Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D.
The genes encoding a thermally stable and regio-selective nitrile hydratase (NHase) and an amidase from Comamonas testosteroni 5-MGAM-4D have been cloned and sequenced, and active NHase has been over-produced in Escherichia coli. Maximal activity requires co-expression of a small open reading frame immediately downstream from the NHase beta subunit gene. Compared to the native organism, the E. coli biocatalyst has nearly threefold more NHase activity on a dry cell weight basis, and this activity is significantly more thermally stable. In addition, this biocatalyst converts a wide spectrum of nitrile substrates to the corresponding amides. Such versatility and robustness are desirable attributes of a biocatalyst intended for use in commercial applications.
Petrillo
Kelly L
KL
Central Research and Development Department, E.I. du Pont de Nemours and Co., Experimental Station, P.O. Box 80328, Wilmington, Delaware 19880-0328, USA.
Wu
Shijun
S
Hann
Eugenia C
EC
Cooling
Frederick B
FB
Ben-Bassat
Arie
A
Gavagan
John E
JE
DiCosimo
Robert
R
Payne
Mark S
MS
D000581:Amidohydrolases / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D021122:Protein Subunits / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021122:Protein Subunits; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019665:amidase; D006836:Hydro-Lyases; C048831:nitrile hydratase