Karpushova A, Brümmer F, Barth S, Lange S, Schmid R D
Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Appl Microbiol Biotechnol. 2005 Apr;67(1):59-69. doi: 10.1007/s00253-004-1780-6. Epub 2004 Dec 22.
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.
从与海洋海绵嗜硫放线菌相关的芽孢杆菌属基因组文库中分离出两种新型酯酶（EstB1和EstB2）。EstB1与已发表的芽孢杆菌属水解酶的同一性较低（26 - 44%），而EstB2与蜡样芽孢杆菌和炭疽芽孢杆菌的羧酸酯酶具有较高的同一性（73 - 74%）。使用载体pET - 22b(+)，在T7启动子的控制下，两种酯酶均在大肠杆菌中高效表达。重组EstB1通过IMAC一步纯化至电泳纯。建立了一种重组EstB2形成的包涵体复性方法以获得活性酶。测定了这两种酶对对硝基苯酯和甲酯的底物特异性以及各自的动力学参数K(m)和V(max)。EstB1和EstB2的最适温度分别确定为30 - 50℃和20 - 35℃。最适pH分别为6.5 - 7.5和6.5 - 8.0。两种酶在高达50%（v/v）的二甲基亚砜中表现出最高稳定性，其次是甲醇、乙醇和2 - 丙醇。测试了高浓度NaCl和KCl的影响。观察到10 - 50 mM Zn(2+)以及50 mM Mg(2+)和Ca(2+)离子对两种酯酶均有抑制作用。1 - 5 mM苯甲基磺酰氟使酶失活，而β - 巯基乙醇、二硫苏糖醇和乙二胺四乙酸对酶活性无影响。
