表面展示南极假丝酵母脂肪酶B的重组毕赤酵母高密度发酵条件优化  

High-density Fermentation Optimization of Candida antarctica Lipase B-displaying Pichia pastoris

作　　者：冯琮[1] 林影[1] 梁书利[1] 苏国栋[1] 韩双艳[1] 

机构地区：[1]华南理工大学生物科学与工程学院广东省发酵与酶工程重点实验室

出　　处：《食品与发酵工业》2010年第9期29-34,共6页Food and Fermentation Industries

基　　金：广东省工业攻关重点项目(2008A01090002)资助

摘　　要：以Invitrogen公司提供的毕赤酵母发酵工艺手册为依据,先用摇瓶模拟甘油分批发酵,确定了表面展示南极假丝酵母脂肪酶B(Candida antarcticlipase B,CALB)的毕赤酵母在BSM培养基中生长所需几种重要配方的最佳浓度为:甘油含量40g/L、硫酸铵4g/L、PTM14mL/L和CaSO410mmol/L;然后利用多功能补料摇床确定了菌株最适生长和产酶的pH分别为5.5和7.0,最适生长和产酶温度为30℃;再在2L发酵罐上放大,并对甘油流加模式和甲醇流加模式进行了优化筛选,确定了以9.6mL/(L.h)的流速流加甘油12h能有效提高菌体密度;以6.4mL/(L.h)的流速恒速流加甲醇比恒定溶氧流加甲醇诱导产酶更佳。最终单位菌体酶活力可达348.4U/g,比甘油含量40g/L、硫酸铵10g/L、PTM14.35mL/L、CaSO40.93g/L,以6.4mL/(L.h)的流速流加甘油4h和以恒定溶氧流加甲醇的未优化条件下提高34.8%。最后通过细胞表面荧光强度检测和全细胞催化合成活力对比,发现优化后单位菌体展示的蛋白表达量得到提高。According to Pichia fermentation manual provided by Invitrogen, we identified the first several important composition of BSM medium of Candida antarctica Lipase B-displaying Pichia pastoris in glycerol batch using flask. The optimal levels were glycerol 40g/L, ammonium sulfate 4g/L, PTM1 dmL/L and CaSO410mmol/L. Then the optimal growth and enzyme production pH were identified as 5.5 and 7.0 respectively. The optimal temperature for growth and enzyme production was 30℃, determined by multi - fed shaker. The above described optimal conditions were used for scaling up in 2L fermentation tank. Cell density and CALB activity were increased when the glycerol fed-batch feeding flow rate was set at 9.6mL/（L · h） for 12h. After that methanol fed - batch feeding flow rate was set at 6.4mL/（L · h） fixed. At the end of the experiment, the activity of enzyme on dry cell increased by 348.4U/g or by 34.8%. Improvement of protein expression was verified by using cell surface fluorescence intensity and synthesis viability assay.

关 键 词：发酵优化 酵母表面展示 南极假丝酵母脂肪酶B 高密度 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]