一株产环糊精葡萄糖基转移酶的地衣芽孢杆菌的选育、产酶条件及酶学特性

Breeding, optimized fermentation and enzymatic properties of a Bacillus licheniformis mutant producing cyclomaltodextrin glucanotransferase

从土壤分离物中筛选到一株环糊精葡萄糖基转移酶 (CGTase)产生菌 4 0 3,96h发酵酶活为 0 95U mL。经紫外辐射和硫酸二乙酯复合诱变而获得突变株CLS4 0 3,96h发酵酶活达 1 36U mL ,提高 4 3%。该突变菌株被鉴定为地衣芽孢杆菌 (Bacilluslicheniformis) ,产CGTase的最佳碳源为可溶性淀粉 ,最佳氮源为硝酸铵 ,最适初始pH为 6 5 ,最适培养温度为 35℃ ,发酵期间CGTase的产生高峰 (第 96h)滞后于菌体生物量高峰 (第 4 8h) 2d。菌株所产CGTase的最适反应pH为 6 0 ,最适温度为 5 5℃ ,在pH 6 0～ 7 5间和 5 0℃下保持 1h后的剩余酶活均达 90 %以上 ;酶液中适量添加Ca2 + 能大幅提高CGTase在 5 5℃下的稳定性。经高效液相色谱分析 ,CGTase作用于淀粉后的产物以α 环糊精为主 ,β 环糊精为次 ,二者比例为 2 4 7∶1,环糊精总产率达 2 9 8% ,但产物中不含γ

A bacterial strain 403 selected from various soil was found to produce cyclomaltodextrin glucanotransferase (CGTase) with an activity of 0.95 U/mL after 96h submerged incubation. A mutant, Bacillus licheniformis CLS403, obtained by means of ultraviolet radiation and diethyl sulfate, enhanced CGTase production by 43%(1.36U/mL). The optimized conditions for CGTase production of mutant were soluble starch as carbon source, ammonium nitrate as nitrogen source, 35℃ and pH6.5 for incubation. Under these conditions, the production of CGTase by the mutant in submerged peaked at the period of 96h incubation, two days later than bacterial biomass peak. The resultant CGTase reacted best at 55℃ and pH 6.0, and had activities >90% after 1h maintenance at 50℃ with pH 6.0～7.5. Addition of Ca 2+ in the reaction largely enhanced stability of the CGTase activity at 55℃. Based on HPLC analysis, the products of starch hydrolized by the CGTase of B. licheniformis CLS403 included much more α-cyclodextrin than β-cyclodextrin but no γ-cyclodextrin. The total yield of cyclodextrin from starch reached 29.8% with the ratio of the two cyclodextrins 2.47∶1.