普鲁兰多糖高产菌株Y68葡萄糖基转移酶的纯化及特性研究

Purification and Features of Glucosyltransferase from Pullulan High-Yielding Strain Y68

运用离子交换层析法和凝胶过滤层析法分离纯化普鲁兰多糖高产菌株Y68胞内葡萄糖基转移酶(简称GTF),并以SDS-PAGE、Native-PAGE对蛋白质进行量化、比较及特性鉴定,研究其酶学特性。结果表明短梗霉Y68中GTF被分离纯化,纯化酶在SDS-PAGE凝胶电泳上显示分子量50.8 ku单一条带,而在Native-PAGE凝胶电泳上显示分子量350 ku单一条带。纯化酶的最适酶反应pH和最适酶反应温度分别为6.0和40℃,酶对pH十分敏感,稳定性较差,对温度的稳定性则稍好。GTF为金属酶,Na+和K+对酶有激活作用,Ca2+、Mn2+、Mg2+、Ba2+、Cu2+、Fe2+、Hg2+、Co2+对酶活性有抑制作用,Hg2+对酶活性的抑制作用说明酶活性中心含有二锍键。EDTA、PMSF、SDS和碘乙酸对GTF活性有很大的抑制作用,而二硫苏糖醇(DTT)对酶活性有保护作用。

Intracellular glucosyltransferase（GTF） from pullulan high-yielding strain Y68 was isolated and purified by ion exchange chromatography and gel filtration chromatography,the protein was qualified,compared and characterized to study its enzymological features by SDS-PAGE and Native-PAGE.The results showed that GTF from Aureobasidium pullulans was isolated and purified and its molecular mass 350 ku with single band on the Native-PAGE gel,but 50.8 ku with single band on the SDS-PAGE gel which indicated the enzyme was composed of several subunits.The most suitable reaction pH and temperature of purified GTF was 6.0 and 40 ℃ respectively,and the enzyme was very sensitive to temperature and had poor stability.GTF was an metallic enzyme,Na＋ and K＋ could activate it.However,Ca2＋,Mn2＋,Mg2＋,Ba2＋,Cu2＋,Fe2＋,Hg2＋,and Co2＋ inhibited its activity.The inhibition of Hg2＋ against it indicated that the activity core of the enzyme contained bi-sulphonium bond.EDTA,PMSF,SDS,and iodo-acetidin greatly inhibited GTF＇s activity;but dithiothreitol（DTT） had protective role of its activity.