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 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 degrees 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-terminus were determined. These data, together with substrate preference and inhibition pattern, allowed us to classify this enzyme as a B-type carboxylesterase (EC 3.1.1.1).
Manco
G
G
Institute of Protein Biochemistry and Enzimology, CNR, Naples, Italy.
Di Gennaro
S
S
De Rosa
M
M
Rossi
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology*; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D004952:Esters; D005227:Fatty Acids; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase