重组点状产气单胞菌脯氨酰内肽酶的纯化和鉴定

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.coli BL21/pKKH\|PEP表达产物apPEP为可溶性蛋白，在NBS BioFlo 3000型5L自控发酵罐中经14h培养每升发酵液可达到22.5g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Sephadex G-25、High performance Q sepharose FF(HP\|Q)、Phenyl separose 6 FF柱层析分离纯化，每升发酵产物最终可得0.86g纯度达96%的重组apPEP，比活力达到65.5u/mg，整个纯化工艺的蛋白回收率为8.2%，活力回收率为24.4%。纯化的apPEP经电喷雾质谱测定分子量为76464±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Aeromonas hydrophila来源的PEP(pI=5.5)相近。

Purification and Characterization of Recombinant Aeromonas punctata Prolyl Endopeptidase

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 5 L fermentor was achieved, the final cell density was 22.5 g (DCW)/L after 14 h cultivation, the yield of apPEP expressed in soluble protein was 3.0 g 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 65.5 u/mg, apPEP yield reached 0.86 g/L broth. Total recovery of enzyme protein was 8.2%, actviity recovery was 24.4%. The molecular weight of apPEP was 76,464 +/- 30 Da 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.