K_(123)菌株普鲁兰酶(Pullulanase)的合成及其酶性质的研究

STUDY ON THE SYNTHESIS AND CHARACTERS OF PULLULANASE FROM K 123 STRAIN

研究了一株从白酒发酵现场分离得到的Ｋ１２３菌株普鲁兰酶的合成及其部分酶性质．结果是Ｋ１２３菌株的异淀粉酶在培养液中的大量积累发生在稳定期后期，菌的最适生长温度为３５℃，产酶的最适温度则为３０℃；菌的最适生长ｐＨ和产酶的最适ｐＨ均在７．０～７．５；产酶的最佳碳源为可溶性淀粉，有效碳源是糯米淀粉、糊精、支链淀粉、玉米面、茁霉多糖、麦芽糖，而葡萄糖则抑制产酶；产酶的最佳氮源是蛋白胨、酪素水解物，无机氮源则使得Ｋ１２３菌株的产酶量略低于有机氮源．通气量对菌体的生长影响较大，但菌体的生长达稳定期后，通气量的改变对菌的产酶影响不大．Ｋ１２３菌株所产的异淀粉酶水解茁霉多糖的产物为麦芽三糖，不水解动物淀粉，是普鲁兰酶（ｐｕｌｕｌａｎａｓｅ）．该酶反应的最适温度为５０℃，最适ｐＨ５．８，受Ｃａ２＋，Ｍｎ２＋等离子激活，受Ｃｕ２＋，Ｚｎ２＋等离子抑制；该酶的热稳定性较差，５０℃以上迅速失活；ｐＨ稳定性较好，中性，微酸性条件下，冰箱中可长期保存．该酶应用于固态白酒发酵可提高出酒率平均４％以上．

The growth conditions for pullulanase production of a bacterium K 123 which isolated from solid fermenting site were researched.A large enzyme accumulation in the culture took place in the late stationary phase of growth.The maximal enzyme activity was achieved after 48 h of shaking cultivation at 30℃ and an initial pH 7.0～7.5 on a medium composed of 1.2%(w/v) soluble starch ,0.8% peptone,0.2% yeast extract,0.05% MgSO 4 and 0.1%K 2HPO 4. Pullulanase synthesis was significantly enhanced by polished glutinous rice starch,dextrin,amylopectin,corn starch,pullulan,maltose as well as solube starch but not at all by glucose.NH + 4 and NO - 3 could replace peptone as nitrogen source for the enzyme production. The extracellular pullulance activity of K 123 was characterized in a crude enzyme production.The enzyme hydrolyzed α 1,6 glucosidic linkages both in starch and in pullulan,but not in glycogen,and the pullulan hydrolysis product was maltotiose,so it is pullulanase.The activity responds to temperature and pH,with optima at 50℃ and pH 5.8,and the activity is stimulated by Ca 2+ 、Mn 2+ 、Mg 2+ ions and inhibited by Cu 2+ 、Zn 2+ ions.The thermostability of the enzyme is low,it is inactive fast up 50℃.By making use of K 123 leaven the output of solid fermenting spirits is raised above the average rate of 4%.