PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE CARBOXYLESTERASE FROM THE THERMOACIDOPHILIC EUBACTERIUM BACILLUS-ACIDOCALDARIUS

A thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius was isolated; purified 1800-fold to homogenei ty, and characterised. The apparent molecular mass was 36.5 +/- 2.5 kD a when determined by SDS/PAGE and 37.5 kDa when determined by analytic al gel filtration, suggesting a monomeric structure. The pure enzyme r egained activity on removal of SDS after SDS/PAGE. Several esterase ac tivities were revealed in crude extracts by PAGE and activity staining , although only one was detected after SDS/PAGE and detergent removal. The esterase showed optimal activity at around 70 degrees C and pH 8, and was thermostable. p-Nitrophenyl esters of fatty acids from C-2 to C-12 were used as substrates; V-max and K-m values were determined at three different temperatures. The enzyme was able to hydrolyse tribut yrylglycerol and trihexanoylglycerol dissolved in 0.8% acetonitrile, b ut neither lipase activity toward [C-14]trioleoylglycerol nor proteoly tic activity could be detected. Inactivation by diethyl p-nitrophenyl phosphate, by phenyl-methansulfonyl fluoride and physostigmine, and by diethylpyrocarbonate suggested that the enzyme contained a catalytic triad Ser-His-Asp/Glu in the active site, similar to that demonstrated for other serine-type enzymes. The amino acid composition and the seq uence of 19 amino acid residues at the N-terminus were determined. The se data, together with substrate preference and inhibition pattern, al lowed us to classify this enzyme as a B-type carboxylesterase (EC 3.1. 1.1).