Cloning, recombinant expression and biochemical characterisation of novel esterases from Bacillus sp. associated with the marine sponge Aplysina aerophoba.
Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity (26-44%) with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73-74%) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards p-nitrophenyl and methyl esters and the respective kinetic parameters K(m) and V(max) were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30-50 degrees C and 20-35 degrees C, respectively. The pH optima were found to be in the range of 6.5-7.5 and 6.5-8.0, respectively. Both enzymes showed the highest stability in up to 50% (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10-50 mM Zn(2+) and 50 mM Mg(2+) and Ca(2+) ions was observed for both esterases. One to five millimolar PMSF deactivated the enzymes, whereas beta-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.
Karpushova
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Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. itbrsc@itb.uni-stuttgart.de
Brümmer
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Barth
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Lange
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Schmid
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D000818:Animals; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004269:DNA, Bacterial / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011161:Porifera / Q000382:microbiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D008670:Metals; D011994:Recombinant Proteins; D004950:Esterases