title: Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach.
abstract: This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Shuying
authors.initials: S
authors.lastname: Fang
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D000468:Alkalies / Q000378:metabolism*; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D061249:Batch Cell Culture Techniques; D018533:Biomass; D001709:Biotechnology / Q000379:methods*; D002244:Carbon / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D005990:Glycerol / Q000494:pharmacology*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010368:Pectins / Q000096:biosynthesis; D010957:Plasmids / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000096:biosynthesis*; D011995:Recombination, Genetic / Q000187:drug effects; D013696:Temperature; D013997:Time Factors
chemical_list: D000468:Alkalies; D003470:Culture Media; D007544:Isopropyl Thiogalactoside; D002244:Carbon; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D005990:Glycerol; C003181:polygalacturonic acid