Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D

• Published in: Applied microbiology and biotechnology Vol. 67; no. 5; pp. 664 - 670
• Main Authors: PETRILLO, Kelly L, SHIJUN WU, HANN, Eugenia C, COOLING, Frederick B, BEN-BASSAT, Arie, GAVAGAN, John E, DICOSIMO, Robert, PAYNE, Mark S
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01.06.2005; Springer Nature B.V
• Subjects: Amides; Amidohydrolases - genetics; Amino Acid Sequence; Bacteria; Bacterial Proteins - genetics; Bacteriology; Base Sequence; Biocatalysts; Biological and medical sciences; Biotechnology; Cloning, Molecular; Comamonas testosteroni - enzymology; Comamonas testosteroni - genetics; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Enzyme Stability; Enzymes; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic engineering; Genetic technics; Hydro-Lyases - biosynthesis; Hydro-Lyases - chemistry; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Open Reading Frames; Protein Subunits - genetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Substrate Specificity; Temperature; Comamonas testosteroni; Regioselectivity; Enzyme; Escherichia coli; Comamonadaceae; Gene overexpression; Lyases; Genetically modified microorganism; Thermal stability; Biocatalyst; Nitrile hydratase; Bacteria; Carbon-oxygen lyases; Enterobacteriaceae; Hydro-lyases
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-004-1842-9

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.