摘要：通过对不同地区长期被牛奶浸润的土壤样品的筛选.docVIP

具有转糖苷活性的β-半乳糖苷酶产生微生物的筛选及应用 邢澍 詹妮 田加强 指导教师：肖敏 摘要：通过对不同地区长期被牛奶浸润的土壤样品的筛选，从中选出β-半乳糖苷酶活力较高的产生菌2株（编号分别为LB5和LB30），纯化后菌株进行产酶发酵培养条件和β-半乳糖苷酶反应条件的优化。LB5菌株β-半乳糖苷酶产酶培养基的碳源为乳糖1%，氮源为蛋白胨0.5%+酵母粉0.3%+硝酸铵0.2%，LB30菌株β-半乳糖苷酶产酶培养基的碳源为乳糖1%，氮源为蛋白胨0.5%+硝酸铵0.5%，两菌株培养基pH6.5，30℃下摇床培养16h。确定LB5菌株的β-半乳糖苷酶转糖基反应体系为pH7.8的磷酸缓冲液配制40%的乳糖溶液，55℃下转糖苷反应12h; LB30菌株的β-半乳糖苷酶转糖基反应体系为pH7.0的磷酸缓冲液配制35%的乳糖溶液，55℃下转糖苷反应12h。两菌株转糖基反应体系中粗酶液∶乳糖溶液＝1∶3（v/v） 关键词：β-半乳糖苷酶 转糖基活性 菌株 筛选 Abstact: Two bacterium strains LB5 and LB30 with highβ-galactosidase activity were selected from multiple soil samples from different regions soaked in milk for a long period of time. Optimal conditions for its enzyme producing and β-galactosidase-catalyzed transgalactosylation reaction conditions were studied. The optimal carbon sourse and nitrogen sourse for LB5 were 1% lactose and 0.5% peptone plus 0.3% yeast extact plus 0.2% ammonium nitrate, respectively, and the optimal carbon source and nitrogen source for LB30 were 1% lactose and 0.5% peptone plus0.5% ammonium nitrate, respectively. High yields of the enzyme for two strains were obtained at 30℃ for 16 hours at pH 6.5. The optimal transgalactosylation reaction conditions were analysed by thin layer chromatography (TLC). The optimal reaction system for LB5 was pH 7.8 phosphate buffer containing 40% lactose , reacted at 55℃ with shaking for 12 hours，while optimal reaction system for LB30 was pH 7.0 phosphate buffer containing 35% lactose, reacted at 55℃ with shaking for 12 hours. Two reaction systems contained enzymes as rough enzyme : solution of lactose = 1∶3(v/v). Keywords: β-galactosidase, transgalactosylation , bacteria strains, isolation 半乳糖寡糖是天然属性的寡糖，作为双歧因子能促进人体内双歧杆菌的繁殖，对人类的健康有多种益处，能改善便秘、降低血压血脂、增强机体免疫力等。目前国内外多通过β-半乳糖苷酶催化乳糖发生转糖苷反应获得半乳糖寡糖，具体来说，即在乳糖的半乳糖一侧又以β（1→4），β（1→3）或β（1→6）键连接半乳糖，其中以β（1→4） 键为主，但是适合大规模生产的β-半乳糖苷酶的转糖苷活性很低，这导致了半乳糖寡糖的价格居高不下[1，2]。 通过筛选β-半乳糖苷酶的菌株获得转糖苷活性较高的β-半乳糖苷酶，以提高半乳糖寡糖产率，这一方面具有较高经济价值，另一方面有益于人类的健康。获得β-半乳糖苷酶还有其他广泛用处，例如利用β-半乳糖苷酶的转糖苷作用人工合成糖苷化合物，这对食品添加剂、药品和其他生理