title: Enhanced Production of Recombinant Thermobifida fusca Isoamylase in Escherichia coli MDS42.
abstract: Isoamylase, an industrially significant enzyme used primarily in the saccharification of starch, is commonly obtained through recombinant expression in Escherichia coli. To improve the yield of this important enzyme, the isoamylase from Thermobifida fusca was expressed in the reduced-genome E. coli strain MDS42. Expression conditions were initially optimized in shake flasks. The optimal induction temperature was 37 °C and IPTG was superior to lactose as an inducer due to the low intracellular β-galactosidase activity in E. coli MDS42 expression system. Then, expression conditions were optimized in a 3.6-L fermenter. In the fermenter, optimal isoamylase expression was obtained when cells were grown at 37 °C and expression was induced at mid-log phase using 0.25 mM IPTG. The greatest isoamylase activity (22,983.0 U/mL of culture) and production (18.8 mg/mL) were obtained 24 h after induction of expression. These values are the highest ever reported, suggesting that E. coli MDS42 is a suitable host for the production of enzymes for industrial use.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Ran
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. xgduan@jiangnan.edu.cn.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. xgduan@jiangnan.edu.cn.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007517:Isoamylase / Q000096:biosynthesis*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007785:Lactose / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D007517:Isoamylase; D007785:Lactose
keywords: Escherichia coli BL21(DE3); Escherichia coli MDS42; Fermentation optimization; Isoamylase; β-galactosidase