阴沟肠杆菌过氧化氢酶基因的重组表达及酶学性质研究

摘要

Hydrogen peroxidase, commonly named catalase, has been widely used in food, textile and paper industries. A catalase gene cat1 was cloned from Enterobacter cloacae by using touch-down PCR and TAIL-PCR. Length of cat1 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℃ . 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 catalase.%　　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2250 bp,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300 U/ mL。重组 CAT1最适温度为37℃,最适 pH 为6.5,比活力为1667 U/ mg；50℃处理2 h 或 pH 5~8处理1 h 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。