PURIFICATION AND PARTIAL CHARACTERIZATION OF A NOVEL THERMOPHILIC CARBOXYLESTERASE WITH HIGH MESOPHILIC SPECIFIC ACTIVITY

An esterase activity obtained from a strain of Bacillus stearothermoph ilus was purified 5,133-fold to electrophoretic homogeneity with 26% r ecovery. The purified esterase had a specific activity of 2,032 mu mol min(-1) mg(-1) based on the hydrolysis of p-nitrophenyl caproate at p H 7.0 and 30 degrees C. The apparent molecular mass was 50,000 +/- 2,0 00 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophore sis and 45,000 +/- 3,000 daltons from gel filtration. Native polyacryl amide gels stained for esterase activity showed three bands, The isoel ectric points were estimated to be 5.7, 5.8, and 6.0. Ferry amino acid residues were sequenced at the N-terminus. The sequence showed no deg eneracy, suggesting that the three esterases are functionally identica l carboxylesterases differing by a limited number of amino acids. The enzyme showed maximum activity al pH 7.0 and was very stable at pH 6.0 -8.9 with optimum stability at pH 6.0. At this pH and 60 degrees C the half-life was 170 h. Esterase activity was totally inhibited by pheny lmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and to syl-L-phenylalanine, but not by ethylendiaminetetra acetic acid. The e sterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrop henyl esters, but both V-max and K-M were protein concentration-depend ent, The esterase was able to hydrolyse a number of p-nitrophenyl deri vatives (amino acid derivatives and aliphatic acids with different cha in lengths).