Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

CORC > 天津工业生物技术研究所 > 结构生物学与蛋白酶学实验室郭瑞庭 > 期刊论文

	Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop
	Cheng, Ya-Shan 1; Ko, Tzu-Ping 2; Huang, Jian-Wen 3,4; Wu, Tzu-Hui 1; Lin, Cheng-Yen 3,4; Luo, Wenhua 5; Li, Qian 6; Ma, Yanhe 6; Huang, Chun-Hsiang 6; Wang, Andrew H. -J.2
刊名	APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
	2012-08-01
卷号	95 期号:3 页码:661-669
关键词	Thermostability Endoglucanase Crystal structure Protein engineering Enzyme kinetics
英文摘要	Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable beta-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k (cat) and K (m) values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Biotechnology & Applied Microbiology
研究领域[WOS]	Biotechnology & Applied Microbiology
关键词[WOS]	SITE-DIRECTED MUTAGENESIS ; THERMAL-STABILITY ; ENZYMES ; XYLANASE ; THERMOSTABILITY ; ENDOGLUCANASE ; BIOTECHNOLOGY ; MECHANISMS ; CHEMISTRY
收录类别	SCI
语种	英语
WOS记录号	WOS:000306421500010
内容类型	期刊论文
源URL	[http://124.16.173.210/handle/834782/1327]
专题	天津工业生物技术研究所_结构生物学与蛋白酶学实验室郭瑞庭_期刊论文
作者单位	1.Natl Taiwan Univ, Inst Biotechnol, Taipei 106, Taiwan 2.Acad Sinica, Inst Biol Chem, Taipei 115, Taiwan 3.Genozyme Biotechnol Inc, Taipei 114, Taiwan 4.AsiaPac Biotechnol Co Ltd, Dongguan 523808, Peoples R China 5.S China Agr Univ, Coll Food Sci, Guangzhou 510642, Guangdong, Peoples R China 6.Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Ind Enzymes Natl Engn Lab, Tianjin 300308, Peoples R China 7.Natl Taiwan Univ, Dept Anim Sci & Technol, Taipei 106, Taiwan
推荐引用方式 GB/T 7714	Cheng, Ya-Shan,Ko, Tzu-Ping,Huang, Jian-Wen,et al. Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop[J]. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY,2012,95(3):661-669.
APA	Cheng, Ya-Shan.,Ko, Tzu-Ping.,Huang, Jian-Wen.,Wu, Tzu-Hui.,Lin, Cheng-Yen.,...&Guo, Rey-Ting.(2012).Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop.APPLIED MICROBIOLOGY AND BIOTECHNOLOGY,95(3),661-669.
MLA	Cheng, Ya-Shan,et al."Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop".APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 95.3(2012):661-669.