木霉 -1,3-1,4-葡聚糖酶性质及其cDNA片段克隆

摘要

本研究探讨了里氏木霉GXC的 -1,3-1,4-葡聚糖酶特性，克隆和分析了酶基因片段。结果表明，粗酶液经硫酸铵沉淀、Sephadex G-25、Sephadex G-100和DEAE-Sephadex A-50 柱层析得到纯 -1,3-1,4-葡聚糖酶；经12.5%SDS-PAGE凝胶电泳表明，该酶的分子量为35.21 kD；酶最适反应pH5.0，最适反应温度为60℃；Michaelis-Menten 动力学分析表明， -1,3-1,4-葡聚糖酶的Km 和 Vmax 分别为10.86 mg/mL和 14 286 mol/(min mg)。通过RT- PCR方法扩增并克隆了 -1,3-1,4-葡聚糖酶cDNA片段，测序表明,该片段长度为280 bp；同源性分析显示，该cDNA片段与水解淀粉芽孢杆菌、厌氧真菌 Orpinomyces strain PC-2、枯草芽孢杆菌中的 -1,3-1,4-葡聚糖酶的基因片段有较高的同源性，分别为90%，79%，91%。%-1,3-1,4-glucanase was purified from a solid-state culture of Trichoderma reesei GXC on wheat bran in three steps which comprisedammonium sulfate precipitation,Sephadex G-100 chromatography,and DEAE-Sephadex A-50 chromatography.The molecular mass was determined to be 35.21 kilodaltons by 12.5%sodium dodecyl sulfate polyacrylamide gel electrophoresis.The optimal temperature and pH for purified　-1,3-1,4-glucanase reaction were 50℃ and 6.0,respectively.The Km of the enzyme on 　-glucan was 10.86 mg/mL,and the Vmax on 　-glucan was 14 286 　molof glucose equivalents per mg of the pure enzyme per min.A partial 　-1,3-1,4-glucanase cDNA Fragment from Trichoderma reesei GXC was amplified and cloned by Reverse Transcription-PCR strategy. Sequence analysis showed that the cDNA Fragment was 280 bp,it had 90%,79%,91% identity with the sequences of 　-1,3-1,4-glucanase from Bacillus amyloliquefaciens,Orpinomyces Strain PC-2 and Bacillus subtilis,respectively.