Identification and thermoadaptation engineering of thermostability conferring residue of deep sea bacterial alpha-amylase AMY121
Yang, Jian; Li, Lizhen; Xiao, Yunzhu; Li, Jie; Long, Lijuan; Wang, Fazuo; Zhang, Si; Zhang, S (reprint author), Chinese Acad Sci, South China Sea Inst Oceanol, CAS Key Lab Trop Marine Bioresources & Ecol, RNAM Ctr Marine Microbiol,Guangdong Key Lab Marin, Guangzhou 510301, Guangdong, Peoples R China.
2016
发表期刊JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
卷号126页码:56-63
摘要Protein engineering on hotspot residues is acknowledged as an effective way to improve the stability and activity of enzymes. Searching for the key spot of a protein is a critically important but time-consuming process. In our previous study, an InDel site neighbouring residue Lys209 of the deep sea bacterial amylase AMY121 was proposed as one putative determinant for thermostability by homology sequence comparison and biochemical analysis [1]. Here, the structural stabilizing role of residue Lys209 was verified, and semi-rational protein design was applied to further improve capacity of higher temperature adaptation. Systematic mutagenesis study on Lys209 of amylase AMY121 revealed that all of the nineteen substitution mutants on the 209th site led to stability loss compared with wild type enzyme, indicating the crucial role of residue Lys209 in protein structural stabilizing. Four Lys209 neighboring sites were then selected and site directed saturation mutagenesis libraries were constructed for high through put thermostability screening. Two thermostability enhanced mutants Y187E and K205L were obtained from 1600 mutant colonies. Temperature properties comparison of wild type AMY121 and its variants showed Y187E and K205L possessed better kinetic thermal stability. The optimum temperature Y187E and K205L both increased by 5 degrees C. T-50(15) values of Y187E and K205L also increased to 0.63 and 8.52 degrees C, respectively. Structural basis for the difference in thermostability of wild type enzyme and its variants was further analyzed by computer modeling, and increasing in number of salt bridges and hydrophobic interactions around Lys209 was proposed as the main driving force for better compact protein structure of the enzyme. (C) 2015 Elsevier B.V. All rights reserved.
部门归属[Yang, Jian; Li, Lizhen; Xiao, Yunzhu; Li, Jie; Long, Lijuan; Wang, Fazuo; Zhang, Si] Chinese Acad Sci, South China Sea Inst Oceanol, CAS Key Lab Trop Marine Bioresources & Ecol, RNAM Ctr Marine Microbiol,Guangdong Key Lab Marin, Guangzhou 510301, Guangdong, Peoples R China; [Li, Lizhen; Xiao, Yunzhu; Wang, Fazuo] Univ Chinese Acad Sci, Beijing 100049, Peoples R China ; LMB
关键词Deep Sea Bacterial Amylase Thermostability Hotspot Residue Semi-rational Design
学科领域Biochemistry & Molecular Biology ; Chemistry
文献类型期刊论文
条目标识符http://ir.scsio.ac.cn/handle/344004/15475
专题中科院海洋生物资源可持续利用重点实验室
通讯作者Zhang, S (reprint author), Chinese Acad Sci, South China Sea Inst Oceanol, CAS Key Lab Trop Marine Bioresources & Ecol, RNAM Ctr Marine Microbiol,Guangdong Key Lab Marin, Guangzhou 510301, Guangdong, Peoples R China.
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Yang, Jian,Li, Lizhen,Xiao, Yunzhu,et al. Identification and thermoadaptation engineering of thermostability conferring residue of deep sea bacterial alpha-amylase AMY121[J]. JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC,2016,126:56-63.
APA Yang, Jian.,Li, Lizhen.,Xiao, Yunzhu.,Li, Jie.,Long, Lijuan.,...&Zhang, S .(2016).Identification and thermoadaptation engineering of thermostability conferring residue of deep sea bacterial alpha-amylase AMY121.JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC,126,56-63.
MLA Yang, Jian,et al."Identification and thermoadaptation engineering of thermostability conferring residue of deep sea bacterial alpha-amylase AMY121".JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 126(2016):56-63.
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