Esterases from Bacillus subtilis and B. stearothermophilus share high sequence homology but differ substantially in their properties.
A novel esterase from Bacillus subtilis (BsubE) was cloned, functionally expressed in Escherichia coli and biochemically characterized. BsubE shows high homology (74% identity, >95% homology) to an esterase from the thermophilic B. stearothermophilus (BsteE). Both enzymes were efficiently expressed in E. coli, using a L-rhamnose-expression system [11,500 units/l (BsteE), 3,400 units/l (BsubE)] and were purified by Ni-nitrilotriacetic acid chromatography, yielding specific activities of 70 units/mg (BsteE) and 40 units/mg (BsubE), as determined by the hydrolysis of p-nitrophenyl acetate. Despite the high homology, both esterases revealed remarkable differences in their properties. As expected, the esterase from the thermophilic organism showed significantly higher temperature stability. Whereas BsteE showed highest activity at 65-70 degrees C, BsubE was almost inactivated at 50 degrees C. Moreover, both enzymes showed quite different substrate patterns in the hydrolysis of various esters. Whilst the B. subtilis esterase accepted esters with a branched alcohol moiety well, the B. stearothermophilus esterase was more useful in the hydrolysis of substrates with a sterically demanding carboxylic acid group. BsteE showed excellent enantioselectivity ( E>100) in the kinetic resolution of menthyl acetate and even accepted the bulky menthyl benzoate as substrate ( E=19). In contrast, BsubE converted 1-phenethylacetate with higher selectivity ( E>150 vs E=8).
Henke
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Institute of Chemistry and Biochemistry, Department of Technical Chemistry and Biotechnology, Greifswald University, Soldmannstrasse 16, 17487 Greifswald, Germany.
Bornscheuer
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UT
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017385:Sequence Homology; D013696:Temperature
D004950:Esterases