重组点状产气单胞菌脯氨酰内肽酶的纯化和鉴定  

Purification and Characterization of Recombinant Aeromonas punctata Prolyl Endopeptidase

作　　者：李民[1] 陈常庆[1] 王德宝[2] 

机构地区：[1]中国科学院上海生物工程研究中心,上海200233 [2]中国科学院上海生物化学研究所,上海200031

出　　处：《生物工程学报》2000年第3期345-348,共4页Chinese Journal of Biotechnology

基　　金：中国科学院上海分院择优项目!(沪院 98 0 2 0 6)

摘　　要：报道重组点状产气单胞菌脯氨酰内肽酶 (简称apPEP)的基因工程下游工艺研究。工程菌株E .coliBL2 1/ pKKH PEP表达产物apPEP为可溶性蛋白 ,在NBSBioFlo 30 0 0型 5L自控发酵罐中经 14h培养每升发酵液可达到 2 2 5 g干重菌体 ,含apPEP 3 0g左右。发酵菌体经超声破碎、硫酸铵沉淀后 ,依次经SephadexG 2 5、HighperformanceQsepharoseFF(HP Q)、Phenylseparose 6FF柱层析分离纯化 ,每升发酵产物最终可得 0 86 g纯度达96 %的重组apPEP ,比活力达到 6 5 5u/mg ,整个纯化工艺的蛋白回收率为 8 2 % ,活力回收率为 2 4 4%。纯化的apPEP经电喷雾质谱测定分子量为 76 46 4± 30D ,N端氨基酸序列与基因序列推导的一致。等电点为 pI6 0左右。与Aeromonashydrophila来源的PEP(pI =5 5 )相近。The study of down\|stream techniques of recombinant Aeromonas punctata prolyl endopeptidase (apPEP) was presented here.High cell\|density fermentation of E.coli BL21/pKKH\|PEP in NBS BioFlo 3000 5L fermentor was achieved,the final cell density was 22\^5g (DCW)/L after 14h cultivation,the yield of apPEP expressed in soluble protein was 3\^0g per litter broth.After sonication,the supernatant of free cell extract was purified by ammonium sulfate fractionation,High performance Q sepharose FF,Phenyl sepharose 6 FF,the purity of apPEP reached 96%,enzyme specific activity was 65\^5u/mg,apPEP yield reached 0\^86g/L broth.Total recovery of enzyme protein was 8\^2%,actviity recovery was 24\^4%.The molecular weight of apPEP was 76464±30Da measured by MS,N terminus amino acids sequence consistent with that deduced from DNA sequence.pI 6\^0,which was similar with PEP from Aeromonas hydrophila.

关 键 词：脯氨酰内肽酶 高密度发酵 纯化 比活力 鉴定 

分 类 号：Q55[生物学—生物化学]