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A kind of 1-amino acid ligase mutant, recombinant vector, recombinant bacteria and application thereof

A recombinant vector, amino acid technology, applied in the field of genetic engineering and biology, can solve the problems of substrate specificity, low BacD protease activity, etc.

Active Publication Date: 2022-07-01
JINING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low enzymatic activity of the original BacD protease and poor substrate specificity, there is an urgent need for a L-amino acid ligase mutant with strong substrate specificity and high catalytic activity, and to apply it to actual production

Method used

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  • A kind of 1-amino acid ligase mutant, recombinant vector, recombinant bacteria and application thereof
  • A kind of 1-amino acid ligase mutant, recombinant vector, recombinant bacteria and application thereof
  • A kind of 1-amino acid ligase mutant, recombinant vector, recombinant bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 The construction of the novel L-amino acid ligase of this example, the specific construction process includes:

[0025] ①. Rational transformation of L-amino acid ligase to improve its substrate specificity and catalytic activity

[0026] From the genome of Bacillus amyloliquefaciens (Bacillus subtilis), clones derived from Bacillus subtilis were obtained by PCR cloning L - Amino acid ligase gene (baLal_16), on this basis, a nucleotide sequence (Balal) suitable for expression in E. coli was obtained by codon optimization, and chemical synthesis was carried out. The above-synthesized L-amino acid ligase gene was cloned into the pET28(a) plasmid between the BamH I and Xho I restriction sites to construct a recombinant plasmid. The recombinant plasmid was transformed into E. coli competent cell DH5α, and the colonies growing on the kanamycin plate were picked, the plasmid was extracted, identified by restriction enzyme digestion and sequenced, and the correct...

Embodiment 2

[0052] Example 2 Application example of producing Ala-Gln based on L-amino acid ligase mutant

[0053] ①Add BL001 and NFLY005 to 5 mL of LB medium containing 50 μg / mL kanamycin, respectively, and culture at 37°C and 220 rpm for 8-12 hours. Then transfer 2 mL to 100 mL of LB medium containing 50 μg / mL kanamycin according to 2% of the inoculum.

[0054] ②The concentration of bacteria (OD) 600 ) reached 0.6-0.8, IPTG was added with a final concentration of 0.1 mM, and cultured at 28° C. and 220 rpm for 12 h. The cells were collected by centrifugation at 6000 × g for 10 min at 4°C, and resuspended by centrifugation with 100 mM Tris-HCl (pH 7.0) for 3 times, and then the cells were resuspended with 100 mM Tris-HCl.

[0055] ③ The resuspended cells were placed in an ice-water bath for ultrasonic disruption, centrifuged at 12,000 × g for 10 min at 4°C, and the disrupted supernatant was purified and desalted to verify the ability of wild-type and mutants to produce Ala-Gln. ability...

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Abstract

The invention discloses an L-amino acid ligase mutant, a recombinant vector, a recombinant bacteria and applications thereof. The L-amino acid ligase mutant is obtained by mutating the 110th position of the amino acid sequence shown in SEQ ID NO.2; or the 108th position and the 110th position of the amino acid sequence shown in SEQ ID NO.2 simultaneously. Obtained by mutation. The amino acid sequences of the L-amino acid ligase mutants are shown in SEQ ID No.4 and SEQ ID NO.5. A recombinant vector constructed by using the L-amino acid ligase mutant encoding gene, the L-amino acid ligase mutant expressed by a genetically engineered bacteria containing the recombinant vector has a higher enzymatic activity than wild-type in the process of catalyzing and synthesizing Ala-Gln Type BacD increased by 1.87 times to 232.45±17.4U·(mg·h) ‑1 ; After 26 hours of reaction, the mutant can make the released product phosphorus concentration reach 694.47 μM, which is 21.4% higher than that of wild-type BacD accumulation of 571.95 μM. By calculation, the yield of the catalytic product Ala-Gln of the double mutant NFLY per unit mass can reach 2.59 mM ‑1 ·L ‑1 ·mg ‑1 .

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biotechnology, and in particular relates to an L-amino acid ligase mutant, a recombinant vector, a recombinant bacteria and its application with improved catalytic activity and improved substrate specificity. Background technique [0002] L-amino acid ligase (Lal) is an important member of the ATP-grasp enzyme superfamily. It can synthesize dipeptides using unprotected L-amino acids as substrates, so it has important application value in the biosynthesis of dipeptides. . Currently, Lal from different sources (eg Ywf E, RizA, BL00235, PSPPH 4299 and TabS, etc.) have been found, which can catalyze the synthesis of different dipeptide compounds. [0003] Lals has a very important potential application value in the biosynthesis of dipeptide. Compared with chemical synthesis or other tool enzymes, Lals has the following advantages: (1) Lals is a soluble protease with a small structure. It is e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/70C12N1/21C12P21/02C12R1/19
CPCC12N9/93C12N15/70C12P21/02C07K5/06026C12Y603/02
Inventor 王涛刘晓环宁丽笑张宇菲王一凡
Owner JINING MEDICAL UNIV