synthase A mutant of claim 1, or the gene or recombinant vector of claim 2, or the recombinant cell of claim 3 or 4, or the recombinant enzyme catalyst of claim 5, or the genetically engineered bacteria of claim 8 in the preparation of L-asparagine or products containing L-asparagine.

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