OVEREXPRESSION AND PROPERTIES OF A NEW THERMOPHILIC AND THERMOSTABLE ESTERASE FROM BACILLUS-ACIDOCALDARIUS WITH SEQUENCE SIMILARITY TO HORMONE-SENSITIVE LIPASE SUBFAMILY

We previously purified a new esterase from the thermoacidophilic eubac terium Bacillus acidocaldarius whose N-terminal sequence corresponds t o an open reading frame (ORF3) reported to show homology with the mamm alian hormone-sensitive lipase (HSL)like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enz yme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was est ablished. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized bioche mically. The enzyme, a 34 kDa monomeric protein, was demonstrated to b e a B ''-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP ) esters tested the best substrate was PNP-exanoate with K-m and k(cat ) values of 11 +/- 2 mu M (mean +/- S.D., n = 3) and 6610 +/- 880 s(-1 ) (mean +/- S.D., n = 3) respectively at 70 degrees C and pH 7.1. In s pite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetam ide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantios electivity was observed only in the resolution -)-3-bromo-5-(hydroxyme thyl)-Delta(2)-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able t o synthesize acetyl esters when tested in vinyl acetate and toluene. I nactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labe lling with [H-3]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relat ionship is discussed in relation to those of the homologous members of the HSL group.