Biphasic fermentation is an efficient strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis

Appl Biochem Biotechnol. 2015 Feb;175(3):1519-35. doi: 10.1007/s12010-014-1383-3. Epub 2014 Nov 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacillus thuringiensis / metabolism*
  • Bacillus thuringiensis Toxins
  • Bacterial Proteins / biosynthesis*
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / toxicity
  • Bacterial Proteins / ultrastructure
  • Biotechnology / methods*
  • Crystallization
  • Culture Media
  • Electrophoresis, Polyacrylamide Gel
  • Endotoxins / biosynthesis*
  • Endotoxins / isolation & purification
  • Endotoxins / toxicity
  • Fermentation*
  • Hemolysin Proteins / biosynthesis*
  • Hemolysin Proteins / isolation & purification
  • Hemolysin Proteins / toxicity
  • Hemolysin Proteins / ultrastructure
  • Microbial Viability
  • Mites / drug effects
  • Spores, Bacterial / metabolism
  • Staining and Labeling

Substances

  • Bacillus thuringiensis Toxins
  • Bacterial Proteins
  • Culture Media
  • Endotoxins
  • Hemolysin Proteins
  • insecticidal crystal protein, Bacillus Thuringiensis