| Autor: |
Karpushova, A., Brümmer, F., Barth, S., Lange, S., Schmid, R. D. |
| Předmět: |
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| Zdroj: |
Applied Microbiology & Biotechnology; Apr2005, Vol. 67 Issue 1, p59-69, 11p, 3 Diagrams, 1 Chart, 3 Graphs |
| Abstrakt: |
Two novel esterases (EstB1 and EstB2) were isolated from a genomic library ofBacillussp. associated with the marine spongeAplysina aerophoba. EstB1 shows low identity (26-44%) with the published hydrolases of the genusBacillus, whereas EstB2 shows high identity (73-74%) with the carboxylesterases fromB. cereusandB. anthracis. Both esterases were efficiently expressed inEscherichia coliunder 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 towardsp-nitrophenyl and methyl esters and the respective kinetic parametersKm andVmax were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30-50°C and 20-35°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 Zn2+ and 50 mM Mg2+ and Ca2+ ions was observed for both esterases. One to five millimolar PMSF deactivated the enzymes, whereas ß-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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