Purification and characterization of a thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius

Abstract

A thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius was isolated, purified 1800‐fold to homogeneity, and characterised. The apparent molecular mass was 36.5 ± 2.5 kDa when determined by SDS/PAGE and 37.5 kDa when determined by analytical gel filtration, suggesting a monomeric structure. The pure enzyme regained activity on removal of SDS after SDS/PAGE. Several esterase activities 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°C and pH 8, and was thermostable. p‐Nitrophenyl esters of fatty acids from C2 to C12 were used as substrates; Vmax and Km values were determined at three different temperatures. The enzyme was able to hydrolyse tributyrylglycerol and trihexanoyl‐glycerol dissolved in 0.8% acetonitrile, but neither lipase activity toward [14C]trioleoylglycerol nor proteolytic 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 sequence of 19 amino acid residues at the N‐teminus were determined. These data, together with substrate preference and inhibition pattern, allowed us to classify this enzme as a B‐type carboxylesterase (EC 3.1.1.1). Copyright © 1994, Wiley Blackwell. All rights reserved