Separation of chitosanase from the fermentation broth of Bacillus cereus NTU-FC-4 using reversed micellar extraction

• Main Authors: Ya-Ling Chen, 陳雅苓
• Other Authors: Been-Huang Chiang, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/38327007518804206236

碩士 === 國立臺灣大學 === 食品科技研究所 === 89 === Abstract The chitosanase is an important industrial enzyme, which can be used to hydrolyze chitosan and yield an important functional food ingredient─chitooligosaccharides. Conventional methods for purifying enzyme often involve tedious procedures, such as precipitation, filtration, centrifugation, and chromatography. Reversed micelles are aggregates of surfactsnt containing an inner core of water molecules, dispersed in an organic solvent. Under proper conditions, many biological products, such as enzyme, can be solubilized into the core of water without loss in activity. The procedure involved in reversed micellear extraction is relatively simple, and the technology is easily scaled up. Therefore, in this study, the chitosanase from Bacillus cereus NTU-FC-4 was attempted to be purified by reversed micellar extraction. The fermentation broth of Bacillus cereus NTU-FC-4 was first treated with acetone to obtain crude enzyme. The crude enzyme was then purified by reversed micellar extraction. It was found that the optimal processing condition for purifying chitosanase from crude enzyme were (1) forward extraction : the crude enzyme (1.0 mg /ml) was dissolved with 50 mM phosphate buffer containing 96.03 mM sodium chloride, The aqueous solution was contacted with an equal volume of 102.43 mM AOT in isooctane solution. The mixture was shaked in water bath at 15℃ for 85 minutes to solubilize the enzyme into the reversed micelles. (2) backward extraction: the organic solution from forward extraction was contacted with 50 mM phosphate buffer containing 1.0 M sodium chloride at pH 10. The mixture was shaked in water bath at 40℃ for 40 minutes (150 rpm).Thus the enzyme transferred from the reversed micelles to the aqueous solution. The processes recovered approximately 69.8% of total activity oh chitosanase. The purity of the enzyme was increased to 32.4 fold, and the specific activity of the final product was 64.9 unit/mg protein. The electrophoretic analysis indicated that the purified enzyme contained chitosanase Ⅰ and Ⅱ. Their molecular weights were 69,000 and 46,000, respectively.