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。
Feng Cong
,
Lin Yin
,
Liang Shu-li
,
Su Guo-dong
,
Han Shuang-yanKey Lab of Fermentation and Enzyme Engineering
,
School of Bioscience and Technology SCUT
,
Guangzhou 000
,
China
. 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[J]. 食品与发酵工业, 2010
, 36(09)
: 29
-34
.
DOI: 10.13995/j.cnki.11-1802/ts.2010.09.004
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 4mL/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.
