阴沟肠杆菌过氧化氢酶基因的重组表达及酶学性质研究

Recombinant Expression and Characterization of a Catalase from Enterobacter cloacae

过氧化氢酶又称触酶,在食品、纺织和造纸等领域应用广泛。本研究从阴沟肠杆菌Enterobacter cloacae XTL13中克隆得到一个过氧化氢酶基因cat1,该基因全长2250bp,编码749个氨基酸和一个终止密码子。将cat1基因连接pPIC9载体并转化毕赤酵母GS115,得到高效表达CAT1的重组酵母菌株。在摇床水平过氧化氢酶活性可达300U/mL。重组CAT1最适温度为37℃,最适pH为6.5,比活力为1667U/mg;50℃处理2h或pH5～8处理1h后,仍然能保留80%以上的酶活力。当CAT1与葡萄糖氧化酶(GOD)联合使用(酶活力比例为1:30)时,能够解除过氧化氢对GOD活力的抑制,使得GOD能够更好地发挥其作用。另外,本研究还建立了一种快速筛选过氧化氢酶重组菌株的方法。

Hydrogen peroxidase, commonly named catalase, has been widely used in food, textile and paper industries. A eatalase gene cat1 was cloned from Enterobacter cloacae by using touch-down PCR and TAIL-PCR. Length of catl is 2 250 bp, which encode 749 amino acids and a termination codon. This gene was cloned into an expression vector pPIC9, and overexpressed in Pichia pastoris GS115 with the maximum catalase activity of 300 U/mL at shaker level. The recombinant CAT1 exhibited optimal activity at pH 6. 5 and 37~C. Specific activity of CAT1 was 1667 U/mg, towards hydrogen peroxide as substrate. CAT1 remains above 80% enzyme activity after treated at 50 C for 2 h or stay at pH 5 - 8 for 1 h. The effects of CAT1 on glucose oxidase （GOD） activity was investigated. When the activity ratio of CAT1 to GOD was 1：30, the GOD activity was no longer inhibited by hydrogen peroxide. This study also introduced a rapid screening method for P. pastoris with recombinant eatalase.