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Recombinant aspartase mutant, coding gene and application of recombinant aspartase mutant

An aspartase and mutant technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of low yield, unfriendly environment, harsh reaction conditions, etc., and achieve high catalytic efficiency and environmental friendliness. , the effect of mild reaction conditions

Active Publication Date: 2020-08-28
ZHEJIANG HAISEN PHARMACY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing chemical synthesis method has harsh reaction conditions, low yield, and easy to produce waste liquid and wastewater, which is not friendly to the environment

Method used

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  • Recombinant aspartase mutant, coding gene and application of recombinant aspartase mutant
  • Recombinant aspartase mutant, coding gene and application of recombinant aspartase mutant
  • Recombinant aspartase mutant, coding gene and application of recombinant aspartase mutant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 produces wild-type aspartase LBS

[0029] The synthetic gene LBS was connected to the expression vector pET-28a(+), so that it could be expressed in Escherichia coli JM109(DE3).

[0030] The experimental operation process is as follows:

[0031] a) Use the same restriction enzymes to double-enzyme digest the nucleotide sequence SEQ ID No.4 encoding the amino acid sequence of SEQ ID No.1 and the expression vector pET-28a(+) to obtain sticky end fragments. After cutting for 100 min, mix with 10X Loading Buffer, pipette evenly, and then perform agarose gel electrophoresis. The 20 μL enzyme cutting system is shown in Table 1.

[0032] Table 1 Restriction enzyme digestion system

[0033]

[0034] b) Purify and recover the LBS gene fragment and pET-28a(+) vector fragment using the DNA gel recovery kit, see the instructions for the specific operation steps;

[0035] c) The recovered product was ligated into a circular plasmid using Ligation high (purchased f...

Embodiment 2

[0045] Embodiment 2 produces aspartase mutant LBS-2 and LBS-3

[0046] The synthetic gene LBS-2 was connected to the expression vector pET-28a(+), so that it could be expressed in Escherichia coli JM109(DE3).

[0047] The experimental operation process is as follows:

[0048] a) Use the same restriction enzymes to double-enzyme digest the nucleotide sequence SEQ ID No.5 encoding the amino acid sequence of SEQ ID No.2 and the expression vector pET-28a(+) to obtain sticky end fragments, and enzyme at 37°C After cutting for 100 min, mix with 10X Loading Buffer, pipette evenly, and run on agarose gel. See Table 3 for the 20 μL enzyme digestion system.

[0049] Table 3 Restriction enzyme digestion system

[0050]

[0051] b) Purify and recover the LBS-2 gene fragment and the pET-28a(+) vector fragment using a DNA gel recovery kit, see the instructions for the specific operation steps;

[0052] c) The recovered product was ligated into a circular plasmid using Ligation high (p...

Embodiment 3

[0063] Embodiment 3 wild aspartase LBS and recombinant aspartase mutant LBS-2 and LBS-3 protein purification

[0064] After equilibrating the protein Ni+ column with buffer A, add the crude enzyme solution prepared in Example 1, fully shake for 30 minutes, let go of the waste liquid, wash with buffer A three times, add buffer B and shake evenly for 20 minutes, and then collect the effluent , add excess ammonium sulfate to the effluent until it cannot be dissolved, centrifuge at 3000rpm for 10min, collect the precipitate, add 15% glycerol aqueous solution to dissolve the precipitate, and obtain a protein solution; The A280 absorption value of the solution was collected, and the part with an A280 absorption value greater than 0.1 was collected to obtain the purified wild-type aspartic acid LBS enzyme solution; buffer A: NaCl 140mmol / L, KCl 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 1.8mmol / L, adjust the pH value to 8.0 with 5M NaOH, the solvent is water; buffer B: Na 2 HP...

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Abstract

The invention provides a recombinant aspartase mutant, a coding gene and an application of the recombinant aspartase mutant in synthesis of R-3-aminobutyric acid, and belongs to the technical field ofgene engineering and biosynthesis. The recombinant aspartase mutant disclosed by the invention can take crotonic acid as a substrate, and has high regioselectivity and stereoselectivity in catalyticsynthesis reaction of R-3-aminobutyric acid; and the catalytic efficiency is high and the cost is lower. The reaction condition for synthesizing the R-3-aminobutyric acid by using the recombinant aspartic acid mutant provided by the invention is mild, and the application is environment-friendly.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and biosynthesis, and in particular relates to recombinant aspartase mutants, coding genes and applications thereof. Background technique [0002] R-3-aminobutyric acid (R-3-aminobutyric acid), CAS: 3775-73-3, is mainly used as the precursor of pharmaceutical intermediate R-3-aminobutyric acid. R-3-amino-1-butanol, CAS: 61477-40-5, is a key intermediate used in the treatment of AIDS drug Dolutegravir. Therefore, R-3-aminobutyric acid is one of the important raw materials for the synthesis of dolutegravir. [0003] The development of the synthetic route of R-3-aminobutyric acid has important application value. The development of high-efficiency and low-cost route to synthesize high-purity R-3-aminobutyric acid is to reduce the cost of dolutegravir raw materials and promote its application range key steps. However, the existing chemical synthesis method has harsh reaction conditions, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/88C12N15/70C12P13/005C12Y403/01001
Inventor 赖敦岳周硕叶涛雷军林张壹腾汪钱
Owner ZHEJIANG HAISEN PHARMACY CO LTD
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