放射菌NCTU3中幾丁質酵素之篩選、純化及性質的研究

Abstract

本研究以37℃篩選出具有幾丁質酵素活性的菌株，經鑑定後定名為Streptomyces sp. NCTU3。 此菌株生產幾丁質酵素的最佳條件以M9 medium中添加1%膠狀幾丁質、yeast extract 1g/L、2ml/L 1M硫酸鎂及0.1ml/L 1M氯化鈣，起始pH值為6.5，接入OD600為8的30 ml/L的菌母，在37℃下以180rpm震盪培養。在培養第三天可得最高活性為0.2unit/ml。 培養液經離心去除菌體後的粗酵素液，濃縮10倍體積，經過80％的硫酸銨沈澱、Hitrap Q及HIC管柱純化後，可得90%以上純度的幾丁質酵素(chitinase)，經蛋白質電泳鑑定其分子量為40 kDa，又經G-75凝膠體管柱層析鑑定為32 kDa，故是一單分子(monomer)，純化後的幾丁質酵素其酵素活性增加54倍；回收率為14%。 純化所得的幾丁質酵素其最適反應溫度為60℃，最適反應的pH值為6.5，酵素在pH5.5~7.0穩定性良好，若在50℃下反應，則酵素可維持4個小時以上的穩定性。此幾丁質酵素水解膠狀幾丁質及N-乙醯幾丁寡醣所得之產物為單醣及雙醣，但是並不水解p-nitrophenyl-β-D-glucosaminide。由這些屬性可判斷是一exo-幾丁質酵素。

A chitinase-producing bacterium strain NCTU3 was screen from nature. It was identified as a Streptomyces sp. according to the basic morphological and biochemical characteristics. This strain grew in a simple medium, contain M9 medium、1% colloidal chitin, yeast extract 1g/L, 1M MgSO4 2ml/L and 1M CaCl2 0.1ml/L, 30ml/L inoculum size of 8 of OD600 with the initial pH 6.5. The culture was incubated at 180 rpm,37℃, the selection of the enzyme reached its peak ( 0.2 unit of activity/ ml of cultural broth) in 3 day. Chitinase was purified by concentrating the supernatant medium to 1/10 volume, 80% ammonium sulfate precipitation, Hitrap Q and HIC (butyl-) column chromatography. The purified enzyme is a monomeric protein with a Mr value of 40 kDa and 32 kDa estimated by SDS-PAGE and gel filtration, respectively. The activity of the purified enzyme increased 54 folds with 14% recovery yield。 The optimal temperature of this enzyme was 60℃ and the optimal pH was 6.5. The enzyme was stable at pH 5.5~7.0, while it was unstable at 65℃ or high. The major products of enzymatic hydrolysis of colloidal chitin and N-acetyl-chitooligosaccharides with this purified enzyme were N-acetyl-glucosamine and N, N'-diacetyl- chitobiose. It was inactive toward p-nitrophenyl-β-D-glucosaminide. We concluded that the purified enzyme is an exo-chitinase.