title: Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-Glucanase (CenC) from Ruminiclostridium thermocellum in a Mesophilic Host Through Various Cultivation and Induction Strategies.
abstract: Commonly, unintentional induction and inadvertently preparing medium for engineered Escherichia coli BL21 CodonPlus (DE3)-RIPL, give poor or variable yields of heterologous proteins. Therefore, to enhance the activity and production of an industrially relevant recombinant processive endo-1,4-β-glucanase (CenC) propagated in Escherichia coli BL21 CodonPlus(DE3)-RIPL through various cultivation and induction strategies. Investigation of various growth media and induction parameters revealed that high-cell-density and optimal CenC expression were obtained in ZYBM9 medium induced either with 0.5 mM IPTG/150 mM lactose, after 6 h induction at 37 °C; and before induction, bacterial cells were given heat shock (42 °C) for 1 h when culture density (OD600nm) reached at 0.6. Intracellular enzyme activity was enhanced by 6.67 and 3.20-fold in ZYBM9 and 3×ZYBM9 medium, respectively, under optimal conditions. Using YNG auto-induction medium, activity was 2.5-fold increased after 10 h incubation at 37 °C. Approximately similar results were obtained by transferring the optimized process at the bioreactor level. Results showed that the effective process strategy is essential to enhance recombinant bacterial cell mass and enzyme production from small to large-scale. To the best of our knowledge, this is the first ever report on enhanced production of thermostable processive endo-1,4-β-glucanase cloned from Ruminiclostridium thermocellum, which is a suitable candidate for industrial applications. Graphical Abstract Flow Chart Summary of Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-glucanase from Ruminiclostridium thermocellum.
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, -54000, Pakistan.
authors.forename: Ikram Ul
authors.initials: IU
authors.lastname: Haq
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, -54000, Pakistan. fatima_iib@yahoo.com.
authors.forename: Fatima
authors.initials: F
authors.lastname: Akram
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002480:Cellulase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D000068016:Eubacteriales / Q000201:enzymology / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002480:Cellulase
keywords: Endo-1,4-β-glucanase; Heterologous protein expression; Induction strategy; Ruminiclostridium themocellum; Thermostable