Abstract: E.coli engineering-bacterium that expressed 1,3-propanediol dehydrogenase(PDH) gene from Klebsiella.p was cultured in 5 L fermentor at 30℃，pH7.0，200 r/min，microaerobic condition for 20h，after the fermented cells were crushed and separated，the acquired sample was then one-step purified to homogeneity by affinity chromatography with HisTrapHP column，this purification process resulted in a 2.94 fold purification of PDH with 50% final yield. One clarity strip was acquired and the relative molecular weight of expression PDH subunits was determined to be approximately 41 kDa using SDS-PAGE. The purified PDH was immobilized by reformative sol-gel (composite gelation) and the immobilized effect was affected by the concentration of tetraethyl orthosilicate (TEOS)，alginate (ALG)，CaCl2 and volume ratio of ALG and TEOS. The result showed that optimized immobilized condition was ρ(TEOS)=20 g/L，ρ(ALG)=30 g/L，ρ(CaCl2)=40 g/L，V(ALG)∶V(TEOS)=3∶1. The ALG-SiO2 composite gelation of PDH was stored for 60 days，and immobilized enzyme activity was preserved 80%. When it reacted for batchs，the immobilized enzyme activity was preserved 70.3% after it reacted for 3 batchs，but was preserved 29.8% after it reacted for 6 batchs.

摘要： 转录表达来源于Klebsiella.p1,3-丙二醇脱氢酶（PDH）基因片段的E.coli 工程菌，在5 L发酵罐、30℃、pH 值7.0、200 r/min（微氧）条件下培养20 h；发酵菌体经破碎分离后所得样品通过HisTrap HP柱亲和层析一步纯化得到电泳纯的PDH，纯化倍数为2.94倍，活性回收率为50%，经SDS-PAGE电泳鉴定获得一条清晰条带，表达蛋白亚基相对分子质量约为41 kDa。用改进的溶胶-凝胶法（杂化凝胶）包埋纯化酶，考察正硅酸乙酯（TEOS）浓度、海藻酸盐（ALG）浓度、CaCl₂浓度及ALG与TEOS体积比等因素对酶固定化效果的影响，结果表明，较优包埋条件为：ρ(TEOS)=20 g/L，ρ(ALG)=30 g/L，ρ(CaCl₂)=40 g/L，V(ALG)∶V(TEOS)=3∶1；由此制备的PDH的ALG-SiO₂杂化凝胶保藏60天后酶活仍保持80%，进行批次反应时，反应3批后酶活保持70.3%，反应第6批时酶活剩余29.8%。