Vaporophage bacterium cgmcc4969 and its application in the biotransformation of 3-cyanopyridine to nicotinamide

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

Inactive Publication Date: 2011-12-21
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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  • Vaporophage bacterium cgmcc4969 and its application in the biotransformation of 3-cyanopyridine to nicotinamide
  • Vaporophage bacterium cgmcc4969 and its application in the biotransformation of 3-cyanopyridine to nicotinamide
  • Vaporophage bacterium cgmcc4969 and its application in the biotransformation of 3-cyanopyridine to nicotinamide

Examples

Experimental program
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Effect test

example 1

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

[0017] 1. Strain isolation

[0018] Collect soil from the Xianlin area of ​​Nanjing City, Jiangsu Province, add 1g of soil to 19mL sterile water containing 5 glass beads, shake for 5min, let stand for 10min, take 1ml suspension and add 19ml mineral containing 1% 3-cyanopyridine 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 ion solution, pH 7.5. The composition of the metal ionic liquid: 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 cultured in a shaker at 30°C and a rotating speed of 220 rpm for 3 days. Dilute 100μl sample to 10 -3 -10 -5 After that, a plate of...

example 2

[0024] Example 2: V. boronicumulans CGMCC 4969 nitrile hydratase gene cloning

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

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

[0027] First clone with degenerate primers V. boronicumulans CGMCC 4969 nitrile hydratase alpha subunit gene fragment. The sequence of the synthesized primer NHCo-f is: 5'-GTSGTGGCSARGGCCTGG -3' (SEQ I...

example 3

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

[0033] 1. Connect the DNA fragment of the nitrile hydratase gene to the plasmid pET28a

[0034] Design primer NHCo-Ef containing EcoRI restriction site and primer NHCo-Er containing XhoI restriction site. The sequence of primer NHCo-Ef consists of 28 nucleotide residues, in order: 5′-GGGGAATTCATGACCGGCCATGACCACT-3 '(SEQ ID No: 13); The sequence of primer NHCo-Er consists of 27 nucleotide residues, in order: 5'-GGGCTCGAGTGCCGCG GGCTCCAGGTA-3' (SEQ ID No: 14). In a sterile 0.2mL PCR thin-walled tube, add 10.8μL sterile water, 2μL amplification buffer, 2μL four deoxynucleotides, 0.5μL primer one, 0.5μL primer two, and 3μL. The genomic DNA solution prepared in step 1, 1μL of DMSO, 0.2μL of Angel DNA polymerase, the total volume is 20μL; put the PCR tube in the PCR machine, and set the conditions as follows: increa...

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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 Applications(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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