摘要:偶氮还原酶（Azoreductase）是降解偶氮染料的关键酶，在染料污染区域的生物修复方面具有独特优势。而对于可以在高温环境中处理工业废水的热稳定性偶氮还原酶，更具有研究应用价值。但目前国内外对热稳定性偶氮还原酶结构和其热稳定性的研究较少。本研究基于蛋白质结构特征分析热稳定偶氮还原酶BC（AzoreductaseBC，简称AzoBC）的特性，预测确定对偶氮还原酶BC热稳定性影响因素，拟通过选择性变性来测定各因素对偶氮还原酶BC热稳定性的影响大小，从而为偶氮还原酶热稳定性的改进提供理论基础。

本实验通过大肠杆菌表达菌株BL21将带有His-tag偶氮还原酶BC基因（azoBC）进行表达，后经过固定镍离子亲和层析柱（Nickel-Immobilizedmetalionaffinitychromatography，简称Ni-IMAC）分离纯化得到偶氮还原酶BC纯酶液。对偶氮还原酶BC进行基本酶学性质研究发现，其最适反应达70℃，是一种热稳定的偶氮还原酶，是研究偶氮还原酶热稳定性同结构关系的理想材料。

用5%甲醇、1mM十二烷基磺酸钠（SDS）、2%氯化钾三种变性剂分别对偶氮还原酶BC的氢键（Hydrogenbond）、疏水作用（Hydrophobicinteraction）、离子键（Ionicbond）进行选择性变性处理，并通过温差脱色试验，测定偶氮还原酶BC的失活动力学参数。结果表明，在70℃时，经甲醇和SDS处理的偶氮还原酶BC失活半衰期由9.56min分别降低到7.11min和6.93min；在80℃时，经甲醇、SDS和氯化钾处理的偶氮还原酶BC失活半衰期分别降低了0.76min，2.48min和2.14min；在90℃时，经SDS处理的偶氮还原酶BC，其失活半衰期降低到只有2.35min。经过5%甲醇、1mMSDS、2%氯化钾处理的偶氮还原酶BC失活活化能由原来的70.84kJ/mol分别降低到51.93kJ/mol、48.52kJ/mol、67.76kJ/mol。这表明了氢键、疏水作用、离子键对偶氮还原酶BC稳定性均有贡献，其中疏水作用对偶氮还原酶BC稳定性的贡献较大。

关键词：偶氮还原酶BC；热稳定性；热失活

Abstract:Azo reductase is the key enzyme in degradation of azo dye and has unique significance in the bioremediation of azo dye-contaminated areas. The thermostable azo reductase has great research value because of its potential application in the treatment of high temperature industrial wastewater. However, there are few studies concerned about the structure and thermostability of thermostable azo reductase at home and abroad. The analysis the 3D- stucture of azo reductase BC (azoBC). Predicted the factors affecting its thermal stability. In present study, we intended to investigate how these factors affect the thermal stability of azoBC by selective denaturation assays. These results will give some clue for the improvement of thermal stability of other azo reductases in the future.

In this study, azo reductase BC gene (azoBC) with His-tag was expressed by Escherichia coli (E. coli) BL21. The expression products were then purified by Nickel- Immobilized metal ion affinity chromatography (Ni-IMAC) to obtain azoBC solution. The results of the enzymatic assay of azoBC showed that the optimum reaction temperature of azo-BC was up to 70 ℃. The results suggested that azoBC is a thermostable azo reductase and it is an ideal material for studying the relationship between the thermal stability and the structure of azo reductases.

The hydrogen bond, hydrophobic interaction and ion bond of azo BC were selectively denatured with 5% methanol, 1 mmol / L SDS and 2% KCl, respectively. The inactivation kinetic parameters of azoBC were measured by decoloration experiment at different temperatures. The results showed that the inactivation half-life of azoBC was decreased from 9.56 min to 7.11 min or 6.93 min by adding of methanol or SDS respectively at 70 ℃. At 80 ℃, the inactivation half-life was reduced by 0.76 min, 2.48 min, and 2.14min treated by methanol, SDS and KCl respectively. At 90 ℃, inactivation half-life of azoBC was only 2.35 min after treating with SDS. The deactivation activation energy of azoBC treated by 5% methanol, 1 mmol / L SDS and 2% KCl was reduced from 70.84 kJ / mol to 51.93 kJ / mol,48.52 kJ / mol, 67.76 kJ / mol respectively. The results indicated that the hydrogen   bonds,the hydrophobic interactions, and the ionic bonds all contribute to the thermal stability of azoBC, and the hydrophobic interaction contributes most to the stability of azoBC.