title: Biphasic fermentation is an efficient strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis.
abstract: This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.
authors.affiliation: Enzyme Technology Laboratory, Biotechnology Division, Department of Botany, University of Calicut, Kerala, 673635, India.
authors.forename: Veloorvalappil Narayanan
authors.initials: VN
authors.lastname: Jisha
authors.forename: Robinson Babysarojam
authors.initials: RB
authors.lastname: Smitha
authors.forename: Prakasan
authors.initials: P
authors.lastname: Priji
authors.forename: Sreedharan
authors.initials: S
authors.lastname: Sajith
authors.forename: Sailas
authors.initials: S
authors.lastname: Benjamin
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity / Q000648:ultrastructure; D001709:Biotechnology / Q000379:methods*; D003460:Crystallization; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity; D005285:Fermentation; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity / Q000648:ultrastructure; D050296:Microbial Viability; D008925:Mites / Q000187:drug effects; D013171:Spores, Bacterial / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D003470:Culture Media; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis