Isolation and expression of a gene for a nitrilase from acidovorax facilis 72W

A nitrilase and chimeric gene technology, applied in the field of molecular biology, can solve the problems of lack of thermostable high-yield nitrilase, thermal instability limiting industrial application and the like

Inactive Publication Date: 2009-11-11
THE CHEMOURS CO FC LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inherent thermal instability of mesophilic nitrilases has been reported to limit their industrial applications (Cramp et al., Microbiol. (1997) 143:2313-2320)
[0013] The problem at hand is: applications (e.g. regioselective hydrolysis of aliphatic dinitriles to cyanocarboxylic acids) in which high yields of products are obtained under mild reaction conditions (including room temperature and without excessive acid or base conditions) without generating relatively large amounts of unwanted waste products Among them, there is a lack of industrially practical thermostable high-yield nitrilases suitable for use as catalysts for nitrile-containing substrates

Method used

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  • Isolation and expression of a gene for a nitrilase from acidovorax facilis 72W
  • Isolation and expression of a gene for a nitrilase from acidovorax facilis 72W
  • Isolation and expression of a gene for a nitrilase from acidovorax facilis 72W

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0196] Purified nitrilase protein

[0197] All steps in this method were performed at 5°C, pH 7.5, unless otherwise stated.

[0198]A 25% by weight suspension of wet cell pulp of A. facilis 72W (ATCC 55746) was prepared with 20 mM Tris pH 7.5, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 2.0 mM dithiothreitol.

[0199] Extracts of the suspension were prepared by a French cell press (American Instrument Co., Silver Springs, MD, USA) according to methods known in the art. After centrifugation at 27,500g for 30 minutes to remove cellular debris, a 20-55% ammonium sulfate fraction of the extract was prepared and then concentrated by overnight precipitation after addition of solid ammonium sulfate to 65% saturation. The concentrated protein pellet was dissolved using a minimal volume of 20 mM Tris pH 7.5 (buffer A) and desalted over a PD10 column containing Sephadex G-25 resin (Pharmacia).

[0200] After desalting, the concentrated protein extract was separated by anion exc...

Embodiment 2

[0202] Isolation of DNA fragments showing high homology to known nitrilases

[0203] Acidovorax facilis 72W (ATCC 55746) genomic DNA was isolated by a modified standard method (Maniatis) adding two rounds of phenol-chloroform extraction. The DNA thus isolated was used as a target for PCR amplification using primers 1F (SEQ ID NO: 1) and 7R (SEQ ID NO: 2). The obtained amplified products were cloned into pGem-T vector (Promega, Madison, WI), and sequenced using methods commonly used in the art. The identity of the 385bp DNA fragment (SEQ ID NO: 3) produced by cloning pJJ28-5 with the DNA sequence of Rhodococcus rhodochrous K22 nitrilase (GenBank accession number D12583) was 74.1%.

Embodiment 3

[0205] Locate the chromosomal segment containing a sequence highly homologous to a 385bp partial gene segment

[0206] 1-3 μg of high molecular weight genomic DNA samples of A. facilis 72W were digested with the restriction enzyme Pstl in buffer provided by the manufacturer (Life Technologies (Gibco-BRL), Gaithersburg, MD, USA). Digested samples were separated on agarose gels and Southern blotted on nylon membranes. A probe containing the 385 bp nitrilase gene fragment (SEQ DI NO: 3) was prepared by cutting pJJ28-5 at the restriction sites flanking the insert. Hybridization was performed at 60°C, followed by high stringency washing (0.1×SSC, 0.1% SDS, 60°C, 15 minutes). Probe labeling, hybridization and dot blot detection and subsequent Southern blotting experiments were performed using the ECL Random Primer Labeling and Detection System Type II (Amersham International plc, Buckinghamshire, England).

[0207] The PstI digest yielded a single 4.1 kb band when hybridized to ...

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Abstract

Recombinant microbial strains expressing nitrilase enzymes and useful as biocatalysts for hydrolyzing nitrile-containing substrates are provided. The recombinant cells were transformed with an exogenous gene isolated from Acidovorax facilis 72W encoding a thermostable nitrilase that catalyzes the hydrolysis of nitrile-containing substrates to carboxylic acids under mild reaction conditions. The nucleotide sequence of the nitrilase gene and the putative amino acid sequence encoded by the nitrilase gene are provided.

Description

field of invention [0001] The invention relates to the field of molecular biology and the use of recombinant microorganisms to express target genes and gene products. More specifically, the gene used in the present invention is a novel nucleic acid fragment encoding a nitrilase that catalyzes the hydrolysis of a wide variety of nitrile-containing substances to produce the corresponding carboxylic acids. The present invention also provides recombinant strains expressing nitrilase activity and useful as biocatalysts for the hydrolysis of nitrile-containing substrates. In addition, the present invention relates to specific nucleic acids that facilitate the isolation of said nitrilase gene. Background of the invention [0002] Nitriles are readily converted to the corresponding carboxylic acids by various chemical methods, but these methods usually require strongly acidic or basic reaction conditions and high reaction temperatures, and often produce unwanted by-products and / or ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/09C12N1/15C12N1/19C12N1/21C12N5/10C12N9/78C12P7/40C12P13/00C12P17/10C12R1/19
CPCC12P13/00C12P13/02C12N9/78C12P7/40C12P13/002C12N15/52
Inventor R·D·法伦M·S·佩尼S·乔汗R·迪科西莫约翰E·加瓦甘
Owner THE CHEMOURS CO FC LLC
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