Variovoraxboronicumulans CGMCC 4969 and use thereof in bioconversion of 3-cyanopyridine for forming nicotinamide

A technology of biotransformation and greedy phagocytosis, applied in the field of microorganisms, can solve problems such as the function of nitrile hydratase that has not been experimentally proved

Inactive Publication Date: 2012-10-24
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the nitrile hydratase produced by Variovorax, currently in the Genbank database, there is a 224bp partial nitrile hydratase from Variovorax sp. DSM 11402 submitted by Lourenco et al. Enzyme gene fragment (genbank accession number: AJ577856); derived from V. paradoxus EPS (Genbank accession number: NC_014931) and V. paradoxus S110 (Genbank accession number: NC_012791) The nitrile hydratase gene in the whole genome sequence of the two strains, however, the above-mentioned nitrile hydratase gene is the result of bioinformatics prediction, and no experiment has proved its nitrile hydratase function

Method used

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  • Variovoraxboronicumulans CGMCC 4969 and use thereof in bioconversion of 3-cyanopyridine for forming nicotinamide
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  • Variovoraxboronicumulans CGMCC 4969 and use thereof in bioconversion of 3-cyanopyridine for forming nicotinamide

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0016] Example 1: Isolation, screening, identification and biological characteristics of strains that can biotransform 3-cyanopyridine into nicotinamide

[0017] 1. Strain isolation

[0018] Collect soil from Xianlin area, Nanjing City, Jiangsu Province, take 1g of soil and add it to 19mL sterile water containing 5 glass beads, shake it for 5min, let it stand for 10min, take 1ml of the suspension and add 19ml of mineral containing 1% 3-cyanopyridine in salt medium. The composition of the mineral salt medium is: 1.36 g / L KH 2 PO 4 , 2.13g / L Na 2 HPO 4 , 0.5 g / L MgSO 4 ·7H 2 O and 10ml / L metal ionic liquid, pH 7.5. The metal ionic liquid composition is: 0.40 g / L CaCl 2 2H 2 O, 0.30 g / L H 3 BO 3 , 0.04 g / L CuSO 4 ·5H 2 O, 0.10 g / L KI, 0.20 g / L FeSO 4 ·7H 2 O, 0.40 g / L MnSO 4 ·7H 2 O, 0.20 g / L NaMoO 4 2H 2 O and 10.0 mL / L concentrated hydrochloric acid. The samples were incubated at 30°C for 3 days in a shaker with a rotational speed of 220 rpm. Take 100 μ...

example 2

[0024] Example two: V. boronicumulans Cloning of CGMCC 4969 Nitrile Hydratase Gene

[0025] V. boronicumulans The extraction of genomic DNA of CGMCC 4969 is the same as the extraction method of genomic DNA in Example 1.

[0026] The four nucleotides involved are: deoxyadenine triphosphate (abbreviated as A), deoxythymidine triphosphate (denoted as T), deoxyguanine triphosphate (abbreviated as G) , Deoxycytosine nucleotide triphosphate (abbreviated as C). Design and synthesize the upstream and downstream primers of nitrile hydratase gene. R in the primer indicates that the base at this position is A or G, S in the primer indicates that the base at this position is C or G, Y in the primer indicates that the base at this position is C or T, and K in the primer indicates the base at this position The base is G or T.

[0027] Cloning with degenerate primers V. boronicumulans CGMCC 4969 nitrile hydratase alpha subunit gene fragment. The sequence of the synthesized pri...

example 3

[0032] Example three: expression of nitrile hydratase gene and gene cluster containing recombinant nitrile hydratase E. coli For the biotransformation of 3-cyanopyridine

[0033] 1. The nitrile hydratase gene DNA fragment was connected to the plasmid pET28a

[0034] The primer NHCo-E-f containing the EcoRI restriction site and the primer NHCo-E-r containing the XhoI restriction site were designed. The sequence of the primer NHCo-E-f consists of 28 nucleotide residues, which are: 5′- GGGGAATTCATGACCGGCCATGACCACT-3 ' (SEQ ID No: 13); the sequence of the primer NHCo-E-r consists of 27 nucleotide residues, which are: 5'-GGGCTCGAGTGCCGCG GGCTCCAGGTA-3' (SEQ ID No: 14). In a sterilized 0.2 mL PCR thin-walled tube, add 10.8 μL sterile water, 2 μL amplification buffer, 2 μL four deoxynucleotides, 0.5 μL primer one, 0.5 μL primer two, 3 μL Genomic DNA solution prepared in step 1, 1 μL of DMSO, 0.2 μL of Angel DNA polymerase, the total volume is 20 μL; put the PCR tube in the PCR...

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Abstract

The invention discloses the use of variovoraxboronicumulans CGMCC 4969 in bioconversion of 3-cyanopyridine for forming nicotinamide. The nitrile hydratase gene cluster produced by the strain consists of a DNA sequence represented by SEQ ID No.1 in a sequence table; the DNA consisting of the sequence represented by SEQ ID No.1 is recombined onto a pET28a plasmid and can be induced to express in EscherichiacoliBL21(DE3) strain; and the Escherichia coli cells containing expressed proteins and cell extracting solution can convert 3-cyanopyridine into nicotinamide.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, in particular to greedy phagocytic bacteria V. boronicumulans CGMCC 4969 and its resulting nitrile hydratase gene cluster applied to the bioconversion of 3-cyanopyridine to nicotinamide. Background technique [0002] Nitrile hydratase (nitrole hydratase, EC.4.2.1.84) (abbreviated as NHase) catalyzes the hydration of nitriles to generate corresponding amides. Nitrile hydratase derived from microorganisms has the advantages of mild reaction conditions, high yield, few by-products, strong regio- and stereoselectivity, etc. production, and shows great potential for development. [0003] The currently reported microorganisms producing nitrile hydratase are: Agrobacterium tumefaciens Agrobacterium tumefaciens d3, Arthrobacter Arthrobacter sp. J-1, Bacillus cereus Bacillus cereus , Bacillus Bacillus sp. BR 449, Bacillus stutzeri Bacillus smithii SC-J05-1, Brevibacterium Brevibacter...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/60C12P17/12C12R1/01C12R1/19
Inventor 戴亦军张会娟周倩雯曹玉敏袁生
Owner NANJING NORMAL UNIVERSITY
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