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(+) gamma-lactamase with activity on splitting racemate gamma-lactam as well as coded gene and application thereof

A technology of lactamase encoding and lactamase, which is applied in the field of enzyme engineering, can solve the problems of non-absolute selectivity, high energy consumption, and high optimum reaction temperature in the resolution

Inactive Publication Date: 2012-09-12
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The main disadvantage of the reported γ-lactamase for the resolution of racemic γ-lactam is that γ-lactamase does not have absolute selectivity for the resolution of racemic γ-lactamase substrates , so the resolution effect depends on the overreaction of the resolution reaction, that is, more than 50% reaction conversion is required
This non-absolutely selective enzyme can easily result in reduced optical activity of the product or loss of the desired optical product
In addition, although the γ-lactamase derived from Sulfolobus solfatrobus has absolute selectivity, its optimum reaction temperature is too high (80°C), and the production process using this enzyme consumes a lot of energy

Method used

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  • (+) gamma-lactamase with activity on splitting racemate gamma-lactam as well as coded gene and application thereof
  • (+) gamma-lactamase with activity on splitting racemate gamma-lactam as well as coded gene and application thereof
  • (+) gamma-lactamase with activity on splitting racemate gamma-lactam as well as coded gene and application thereof

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Effect test

Embodiment 1

[0035] Embodiment 1 Obtaining of (+) γ-lactamase MHlac gene of the present invention

[0036] (1) Construction and screening of genome plasmid library of Microbacterium hydrocarbonoxydans

[0037] After the ampere tube nozzle of the preserved Microbacterium hydrocarbonoxydans L29-9 (Microbacterium hydrocarbonoxydans), CGMCC No.2085 (the relevant information and preservation of the Microbacterium have been announced in the Chinese patent application with the publication number CN101113423A) was broken Add 1mL liquid nutrient gravy agar medium (peptone 10g / L, beef extract 3g / L, NaCl 5g / L, distilled water 1L, pH7.0), and then insert the suspension of the bacterial strain into 100mL of the same medium for culture, Shaker conditions are 220 rpm, 30°C, 48 hours. After the cultivation was completed, the bacteria were collected by centrifugation at 12000 rpm.

[0038]Genomic DNA from Microbacterium oxidizers was purified and extracted using a Bacterial Genome Extraction Kit (Shangha...

Embodiment 2

[0051] Embodiment 2 recombinant protein expression and purification

[0052] The constructed plasmid pETMHlac was introduced into Escherichia coli E.coil BL21(DE3) by electrotransformation method to obtain transformant E.coil BL21(pETMHlac). 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, 20mM imidazole, 50mMNaCl, pH8.0,), perform ultrasonic disruption (300W, ultrasonic 3 seconds, 1 second interval, 90 cycles in total), centrifuge at 14000RPM, and collect the supernatant solution, the supernatant was added to 1 mL of Ni-NTA affinity column (Novogen), and after binding, it was wa...

Embodiment 3

[0054] Embodiment 3 (+) Immobilization of γ-lactamase

[0055] The immobilized (+) γ-lactamase is prepared by the following method: the (+) γ-lactamase is immobilized by Ni-NTA agarose method, and the immobilization condition is as follows: (+) ) γ-lactamase is dissolved in binding buffer (50mM TrisHCl, 20mM imidazole, 50mMNaCl, pH8.0), the concentration of (+) γ-lactamase in this buffer is 4g / L, and then Ni-NTA agarose is added in the solution of (+) γ-lactamase, this Ni-NTA agarose is washed repeatedly with above-mentioned binding buffer solution and soaked more than 3 hours before using, and described (+) γ-lactamase The volume ratio of the solution to Ni NTA agarose was controlled at 1:1, the immobilization time was 2 hours, and the immobilization temperature was 4°C.

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Abstract

The invention discloses a (+) gamma-lactamase with activity on splitting racemate gamma-lactam as well as a coded gene and application thereof, wherein the (+) gamma-lactamase and the coded gene thereof derive from Microbacterium hydrocarbonoxydans, and the application refers to hydrolysis and splitting of the racemate gamma-lactam by the (+) gamma-lactamase. The (+) gamma-lactamase refers to a protein in (a) or (b) as follows: (a) a protein formed by the amino acid residue sequence of SEQ ID NO:1 in a sequence table; and (b) the protein which has activity on splitting racemate gamma-lactam and derives from the SEQ ID NO:1 by the steps of carrying out substitution and / or deletion and / or adding of one or more amino acid residue sequences on the amino acid residue sequences of the SEQ ID NO:1 in the sequence table. By utilizing the (+) gamma-lactamase to hydrolyze and split the racemate gamma-lactam, (-) gamma-lactam with the optical purity of 99.5 percent can be obtained with the yield larger than 39 percent.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a (+) gamma-lactamase with the activity of splitting racemate gamma-lactam and its coding gene, and the application of the (+) gamma-lactamase. 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. Currently, methods for synthesizing chiral (-)γ-lactams are mainly divided into chemical synthesis, chiral auxiliary co-crystallization and biological enzymatic conversion. The chemical method has hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/86C12N15/55C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P41/00
Inventor 王建军吴胜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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