重组甘蔗1-氨基环丙烷-1-羧基(ACC)氧化酶的纯化及其特性

在IPTG诱导下，重组甘蔗(Sacharum sinensis)1-氨基环丙烷-1-羧基(ACC)氧化酶在体外以包涵体蛋白形式表达。包涵体蛋白经Ni2+-NTA金属螯合层析柱纯化和复性后，获得酶活力高达132.58 nmolC2H4/ mg/h 蛋白。该蛋白在pH 5.5 ~ 8.0 和20 ~ 45 ℃温度范围内比较稳定，最适pH 6.7，最适温度33 ℃；米氏常数(Km )61.77 μmol/L，酶动力学参数（Vm）值0.9647 μmol/min，被一定浓度的CO2和底物ACC所激活，受Mg2+、Cu2+、Zn2+和EDTA抑制。

Purification and Characterization of the Recombinant 1-aminocyclopropane1-carboxylate (ACC) Oxidase from Sugarcane

The plasmid pET30a-SgACO, which contains 1-aminocyclopropane-1-carboxylate (ACC) oxidase （SgACO） gene from sugarcane (Sacharum sinensis Roxb), was transformed into Escherichia coli BL21 (DE3) plysS successfully, and the SgACO protein was expressed in BL21 (DE3). The SgACO was highly expressed in BL21 (DE3) in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) and most products existed in an inclusion body form. The final SgACO activity of 132.58 nmolC2H4/mg/h was obtained by one-step renaturation and purification of Ni2+- NTA column to the inclusion body protein. The renaturation and purification protein was relatively stable at the pH ranging from 5.5 to 8.0 and at the temperature ranging from 20 ℃ to 45 ℃. Its optimum pH was 6.7, optimum temperature was 33 ℃, Km was 61.77 μmol/L and Vm was 0.9647 μmol/min. The enzyme activity was activated by certain concentration of CO2 and ACC, and inhibited by Mg2+, Cu2+, Zn2+ and EDTA.