Method for preparing (‑) gamma lactam by using members of cysteine ​​hydrolase family

A technology of cysteine ​​and γ-lactam, which is applied in the field of enzyme engineering and can solve the problem of no patent report for the cysteine ​​hydrolase family

Active Publication Date: 2018-02-09
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no patent reports and literature reports on the application of the cysteine ​​hydrolase family in the resolution of racemic γ-lactams

Method used

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  • Method for preparing (‑) gamma lactam by using members of cysteine ​​hydrolase family
  • Method for preparing (‑) gamma lactam by using members of cysteine ​​hydrolase family
  • Method for preparing (‑) gamma lactam by using members of cysteine ​​hydrolase family

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Embodiment 1

[0073] Embodiment 1 The acquisition of the cysteine ​​hydrolase gene of 13 kinds of microbial sources involved in the present invention

[0074] (1) Acquisition of bacterial genomic DNA

[0075] Purchase nitrogen-fixing rhizobia (Azorhizobium caulinodans 1.2548), Bacillus pumilus 1.326, Escherichia coli 1.359, Lactobacillus lactis (Lactococcus lactis 1. .2030), Methylobacterium extorquens4.1476, Pseudomonas aeruginosa1.228, Ralstonia pickettii1.1759, Sinorhizobium fredii1. 535), Streptococcus aureus (Staphylococcus aureus1.363), the bacterial strain ampoule tube of Vibrio parahaemolyticus (Vibrio parahaemolyticus1.1997), add 1mL culture medium after the mouth of the pipe is smashed, and the culture medium is published according to the Microbial Strains Preservation Management Committee The methods in the "Catalogue of Strains" are formulated for different strains. Then, the suspension of the bacterial strain was inserted into 100 mL of the same medium for culture, and the sh...

Embodiment 2

[0093] Example 2 Expression and purification of recombinant cysteine ​​hydrolase protein

[0094] The constructed plasmid pET**ch was introduced into Escherichia coli E.coil BL21(DE3) by electroporation to obtain the transformant E.coil BL21(pET**ch). Inoculate the transformant into a test tube of LB liquid medium (containing kanamycin), culture overnight at 37°C, and transfer to 400 mL of LB liquid medium (containing kanamycin) at a transfer rate of 1%. , cultured at 37°C, and when the OD value was 0.6-0.8, IPTG with a final concentration of 1 mM was added for induction culture for 3 hours, and the cells were collected by centrifugation. Suspend the bacteria in the binding buffer (50mM TrisHCl, pH8.0, 20mM imidazole, 50mMNaCl), perform ultrasonic disruption (300W, ultrasonic 3 seconds, interval 1 second, 90 cycles in total), centrifuge at 14000RPM, and collect the supernatant , the supernatant was added to 1 mL of nickel affinity column (Novogen), combined and washed with 5 ...

Embodiment 3

[0096] The immobilization of embodiment 3 cysteine ​​hydrolase

[0097] The purified protein was immobilized by sodium alginate method. The immobilization method was as follows. Add 0.3 g of sodium alginate (purchased from Bailingwei Company) to 10 mL of water and heat it to 121 ° C to completely dissolve it. Cool to 30°C, add cysteine ​​hydrolase dissolved in pH 7.0 phosphate buffer, the concentration of cysteine ​​hydrolase in the sodium alginate solution is 4g / L, stir the mixture slightly Mix, carefully drop the mixture into ice-cold 0.1mol / L CaCl with a syringe 2 1.5mol / L boric acid solution, the sodium alginate and CaCl 2 The molar ratio of the immobilized enzyme was controlled at 23:10, and the immobilized enzyme was suspended in the above boric acid solution for 3 hours, keeping the temperature at 4°C. After immobilization, the immobilized enzyme was collected by filtration, washed with distilled water and stored in a refrigerator at 4°C.

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Abstract

The invention provide a group of cysteine hydrolases with (+) gamma lactam resolution activity. The cysteine hydrolases come from 13 microorganism species, and have three common conserved structural domains: the conserved structural domain I: *A(V / M / R / G / T)L(V / H / I)L(V / I)L(V / I)V(N / E / D)*(H / I)DM(L / V / I)Q, the conserved structural domain II: *S(G / D / N)*F(W / Y)*, the conserved structural domain III: *(F / V / M)V(A / C / M / T)G*T(A)N(E / H / D / A)*C(G / A / D / P), wherein * stands for any amino acid residue, and the amino acids in the brackets stand for possible amino acid residues at the same amino acid residues. The invention also provides coding genes of the above cysteine hydrolases, and applications of the above cysteine hydrolases in resolution of racemate gamma lactam.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to cysteine ​​hydrolase family members with (+) gamma lactam splitting activity and their coding genes, as well as the application of the enzymes. Background technique [0002] At present, resource, energy and environmental crises have threatened the survival and development of human beings. Biotransformation uses microorganisms or enzymes as catalysts to replace non-renewable resources with renewable resources, and is an effective means for large-scale production of chemicals, medicines, energy, materials, etc. needed by humans. [0003] (-)γ-lactam is an important intermediate in the synthesis of anti-AIDS drug abacavir and anti-influenza A and bird flu drug peramivir. At present, the methods for synthesizing chiral (-) γ-lactams are mainly divided into chemical synthesis, chiral auxiliary co-crystallization and biological enzymatic conversion. The chemical method h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/14C12N15/55C12N15/63C12N1/15C12N1/19C12N1/21C12P17/10
CPCC12N9/14C12P17/10C12P41/001
Inventor 王建军吴胜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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