Application de la chromatographie en phase gazeuse à l'étude des nitrilases et amidases

Allageas, J.C.; Arnaud, A.; Galzy, P.

doi:10.1016/s0021-9673(00)92261-1

Cited by 30 publications

(5 citation statements)

References 16 publications

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“…Nitrile hydratase activity was measured by gas chromatography using intact cells by the previously described technique (Jallageas, Arnaud and Galzy, 1978b;Bui et al, 1984~). Propionitrile was used as the substrate.…”

Section: Nitrile Hydratase Activity Measurementmentioning

confidence: 99%

Optimization of Culture Conditions ofBrevibacterium SP.A4 for the Production of Nitrile Hydratase

Bernet¹,

Arnaud²,

Galzy³

1990

Biocatalysis

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Optimum culture conditions of Breuibacterium sp. A4 for production of nitrile hydratase were determined by two mathematical methods: the Hadamard method and graphic analysis of response areas. A minimal medium was optimized and the basic roles of Fez+ and M$+ were clearly shown.The influence of physico-chemical factors (pH, temperature and light conditions) on the culture and on nitrile hydratase were also studied. Various results permit the production of Breuibucterium sp. A4 cells with low protease and high nitrile hydratase contents.

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Section: Nitrile Hydratase Activity Measurementmentioning

confidence: 99%

Optimization of Culture Conditions ofBrevibacterium SP.A4 for the Production of Nitrile Hydratase

Bernet¹,

Arnaud²,

Galzy³

1990

Biocatalysis

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“…The reaction was stopped by the addition of 40 ml 1·5 mol l −1 HCl. The amount of remaining nitrile in the reaction mixture was determined by gasliquid chromatography as previously described (Jallageas et al 1978). One unit (U) of nitrile hydratase activity was defined as the amount of enzyme which catalysed the transformation of one micromole of propionitrile per minute under the above-described conditions.…”

Section: Nitrile Hydratase Assaymentioning

confidence: 99%

Transcriptional analysis of the nitrile‐degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli – T7 expression system

Bigey¹,

Chebrou²,

Fournand³

et al. 1999

Journal of Applied Microbiology

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. 1999. Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli -T7 expression system by lowering the growth temperature to 29°C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.

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“…The reaction mixture was incubated at 30°C. At regular intervals, a volume of the mixture was removed and the propionamide concentration was measured by GLC as described by Jallageas et al [16]. One unit of amidase activity was defined as the amount of cells required for hydrolyzing 1 p~mol of propionamide per min.…”

Section: Amidase Assaymentioning

confidence: 99%

Cloning of the wide spectrum amidase gene fromBrevibacteriumsp. R312 by genetic complementation. Overexpression inBrevibacteriumsp. andEscherichia coli

Azza¹,

Bigey²,

Arnaud³

et al. 1994

FEMS Microbiology Letters

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The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. Another promoter localized in the pSR 1 fragment of the cloning vector was shown to be able to control amiE gene expression. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli.

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Application de la chromatographie en phase gazeuse à l'étude des nitrilases et amidases

Cited by 30 publications

References 16 publications

Optimization of Culture Conditions ofBrevibacterium SP.A4 for the Production of Nitrile Hydratase

Optimization of Culture Conditions ofBrevibacterium SP.A4 for the Production of Nitrile Hydratase

Transcriptional analysis of the nitrile‐degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli – T7 expression system

Cloning of the wide spectrum amidase gene fromBrevibacteriumsp. R312 by genetic complementation. Overexpression inBrevibacteriumsp. andEscherichia coli

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