耐热碱性纤维素酶分离纯化及酶学特性研究

Purification and enzymatic properties of thermostable alkaline cellulase Ⅲ-3-A

通过离子交换和凝胶过滤两步层析法，从耐碱芽孢杆菌Ⅲ-3的培养液中分离到相对分子质量约为89kD、比活力高达420U／mg的碱性纤维素酶Ⅲ-3-A．质谱分析显示：Ⅲ-3-A酶蛋白与芽孢杆菌KSM-64、KSM-S237等产生的A5家族碱性纤维素酶具有一定的同源性．酶学性质研究结果表明：Ⅲ-3-A的CMC酶反应以pH值为9．0、反应温度45℃左右为宜，在pH值为6～11和50℃以下酶活性稳定，且具有耐金属离子和表面活性剂等特性；Ca^2＋对酶的最适反应温度和热稳定性有显著影响，添加5mmol／L CaCl2可使最适酶反应温度和稳定性分别提高至50℃和55℃．该酶具有耐热、耐金属离子、耐碱及高比活力等特点，在棉织品的水洗整理及洗涤剂工业中具有良好应用前景。

Using a two-step column-chromatographic procedure of ion-exchange and gel filtration, alkaline cellulase Ⅲ- 3 -A, with molecular weight of about 89 kD and specific activity of 420 U/mg, was purified to homogeneity from the culture broth of Bacillus sp. Ⅲ-3. Result of MALDI-TOF mass spectrometry analysis showed that the typical a- m/no acid sequences of Ⅲ-3-A were similar to those of alkaline cellulases from Bacillus sp. 1139 and Bacillus sp. KSM-64, which belonged to family A5. The optimal conditions for the enzymatic reaction were about pH 9. 0 at 45℃. At pH 6 - 11 and below 50℃, the enzyme showed high activity and good stability. The enzyme was resistant to most metallic ions and reagents explored. It was also found that Ca^2 ＋ ion had a significant influence on the optimal temperature for enzymatic reaction and thermostability. 5 mmol/L CaCl2 lifted the optimum temperature and thermostability up to 50℃ and 55℃ respectively. This novel cellulase showed great potential in the textile biofinishing and detergent industry.