Abstract

Gene CA_C0816 codes for a serine hydrolase protein from Clostridium acetobutylicum (ATCC 824) a member of hormone-sensitive lipase of lipolytic family IV. This gene was overexpressed in E. coli strain BL21and purified using Ni²⁺–NTA affinity chromatography. Size exclusion chromatography revealed that the protein is a dimer in solution. Optimum pH and temperature for recombinant Clostridium acetobutylicum esterase (Ca-Est) were found to be 7.0 and 60 °C, respectively. This enzyme exhibited high preference for p-nitrophenyl butyrate. K_M and k_cat/K_M of the enzyme were 24.90 µM and 25.13 s⁻¹ µM⁻¹, respectively. Sequence analysis of Ca-Est predicts the presence of catalytic amino acids Ser 89, His 224, and Glu 196, presence of novel GYSMG conserved sequence (instead of GDSAG and GTSAG motif), and undescribed variation of HGSG motif. Site-directed mutagenesis confirmed that Ser 89 and His 224 play a major role in catalysis. This study reports that Ca-Est is hormone-sensitive lipase with novel GYSMG pentapeptide motif at a catalytic domain.

中文翻译：

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丙酮丁醇梭菌酯酶的生化特征，在催化域具有新的GYSMG五肽基序。

摘要

基因CA_C0816编码丙酮丁醇梭菌（ATCC 824）的丝氨酸水解酶蛋白，该蛋白是脂肪分解家族IV的激素敏感性脂肪酶的成员。该基因在大肠杆菌BL21菌株中过表达，并使用Ni ²⁺ -NTA亲和层析纯化。尺寸排阻色谱法显示该蛋白质为溶液中的二聚体。重组乙酰丙酮丁酸梭菌酯酶（Ca-Est）的最佳pH和温度分别为7.0和60°C。该酶表现出对对硝基苯基丁酸酯的高度偏好。酶的K _M和k _cat / K _M为24.90 µM和25.13 s^-1  µM ^-1。Ca-Est的序列分析预测催化氨基酸Ser 89，His 224和Glu 196的存在，新型GYSMG保守序列的存在（而不是GDSAG和GTSAG基序）以及HGSG基序的未描述变异。定点诱变证实Ser 89和His 224在催化中起主要作用。这项研究报告说Ca-Est是激素敏感的脂肪酶，在催化域具有新的GYSMG五肽基序。