Montoro-García Silvia, Martínez-Martínez Irene, Navarro-Fernández José, Takami Hideto, García-Carmona Francisco, Sánchez-Ferrer Alvaro
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E30011 Murcia, Spain.
J Bacteriol. 2009 May;191(9):3076-85. doi: 10.1128/JB.01060-08. Epub 2009 Mar 20.
The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65 degrees C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new alpha/beta hydrolase family different from IV and VI.
克隆、测序了嗜热栖热芽孢杆菌HTA426的基因GK3045（741 bp），并将其在大肠杆菌Rosetta（DE3）中过表达。推导的蛋白质是一种30 kDa的单体酯酶，与嗜热栖热芽孢杆菌NY的羧酸酯酶具有高度同源性（99%同一性），与嗜热脂肪芽孢杆菌的羧酸酯酶具有97%同一性。该蛋白质在大肠杆菌中发生蛋白水解切割，通过在基因中引入突变（K212R）克服了这个问题，且不影响活性。所得的Est30在65℃显示出显著的热稳定性，高于嗜热栖热芽孢杆菌HTA426的最佳生长温度。该酶的最适pH为8.0。此外，纯化的酶对变性剂如有机溶剂、去污剂和尿素具有稳定性。该蛋白质催化不同酰基链长度的对硝基苯酯的水解，证实了酯酶活性。序列分析表明，该蛋白质包含由Ser93、Asp192和His222形成的催化三联体，活性位点的Ser位于大多数酯酶和脂肪酶所含的保守基序Gly91-X-Ser93-X-Gly95中。然而，这种羧酸酯酶与微生物羧酸酯酶八个家族中最接近的成员的序列同一性不超过17%。通过序列比对构建了三维结构，并与其他羧酸酯酶进行了比较。拓扑差异表明该酶和其他嗜热栖热芽孢杆菌相关的羧酸酯酶可归为不同于IV和VI的新的α/β水解酶家族。
