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.
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Acknowledgments
JVN is grateful to the University Grants Commission, Government of India for granting RGNF, and SRB is grateful to the University of Calicut for granting the University Research Fellowship. The Indian patent application no. 339/DEL/2012 dated February 7 is also acknowledged.
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The authors declare that there exist no competing financial or other interests.
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Jisha, V.N., Babysarojam Smitha, R., Priji, P. et al. Biphasic Fermentation Is an Efficient Strategy for the Overproduction of δ-Endotoxin from Bacillus thuringiensis . Appl Biochem Biotechnol 175, 1519–1535 (2015). https://doi.org/10.1007/s12010-014-1383-3
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DOI: https://doi.org/10.1007/s12010-014-1383-3