Optimum culture conditions for production by Pseudomonas chlororaphis B23 of nitrile hydratase.

Yamada, Hideaki; Ryuno, Koichiro; Nagasawa, Torn; Enomoto, Kanehiko; Watanabe, Ichirô

doi:10.1271/bbb1961.50.2859

Cited by 43 publications

(11 citation statements)

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“…tein was observed in any preparations. These data also Production of NHase in the original P. chlororaphis B23 is indicate that addition of IPTG is essential for production of induced by addition of metacrylamide (24). We could not the NHase proteins, suggesting that a promoter for these expect similar induction by metacrylamide in the E. coli genes does not function in E. coli.…”

Section: Resultsmentioning

confidence: 64%

“…strain N-774 as the probe. Southern hybridization using this probe against chromosomal DNA from P. chlororaphis digested with several restriction enzymes revealed that this 24,545), which was located 42 bp downstream of the above-described sequence, was also found to include amino acid sequences corresponding to those determined by using purified subunit P3. These findings ( Fig.…”

Section: Resultsmentioning

confidence: 94%

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Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23

et al. 1991

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The nitrile hydratase (NHase) of Pseudomonas chlororaphis B23, which is composed of two subunits, a and IA, catalyzes the hydration of nitrile compounds to the corresponding amides. The NHase gene of strain B23 was cloned into Escherichia coli by the DNA-probing method with the NHase gene of Rhodococcus sp. strain N-774 as the hybridization probe. Nucleotide sequencing revealed that an amidase showing significant similarity to the amidase of Rhodococcus sp. strain N-774 was also coded by the region just upstream of the subunit a-coding sequence. In addition to these three proteins, two open reading frames, P47K and OrfE, were found just downstream of the coding region of subunit P. The direction and close locations to each other of these open reading frames encoding five proteins (amidase, subunits a and j1, P47K, and OrfE, in that order) suggested that these genes were cotranscribed by a single mRNA. Plasmid pPCN4, in which a 6.2-kb sequence covering the region coding for these proteins is placed under control of the lac promoter, directed overproduction of enzymatically active NHase and amidase in response to addition of isopropyl-o-D-thiogalactopyranoside.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of subunits a and , of NHase was about 10% of the total cellular proteins and that an additional 38-kDa protein probably encoded by the region upstream of the amidase gene was also produced in a large amount. The 38-kDa protein, as well as P47K and OrfE, appeared to be important for efficient expression of NHase activity in E. coli cells, because plasmids containing the NHase and amidase genes but lacking the region coding for the 38-kDa protein or the region coding for P47K and OrfE failed to express efficient NHase activity.

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Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 94%

Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23

et al. 1991

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“…R312 using oligonucleotide probes designed on the basis of earlier known sequences. This enantioselective amidase amino acid sequence showed significant homology with indole acetamide hydrolases from P. syringae (Yamada et al 1986) and Agrobacterium tumefaciens (Schröder et al 1984), and acetamidase from Aspergillus nidulans (Corrick et al 1987). Nitrile hydratase gene was located 73 bp downstream of amidase gene and considered to be the part of same operon.…”

Section: Molecular Biology Of Amidase Genementioning

confidence: 88%

Amidases: versatile enzymes in nature

Sharma

Bhalla

2009

Rev Environ Sci Biotechnol

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Amidases are ubiquitous enzymes and biological functions of these enzymes vary widely. In past five decades, they turned out to be an attractive tool in industries for the synthesis of wide variety of carboxylic acids, hydroxamic acids and hydrazide, which find applications in commodity chemicals synthesis, pharmaceuticals agrochemicals and waste water treatments etc. Their proteins structures revealed that aliphatic amidases share the typical a/b hydrolase fold (like nitrilase superfamily) and signature amidases are evolutionary related to aspartic proteinases. They hydrolyse wide variety of amides (short chain aliphatic amides, mid-chain amides, arylamides, a-aminoamides and a-hydroxyamides) and can be grouped on the basis of their catalytic site and preferred substrate. They resist denaturation at extreme of pH and temperature because of their strong and compact multimeric structures. Inhibition studies and three-dimensional analysis of the structures identified a Glu59, Lys134, Cys166 catalytic triad and follow ''Bi-bi Ping-Pong'' mechanism reaction for amide hydrolysis and acyl transferase reactions. Many recombinant amidases have been expressed in Escherichia coli as well as in Brevibacterium lactofermentum.

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“…We were interested in the discovery of a regulatory protein that plays a key role in control of the expression of the NHase gene cluster in P. chlororaphis B23 for the following reasons: (i) the induction level of useful NHase is very high (34); and (ii) although a substrate of an inducible enzyme often acts as an inducer in the regulation mechanism, methacrylamide, which is the reaction product of NHase, acts as a strong inducer of NHase in P. chlororaphis B23 (47).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, the NHase of Pseudomonas chlororaphis B23 (33), which was previously used as a catalyst for acrylamide manufacture (42,46), is now used for the production of 5-cyanovaleramide, a herbicide intermediate, at the industrial level (10). Strong induction of NHase formation was observed after addition of methacrylamide as the sole nitrogen source (47). A substrate of an enzyme often acts as an inducer; however, the reaction product of NHase, methacrylamide, serves as a strong inducer of the NHase of P. chlororaphis B23.…”

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confidence: 99%

Transcriptional Regulation of the Nitrile Hydratase Gene Cluster in Pseudomonas chlororaphis B23

et al. 2008

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An enormous amount of nitrile hydratase (NHase) is inducibly produced by Pseudomonas chlororaphis B23 after addition of methacrylamide as the sole nitrogen source to a medium. The expression pattern of the P. chlororaphis B23 NHase gene cluster in response to addition of methacrylamide to the medium was investigated. Recently, we reported that the NHase gene cluster comprises seven genes (oxdA, amiA, nhpA, nhpB, nhpC, nhpS, and acsA). Sequence analysis of the 1.5-kb region upstream of the oxdA gene revealed the presence of a 936-bp open reading frame (designated nhpR), which should encode a protein with a molecular mass of 35,098. The deduced amino acid sequence of the nhpR product showed similarity to the sequences of transcriptional regulators belonging to the XylS/AraC family. Although the transcription of the eight genes (nhpR, oxdA, amiA, nhpABC, nhpS, and acsA) in the NHase gene cluster was induced significantly in the P. chlororaphis B23 wild-type strain after addition of methacrylamide to the medium, transcription of these genes in the nhpR disruptant was not induced, demonstrating that nhpR codes for a positive transcriptional regulator in the NHase gene cluster. A reverse transcription-PCR experiment revealed that five genes (oxdA, amiA, nhpA, nhpB, and nhpC) are cotranscribed, as are two other genes (nhpS and acsA). The transcription start sites for nhpR, oxdA, nhpA, and nhpS were mapped by primer extension analysis, and putative ؊12 and ؊24 54

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Optimum culture conditions for production by Pseudomonas chlororaphis B23 of nitrile hydratase.

Cited by 43 publications

References 1 publication

Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23

Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23

Amidases: versatile enzymes in nature

Transcriptional Regulation of the Nitrile Hydratase Gene Cluster in Pseudomonas chlororaphis B23

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