Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis

doi:10.1016/j.jbiotec.2019.01.007

	Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis
	Xie, Dong-Fang 1; Yang, Jun-Xing 2; Lv, Chang-Jiang 1; Mei, Jia-Qi 3; Wang, Hong-Peng 1; Hu, Sheng 4; Zhao, Wei-Rui 4; Cao, Jia-Ren 1; Tu, Jun-Liang 1; Huang, Jun 1
刊名	JOURNAL OF BIOTECHNOLOGY
	2019-03-10
卷号	293 页码:8-16
关键词	Amine transaminase Consensus mutagenesis Hydrogen bond Molecular dynamics simulation Thermostability
ISSN号	0168-1656
DOI	10.1016/j.jbiotec.2019.01.007
通讯作者	Huang, Jun(huangjun@zust.edu.cn) ; Mei, Le-He(meilh@zju.edu.cn)
英文摘要	Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (ATATA) were constructed by consensus mutagenesis. Using Consensus Finder (http://cbs-kazlab.oit.umn.edu/), six positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these six residues were individually mutated to match the consensus sequence (I77 L, Q97E, H210N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wildtype, the thermostability of all six single variants was improved. The H210N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t(1/2)) at 40 degrees C, and a 4.6 degrees C increase in T-50(10) among the single variants. In addition, the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t(1/2) at 40 degrees C, and a 6.6 degrees C increase in T-50(10). Furtherly, the H210N/I77L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (Delta Delta G(fold)) calculations. Our results showed that the combined variant H210N/I77L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t(1/2) and a 11.8 degrees C higher T-50(10).
资助项目	National Natural Science Foundation of China[31470793] ; National Natural Science Foundation of China[31670804] ; Zhejiang Natural Science Foundation[LZ13B060002] ; Zhejiang Natural Science Foundation[LY16B060008] ; Zhejiang Natural Science Foundation[LQ18B060002] ; National Training Programs of Innovation and Entrepreneurship for Undergraduates[201611057011]
WOS关键词	ASYMMETRIC-SYNTHESIS ; MOLECULAR-DYNAMICS ; CHIRAL AMINES ; SEQUENCE ; AMINATION ; THERMOSTABILITY ; DEHYDROGENASE ; REDUCTION ; PROTEINS ; KETONES
WOS研究方向	Biotechnology & Applied Microbiology
语种	英语
出版者	ELSEVIER SCIENCE BV
WOS记录号	WOS:000460015300002
资助机构	National Natural Science Foundation of China ; Zhejiang Natural Science Foundation ; National Training Programs of Innovation and Entrepreneurship for Undergraduates
内容类型	期刊论文
源URL	[http://ir.igsnrr.ac.cn/handle/311030/49337]
专题	中国科学院地理科学与资源研究所
通讯作者	Huang, Jun; Mei, Le-He
作者单位	1.Zhejiang Univ Sci & Technol, Sch Biol & Chem Engn, Hangzhou 310023, Zhejiang, Peoples R China 2.Chinese Acad Sci, Inst Geog Sci & Nat Resources Res, Beijing 100101, Peoples R China 3.Univ Utah, Dept Chem Engn, Salt Lake City, UT 84102 USA 4.Zhejiang Univ, Ningbo Inst Technol, Dept Biol & Pharmaceut Engn, Ningbo 315100, Zhejiang, Peoples R China
推荐引用方式 GB/T 7714	Xie, Dong-Fang,Yang, Jun-Xing,Lv, Chang-Jiang,et al. Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis[J]. JOURNAL OF BIOTECHNOLOGY,2019,293:8-16.
APA	Xie, Dong-Fang.,Yang, Jun-Xing.,Lv, Chang-Jiang.,Mei, Jia-Qi.,Wang, Hong-Peng.,...&Mei, Le-He.(2019).Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis.JOURNAL OF BIOTECHNOLOGY,293,8-16.
MLA	Xie, Dong-Fang,et al."Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis".JOURNAL OF BIOTECHNOLOGY 293(2019):8-16.