Oral Presentation 6-01

EXPRESSION OF THERMOSTABLE NITRILE
HYDRATASE IN Escherichia coliFOR
ACRYLAMIDE PRODUCTION

Rugmini Padmakumar, Sang-Hoon Kim, and Patrick Oriel

Department of Microbiology
Michigan State University
Giltner Hall
East Lansing, Michigan 48824

Telephone: (517) 353-4664; Fax: (517) 353-8957; E-mail: oriel@pilot.msu.edu

Bioproduction of acrylamide utilized as polyacrylamide in waste treatment and paper coating applications is one of the first examples of "green" production of commodity chemicals. The nitrile hydratase enzyme used as the process catalyst is unusual in that it utilizes Co³⁺ at the catalytic site. Expression of nitrile hydratase genes in Escherichia coli desired for enhancement of enzyme levels and genetic modification has proven difficult due to the formation of inclusion bodies and poor transport of the required cobalt ion. We discuss enzyme properties of the thermostable nitrile hydratase from a moderate thermophile Bacillus that is activated using a novel process of Co²⁺ ion introduction and oxidation following enzyme overexpression in E. coli at 32EC. Initial mass spectroscopy experiments examining alkylation of the catalyst by the acrylonitrile substrate are also presented.