Optimization of reversed micellar extraction of chitosanases produced by Bacillus cereus

The fermentation broth of Bacillus cereus NTU-FC-4 was precipitated with 70% acetone to obtain crude enzyme. Chitosanases in the crude enzyme were then extracted by reversed micelles. It was found that proper amount of crude enzyme should be first dissolved in the 50.0 mM phosphate buffer containing 96.0 mM sodium chloride to make a 1.0 mg/ml protein solution. After adjusting the pH of the crude enzyme solution to a value of 4.0, the aqueous solution was mixed with an organic solution, the isooctane containing 102.3 mM of the anionic surfactant AOT (sodium 1,2-bis(-2-ethylhexyl) sulfosuccinate). The mixture was shaken in reciprocating shaker bath at 15 °C for 85 min to solubilize the target enzymes in the reversed micelles formed in the organic phase, thus completed the forward extraction. Then, the reversed micellar phase was separated from the aqueous phase, and allowed to mixed with 50 mM phosphate buffer containing 1.0 M potassium chloride at pH 10. After mixing the two solutions at 40 °C for 40 min, the target enzymes in the reversed micelles transferred back to the aqueous solution. The processes recovered approximately 70% of total activity of chitosanases. The purity of the chitosanases was increased to 30-fold as compared to that of the fermentation broth, and the specific activity of the final product was 60.3 unit/mg.