(-)-[gamma]-lactamase, gene, mutant, vector as well as preparation method and application of (-)-[gamma]-lactamase

A technology of lactamase and lactam, applied in the field of bioengineering, can solve problems such as difficult large-scale application, low product concentration, and large catalyst usage

Active Publication Date: 2016-09-21
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, these enzymes are difficult to apply on a large scale due to their low activity and poor substrate concentration tolerance.
[0004] Compared with the traditional chemical synthesis method, the method of preparing (+)-γ-lactam by biocatalytic hydrolysis of racemic γ-lactam has the advantages of mild reaction conditions, environmental friendliness, and simple operation, but currently (+)-γ-lactam The biocatalytic synthesis method of lactam is limited to the laboratory scale, and has the defects of low enzyme activity, large amount of catalyst used, and low product concentration, and is not suitable for industrial production

Method used

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  • (-)-[gamma]-lactamase, gene, mutant, vector as well as preparation method and application of (-)-[gamma]-lactamase
  • (-)-[gamma]-lactamase, gene, mutant, vector as well as preparation method and application of (-)-[gamma]-lactamase
  • (-)-[gamma]-lactamase, gene, mutant, vector as well as preparation method and application of (-)-[gamma]-lactamase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning of (-)-γ-lactamase SvGL gene

[0046] The strain Streptomyces viridochromogenes CGMCC 4.692 was cultured in LB medium, and the high-purity and large-fragment genomic total DNA was extracted by the high-salt method, dissolved in TE buffer (pH8.0), and stored at -20°C. The specific method Refer to the "Refined Molecular Biology Experiment Guide" edited by F. Osper et al.

[0047] The extracted total DNA was partially cleaved with restricted enzyme Sau3AI, and the cleaved DNA fragments were purified by electrophoresis, and about 4-6kb fragments were recovered using a gel recovery and purification kit, and the recovered DNA was dissolved in TE buffer (10mM ,pH8.0), stored at -20°C.

[0048] Connect with the vector pUC118 according to the following reaction system:

[0049]

[0050]

[0051] Incubate at 16°C for 6 hours, take 10 μL of the enzyme-linked product and transform 200 μL of Escherichia coli DH5α competent cells (TaKaRa, Code: D9057), pick ...

Embodiment 2188

[0053] Example 2 Site-directed saturation mutation of 188-position phenylalanine

[0054] use II Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522) described the protocol to operate. Primers containing mutation points were designed: forward primer (Primer F) 5'-GCCGTCCGCAACAGCNKAACGTCGCCGCGGGC-3' and reverse primer (Primer R) 5'-GCCCGCGGCGACGTTMNNGCTGTTGCGGACGGC-3'. PCR reaction system (50μL): template 0.5~20ng, 5μL 10×KOD plus buffer, 5μL dNTP (each 2.0mM), 2μL MgSO 4 (25mM), 1 μL (20 μM) of each pair of mutant primers, 1 unit of KOD enzyme (TOYOBO CO., LTD., Osaka, Japan), add sterilized distilled water to 50 μL. The template described therein is the plasmid pET28a-SvGL obtained in Example 1. PCR reaction program: (1) denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 sec, (3) annealing at 55°C for 1 min, (4) extension at 68°C for 7 min, steps (2) to (4) were performed for 30 cycles in total, Finally, extend at 68°C for 10 min, and store the produc...

Embodiment 3

[0055] Example 3 Site-directed saturation mutation of leucine at position 130

[0056] use II Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522) described the protocol to operate. Primers containing mutation points were designed: forward primer (Primer F) 5'-TCGCTGGAGCCCTGNNKCTCAAGTCCGACGAC-3' and reverse primer (Primer R) 5'-GTCGTCGGACTTGAGMNNGCAGGGCTCCAGCGA-3'. PCR reaction system (50μL): template 0.5~20ng, 5μL 10×KOD plus buffer, 5μL dNTP (each 2.0mM), 2μL MgSO 4 (25mM), 1 μL (20 μM) of each pair of mutant primers, 1 unit of KOD enzyme (TOYOBO CO., LTD., Osaka, Japan), add sterilized distilled water to 50 μL. Wherein said template is the plasmid pET28a-SvGL that embodiment 2 obtains F188W . PCR reaction program: (1) denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 sec, (3) annealing at 55°C for 1 min, (4) extension at 68°C for 7 min, steps (2) to (4) were performed for 30 cycles in total, Finally, extend at 68°C for 10 min, and store the produc...

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Abstract

The invention relates to (-)-[gamma]-lactamase, a gene and a mutant of the (-)-[gamma]-lactamase, a recombinant expression plasmid and a recombinant expression transformant containing the gene and the mutant, a preparation method of the (-)-[gamma]-lactamase and an application of the (-)-[gamma]-lactamase in preparing (+)-[gamma]-lactam. Compared with the prior art, the (-)-[gamma]-lactamase disclosed by the invention has the characteristics of being high in enzymatic activity and good in substrate concentration tolerance; the (+)-[gamma]-lactam, which is prepared through enzymatic catalysis, has the advantages of being mild in reaction condition, high in substrate concentration, low in catalyst dosage and the like; therefore, the (-)-[gamma]-lactamase has a good application prospect in industrial production of the (-)-[gamma]-lactamase which is an intermediate product of carbocyclic nucleoside drugs.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a (-)-γ-lactamase, its gene and mutant, a recombinant expression plasmid and a recombinant expression transformant containing the gene and the mutant, the recombinant (-) - Preparation of gamma-lactamase, and application of the (-)-gamma-lactamase in preparing (+)-gamma-lactam. Background technique [0002] γ-lactam is the abbreviation of the compound 2-azabicyclo[2.2.1]hept-5-en-3-one (also commonly known as Wens lactone), and its molecular formula is C 6 h 7 NO. Both enantiomers of γ-lactam are useful pharmaceutical intermediates, among which (-)-γ-lactam can be used to synthesize antiretroviral drug abacavir and anti-influenza virus drug peramivir ; while (+)-γ-lactam can be used to synthesize chemokine receptor CCR2 antagonist MK-0812 and dipeptidyl peptidase inhibitor merolipine. [0003] The preparation of (+)-γ-lactam or (-)-γ-lactam by biocatalysis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12P41/00C12P17/10
Inventor 许建和殷金岗郑高伟潘江
Owner EAST CHINA UNIV OF SCI & TECH
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