title: Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control.
abstract: Pullulanase was extracellularly produced with an engineered Escherichia coli with a combined strategy. When auto-induction instead of isopropyl β-D-1-thiogalactopyranoside (IPTG) induction method was implemented, we observed increased extracellular activity (4.2 U ml(-1)) and cell biomass (7.95 g DCW l(-1)). Subsequent investigation of temperature effect on fermentation showed cultivation performed at 25 °C presented the highest extracellular titer and cell biomass. In order to reduce the extended production period, we developed a two-stage temperature control strategy. Its application not only reduced the production period from 72 to 36 h, but also further enhanced the yield of extracellular pullulanase. Finally, with a view to releasing more intracellular pullulanase, we altered cell membrane permeability with various medium additives. As a result, extracellular titer was elevated to 68.23 U ml(-1), nearly 35-fold higher than that with IPTG induction method. The combined strategy developed here may be useful for the production of other extracellular proteins by recombinant E. coli.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Ave., Wuxi, 214122, Jiangsu Province, China.
authors.forename: Wen-Bo
authors.initials: WB
authors.lastname: Chen
authors.forename: Yao
authors.initials: Y
authors.lastname: Nie
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D021902:Enterobacter aerogenes / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase