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CN-121065113-B - Asparagine synthetase A mutant

CN121065113BCN 121065113 BCN121065113 BCN 121065113BCN-121065113-B

Abstract

The invention discloses an asparagine synthetase A mutant, and belongs to the technical field of bioengineering. The invention takes L-aspartic acid and ATP as raw materials, utilizes an asparagine synthetase A (EcAsnA) mutant with improved activity to carry out biosynthesis of L-asparagine, and has simple and efficient whole catalytic process. Compared with the parent capable of catalyzing L-aspartic acid to generate L-asparagine at present, ecAsnA L109A/T44I/Q66L has higher catalytic efficiency, the catalytic activity is improved to 3222.51U/mg, which is 5.48 times of that of the parent (497.15U/mg), and is improved by 23.77% compared with EcAsnA L109K/K58R . The invention provides a novel synthesis method for rapidly synthesizing the L-asparagine and provides a novel idea for the industrial production of the L-asparagine.

Inventors

  • LIU YANG
  • WU JING
  • WANG RAN
  • SONG WEI
  • LIU LIMING

Assignees

  • 山东凯密斯新材料科技有限公司
  • 江南大学

Dates

Publication Date
20260508
Application Date
20250924

Claims (13)

  1. 1. An asparagine synthetase a mutant, characterized in that the asparagine synthetase a mutant is obtained by mutating leucine at position 109 of asparagine synthetase a with an amino acid sequence shown in SEQ ID No.1 to alanine, mutating threonine at position 44 to isoleucine, and mutating glutamine at position 66 to leucine.
  2. 2. A gene encoding the asparagine synthetase a mutant of claim 1 or a recombinant vector carrying said gene.
  3. 3. A recombinant cell expressing the asparagine synthetase a mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector.
  4. 4. The recombinant cell of claim 3, wherein the recombinant cell is a bacterial or fungal host cell.
  5. 5. A recombinase catalyst comprising an asparagine synthetase a mutant according to claim 1, characterized by being in any one of the following forms: (1) Culturing a recombinant expression transformant containing the asparagine synthetase a mutant, and isolating a transformant cell containing the recombinant asparagine synthetase a mutant; (2) Culturing a recombinant expression transformant containing the asparagine synthetase a mutant, isolating a transformant cell containing the recombinant asparagine synthetase a mutant, disrupting the transformant cell containing the recombinant asparagine synthetase a mutant, and obtaining a cell disruption solution; (3) Culturing a recombinant expression transformant containing the asparagine synthetase A mutant, separating transformant cells containing the recombinant asparagine synthetase A mutant, disrupting the transformant cells containing the recombinant asparagine synthetase A mutant to obtain a cell disruption solution, and freeze-drying the cell disruption solution of the recombinant asparagine synthetase A mutant to obtain freeze-dried enzyme powder.
  6. 6. A method for improving catalytic activity of asparagine synthetase A on substrate and improving specific enzyme activity is characterized in that leucine at 109 th position of asparagine synthetase A with an amino acid sequence shown as SEQ ID NO.1 is mutated into alanine, threonine at 44 th position is mutated into isoleucine, and glutamine at 66 th position is mutated into leucine.
  7. 7. A method for improving the yield of L-asparagine is characterized in that L-asparagine is prepared by reacting L-aspartic acid and NH 4 Cl as substrates with the asparagine synthetase A mutant according to claim 1, the recombinant cell according to claim 3 or 4, or the recombinant enzyme catalyst according to claim 5.
  8. 8. A genetically engineered bacterium, wherein the genetically engineered bacterium uses escherichia coli as a chassis cell, and expresses the asparagine synthetase a mutant of claim 1.
  9. 9. A process for producing L-asparagine, comprising adding the asparagine synthetase A mutant of claim 1, the recombinant cell of claim 3 or 4, the genetically engineered bacterium of claim 8, the recombinant asparagine synthetase A mutant enzyme produced by the genetically engineered bacterium of claim 8, or the recombinase catalyst of claim 5 to a system containing L-aspartic acid and NH 4 Cl, and reacting.
  10. 10. The method of claim 9, wherein the system further comprises MgCl 2 , ATP.
  11. 11. The method of claim 10, wherein the system has a concentration of NH 4 Cl of 150 to 250 mM, a concentration of MgCl 2 of 150 to 250 mM, a concentration of ATP of 150 to 250 mM, and a concentration of L-aspartic acid of 150 to 250 mM.
  12. 12. The method according to claim 11, wherein 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. 13. Use of an asparagine synthetase a mutant according to claim 1, or a gene or recombinant vector according to claim 2, or a recombinant cell according to claim 3 or 4, or a recombinant enzyme catalyst according to claim 5, or a genetically engineered bacterium according to claim 8 for the preparation of L-asparagine or a product containing L-asparagine.

Description

Asparagine synthetase A mutant Technical Field The invention relates to an asparagine synthetase A mutant, and belongs to the technical field of bioengineering. Background Asparagine is one of 20 common amino acids and is widely applied to the fields of medicines, foods and the like. In recent years, research shows that the compound obtained by converting asparagine has high medicinal value, and shows good biological activity in the aspects of diminishing inflammation, reducing blood pressure, stopping bleeding and the like, such As Captopril (ACEI) antihypertensive drugs, endomorphin-2 (EM 2) analgesic drugs, anti-inflammatory drugs N- [ beta- (p-substituted benzoyl) ethyl ] asparagine and the like. The synthesis method of L-asparagine mainly comprises chemical synthesis, plant extraction and biosynthesis, wherein the biosynthesis has the advantages of simple process, low equipment requirement, high production efficiency, low energy consumption, little pollution and the like, but the problems of low key enzyme activity and the like still exist. In view of the importance of L-asparagine, there is a strong need to develop methods for efficient synthesis of L-asparagine, which drive the production and use of L-Asn. In the existing biological synthesis, L-aspartic acid is used as a substrate, and an asparagine synthetase A is utilized to carry out ammonia connection reaction on the side chain carboxyl of the substrate so as to synthesize L-asparagine. Asparagine synthetase A is a multifunctional enzyme which takes ATP as cofactor and performs functionalization on an amino acid C-O bond, and is considered as one of important catalysts for amino acid amidation. Although mutants of the enzyme have been disclosed in the prior art, the catalytic efficiency and specific activity of the presently disclosed enzyme need to be further improved, and in order to solve the above problems, the present invention further modifies asparagine synthetase A mutants EcAsnA L109A/T44I by protein engineering means to obtain asparagine synthetase A with improved activity, thereby completing low-cost industrial synthesis of L-asparagine. Disclosure of Invention Aiming at the defects of the prior art, the invention provides construction and property research of an asparagine synthetase A mutant for producing L-asparagine, and aims to efficiently synthesize L-asparagine by using low-cost substrates L-aspartic acid and cofactor ATP through genetic engineering bacteria, thereby solving the technical problems that the activity of the asparagine synthetase A is lower in the existing biological synthesis, the yield of L-asparagine is too low, and the production strength of the asparagine synthetase A is still to be further improved. The invention provides an asparagine synthetase A mutant, which is obtained by mutating leucine 109, threonine 44 and glutamine 66 of an asparagine synthetase A parent, wherein the amino acid sequence of the asparagine synthetase A (EcAsnA) is shown as SEQ ID NO. 1. MKTAYIAKQRQISFVKSHFSRQLEERLGLIEVQAPILSRVGDGTQDNLSGCEKAVQVKVKALPDAQFEVVHSLAKWKRQTLGQHDFSAGEGLYTHMKALRPDEDRLSPLHSVYVDQWDWERVMGDGERQFSTLKSTVEAIWAGIKATEAAVSEEFGLAPFLPDQIHFVHSQELLSRYPDLDAKGRERAIAKDLGAVFLVGIGGKLSDGHRHDVRAPDYDDWSTPSELGHAGLNGDILVWNPVLEDAFELSSMGIRVDADTLKHQLALTGDEDRLELEWHQALLRGEMPQTIGGGIGQSRLTMLLLQLPHIGQVQCGVWPAAVRESVPSLL In one embodiment, the nucleic acid sequence of the gene encoding EcAsnA is shown as SEQ ID NO. 2. SEQ ID NO.2: atgaaaaccg cttacattgc caaacaacgt caaattagct tcgtgaaatc tcacttttct cgtcaactgg aagaacgtct ggggctgatc gaagtccagg cgccgattct tagccgtgtg ggggatggca cgcaggataa cttgtcgggc tgtgaaaaag cggtgcaggt aaaagtgaaa gctctgcctg atgcccagtt cgaagtggtt cattcactgg cgaagtggaa acgtcagacc ttagggcaac acgacttcag cgcgggcgaa gggctgtaca cgcacatgaa agcccttcgc cccgatgaag accgtctttc tccgttgcac tcggtctatg ttgaccagtg ggactgggaa cgcgtaatgg gcgacggtga gcgtcaattc tcgactctga aaagcacggt agaggcgatc tgggcgggaa ttaaagcaac cgaagctgcg gttagcgaag agtttggcct ggcaccgttc ctgccggatc agatccactt cgtacacagc caggagttac tgtctcgtta tccggatctt gatgccaaag ggcgtgagcg ggcgatagcg aaagatcttg gcgcggtatt ccttgtcggg attggcggca agctgagcga tggtcatcgc cacgacgtgc gcgcaccgga ttatgatgac tggagcaccc cgtcagagct gggccatgcg ggtctgaacg gcgatattct ggtgtggaac ccggtactgg aagatgcgtt tgagctttcc tccatgggga tccgtgtaga tgccgacacg ctgaagcatc aactggcgct gaccggtgac gaagatcgcc tggagctgga gtggcatcag gcgctgctgc gcggtgaaat gccgcagacc atcggcggcg gtatcggcca gtctcgtttg actatgctgc tgctgcaact gccgcatatc ggccaggttc agtgtggagt atggccagct gctgttcgcg agagcgtccc ttctctgctg taa In one embodiment, the mutant is a mutation of the parent as shown in mutation of leucine L at position 109 to alanine A, threonine T at position 44 to isoleucine I and glutamine Q at position 66 to leucine L. In one embodiment, the mutant is mutated at amino acid 109, amino acid 44, and glutamine 66 relative to the EcAsnA parent to obtain mutant EcAsnA L109A/T44I/Q66L. In one embodiment, the amino acid sequence of the mutant EcAsnA L109A/T44I/Q