基因工程菌1020耐热木聚糖酶的纯化与性质

Study on Purification and Properties of Thermo-stable Xylanase from Bioengineering Bacterium 1020

通过热变性处理和Ni-NTA层析分离纯化基因工程菌1020耐热木聚糖酶。对热变性条件优化,70℃处理30min效果,纯化倍数4.9;利用木聚糖酶C端6个His标记对Ni-NTA琼脂糖凝胶的结合性,一步层析得到电泳纯的木聚糖酶。酶的最适反应温度为110℃,具有高度耐热性,在70℃保温8h,酶活基本不变,100℃保温5h后,酶活残余40%;在pH6.0～10.0范围内都有较高的酶活性和稳定性。低浓度的异丙醇对酶反应有促进作用,Hg2+对酶反应有强抑制作用。该酶对燕麦木聚糖的Km为0.55mg/ml,Vmax=14.26μmol/min·mg。

The thermo stable xylanase from bioengineering bacterium 1020 was purified to electrophoretic homogeneity by heat treatment and Ni-NTA chromatography. The optimal conditions are： heat treated at 70℃ for 30min, and purification factor 4.9. Only one step chromatography of Ni-NTA makes xylanase up to electrophoretic homogeneity. The optimal temperature is 110℃, and can hold whole activity for 8h at 70℃ and 40% activity at 100℃ after 5h. Among the ranges from pH6.0 to pH 10.0, the enzyme shows high activity. Low concentration of isopropyl alcohol accelerate enzyme activity while Hg2＋ inhibits it.Mcihaelis constant of the enzyme is 0.55mg/ml, and Vmax is 14.26μ mol/min · mg for the oat spelt xylan.