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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。
Reported the genetic engineering of recombinant Pseudomonas aeruginosa prolyl endopeptidase (apPEP). Engineering strain E. coli BL21 / pKKH PEP expression product apPEP as soluble protein in NBSBioFlo 30 0 0 type 5L controlled fermenter after 14h culture per liter of fermentation broth can reach 225 g dry weight of bacteria, containing apPEP 3 0g or so . After the fermentative bacteria were sonicated and precipitated by ammonium sulfate, they were separated and purified by Sephadex G 2 5, Highperformance Q Sepharose FF (HP Q) and Phenylseparose 6FF column chromatography. The yield of recombinant apPEP was 866% Specific activity reached 655u / mg, the purification of the entire protein recovery rate was 82%, the viability recovery was 24.4%. The purified apPEP had a molecular weight of 76 46 4 ± 30D as determined by electrospray ionization mass spectrometry. The N-terminal amino acid sequence was deduced from the gene sequence. The isoelectric point is around pI60. Similar to PEP derived from Aeromonas hydrophila (pI = 5 5).