保护剂对大肠杆菌重组NAD激酶热稳定性的影响

Effects of the Protective Additives on Thermostability of NAD Kinase Recombinanted in E. coli

摘要:

为了提高烟酰胺腺嘌呤二核苷酸（NAD）激酶的稳定性，研究几种糖类（葡萄糖、麦芽糖、蔗糖）、醇类（甘油、乙醇、山梨醇）和表面活性剂类（吐温20、司盘20）对NAD激酶热稳定性的影响，通过正交实验得到一种复合保护剂，并研究复合保护剂对NAD激酶热稳定性、最适反应温度、最适反应pH、储存稳定性的影响。结果表明，麦芽糖、甘油、乙醇、吐温20能显著提高NAD激酶热稳定性，复合保护剂配方为质量分数0.6%的麦芽糖，体积分数8%的甘油和乙醇，体积分数0.6%的吐温20。添加复合保护剂后，在55 ℃，NAD激酶半衰期提高了16.7倍，由1.1提高到18.4 h;最适反应温度由35提高到50 ℃;pH稳定范围由6.5～9扩大到5.5～9.5;在4 ℃储存28 d后，酶活保留率提高31%。

Abstract:

The effects of several sugars (glucose, maltose and sucrose), polyols (glycerol, ethanol and sorbitol) and surfactants (tween 20 and span 20) on thermostability of nicotinamide adenine dinucleotide (NAD) kinase were studied. In addition, a composite protective additive was obtained by the orthogonal experiment and its effects on the thermostability, optimum reaction temperature, optimum pH and storage stability of NAD kinase were also investigated. The results showed that maltose, glycerol, ethanol, and tween 20 could improve the thermostability of NAD kinase significantly. The obtained composite protective additive included mass fraction of maltose 0.6%, volume fraction of glycerol 8%, volume fraction of ethanol 8%, and volume fraction of tween 20 0.6%. After treatment of the composite protective additive, at 55℃, the half-life of NAD kinase was increased by 16.7 times, and it increased from 1.1 h to 18.4 h. Meanwhile, the optimum reaction temperature increased from 35℃ to 50℃, the range of pH stability expanded from 6.5～9 to 5.5～9.5. The retention of enzyme activity was an improvement of 31% at 4℃ storage for 28 d.