Title: 仙人掌桿菌NCTU4幾丁聚醣酵素之性質研究與應用
Characterization and Application of Chitosanase from Bacillus cereus NCTU4
Authors: 李博仁
Bor-ran Li
Dr.Yaw-kuen Li
Keywords: 幾丁聚醣酵素;仙人掌桿菌;桿菌;chitosanase;Bacillus cereus;Bacillus
Issue Date: 2000
Abstract: 仙人掌桿菌中幾丁聚醣酵素之生產,在添加適量之幾丁聚醣後能提高其產量10 ~ 30 %。此胞外幾丁聚醣內切酵素之分子量約42 kDa,等電點為5.0,最適反應溫度與酸鹼值為60℃及pH = 5 ~ 6。在5 mM濃度之二價金屬離子(Pb2+、Ba2+、Cu2+、Mn2+、Ca2+)環境下,對酵素產生促進活性1.1 ~ 3.5倍的效果,其中以Mn2+最佳,而EDTA在5 mM濃度時則有30%抑制催化之效果。反應最終產物為2、3、4醣。其催化過程屬於位向反轉的機制(inversion)。幾丁聚醣最終水解程度,受溫度之影響,Tg點溫度下,有階段性反應終點。以silica gel為基材之酵素固定化有良好的酵素活性,且為期24小時之批次(Batch)反應中,連續使用7次以上仍可維持100 %之活性。另利用陰離子交換樹脂之可有效回收批次反應中所使用之酵素,活性回收率亦幾近100 %。
Although chitosanase can be produced from Bacillus NCTU4 without adding any inducers, suitable amount (500 ppm) of chitosan results in a 10 ~ 30 % higher yield of the activity. The extracellular chitosanase was purified and characterized. The monomeric enzyme is an endo-chitosanase with a molecular mass of 42 kDa, pI = 5.0. The optimal activity of the purified enzyme was at 60℃, pH = 5 ~ 6. With the presence of 5 mM divalent metal ion (Pb2+, Ba2+, Cu2+, Mn2+ and Ca2+), enzyme activity was found to enhance by a factor of 1.1 ~ 3.5-fold. Among them, Mn2+ was most effective. EDTA (5 mM) inhibited 30 % of the enzyme activity. Chitosan dimmer, trimer and tetramer were found to be the predominate end products. The catalytic reaction of the enzyme was identified as an inversion of the anomeric configuration indicating that a single step mechanism was involved. The end point of the hydrolysis depends on the temperature. Which consequently medicates the molecular structure of polysaccharide chain. Immobilization enzyme and resin-recovering system were developed to reduce chitosanase consumption. Both methods can nearly retain or recover 100 % of the activity. These processes are potentially useful in industry.
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