文献类型：期刊文献

英文题名：Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis

作者：Yang, Jianhua[1,3];Li, Wei[1];Wang, Dezheng[1];Wu, Hui[1];Li, Zhimin[1,2];Ye, Qin[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg Technol, 130 Meilong Rd, Shanghai 200237, Peoples R China;[3]Rhein Westfal TH Aachen, Lehrstuhl Biotechnol, Worringerweg 3, D-52074 Aachen, Germany

年份：2016

卷号：100

期号：14

起止页码：6279

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20161302142741);WOS:【SCI-EXPANDED(收录号:WOS:000378776300015)】；

基金：This study was supported by the project of Shanghai Leading Academic Disciplines (Grant No. B505), the National Science Foundation for Young Scientist of China (Grant No. 21406065), and the Fundamental Research Funds for the Central Universities (Grant No. 222201313007 and 22A201514042). This study was partially supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering and the Foundation of Key Laboratory for Industrial Biocatalysis (Tsinghua University), Ministry of Education (Grant No. 2015101). We thank Professor Ulrich Schwaneberg for the supporting of protein modeling tools, Gaurao V. Dhoke for suggestions of protein modeling, and Dr. Thomas Horn and Dr. Yipeng Wang for reading through the manuscript.

语种：英文

外文关键词：Glutathione; Bifunctional glutathione synthetase; Actinobacillus pleuropneumoniae; Actinobacillus succinogenes; Escherichia coli; One-strain system

摘要：Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, gamma-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production.