脯氨酰内肽酶培养条件的优化及高密度发酵  

Cultivation Optimization of Prolyl Endopeptidase and High Cell Density Fermentation

作　　者：李民[1] 修朝阳[1] 陈常庆[1] 

机构地区：[1]中国科学院上海生物工程研究中心,上海200233

出　　处：《生物工程学报》2000年第2期183-187,共5页Chinese Journal of Biotechnology

摘　　要：基因工程菌E.coliBL21/pGEMPEP可以组成型表达重组的点状产气单胞菌脯氨酰内肽酶(PEP),但培养条件极大地影响着酶的产量,为了获得高效表达,首先测定了工程菌表达PEP的稳定性并考察了培养温度、pH、发酵时间、碳源、氮源、无机盐等对产酶的影响,得到了优化的发酵条件,L9(34)正交试验进一步明确了摇床转速、培养温度、pH值、培养时间对产酶量的影响都有高度的统计学意义。在此基础上利用NBSBioFlo3000型5L自控发酵罐进行了高密度、高表达发酵、经20h培养,最终菌体密度达OD60060(相当于干菌体225g/L),PEP表达量为28%,每升发酵液中含PEP酶315g。Engineered E.coli BL21/pGEM PEP could constitutively express recombinant prolyl endopeptidase from Aeromonas punctata, which was extremely effected by culture conditions,so fermentation conditions were optimized to obtain it's high expression level.Firstly,the stability of BL21/pGEM PEP was investigated,then,culture temperature,pH,time,medium were optimized in shake flaskd.The L 9(3 4) orthogonal experiment confirmed that shaker speed ,pH,culture temperature,culture time had high degree statistical meaning.Based on these data,high cell density fermentation of E.coli BL21/pGEM PEP on NBS BioFlo 3000 5L fermentor was achieved,after 20h cultivation,the final density(dwt) was 60 OD 600 (22.5g/L),the expressed PEP was about 28% of total cellular protein,the yield was 3.15g per litter broth.

关 键 词：脯氮酰内肽酶 高密度发酵 正交实验 条件优化 

分 类 号：Q815[生物学—生物工程]