Cloning of beta-isopropylmalate dehydrogenase gene from Bacillus coagulans in Escherichia coli and purification and properties of the enzyme.

ABSTRACT

The beta-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate NAD+ oxidoreductase, EC 1.1.1.85) gene from Baccilus coagulans was cloned and expressed in Escherichia coli C600, using pBR322 as a vector plasmid. The B. coagulans enzyme was purified to a homogeneous state from the E. coli carrying a pBR322 - the B. coaglulans enzyme gene hybrid plasmid. The enzyme consists of two subunits of equal molecular weight (4.4 X 10(4) ). The enzyme activity was stimulated by 0.5 mM Mn2+, Mg2+ and Co2+. The enzyme was strongly inhibited by 0.2 mM p-chloromercuribenzoate and the inhibition was completely recovered by 1 mM dithiothreitol. The B. coagulans enzyme was thermostabilized by 1.5 M NaCl. The B. coagulans enzyme is a composite of alpha-helix, beta-sheet and remainder. The secondary structure of the enzyme was appreciably altered by 0.5 mM MgCl2 and 1.5 M NaCl.