壳寡糖化学修饰对云芝漆酶催化活性和酶学性质的影响

Effects of Chemical Modification with Chitooligosaccharides on Catalytic Activity and Enzymological Properties of Laccase from Trametes versicolor

利用壳寡糖（COS）对纯化后的云芝漆酶（EC1.10.3.2）进行化学修饰以改善其活性和稳定性.最优化学修饰条件：温度5℃,pH=4.0,100mgNaIO4,20mgCOS.在此条件下得到的修饰酶（COS-TvL）的活性（456.00U/mg）为天然酶（TvL）活性（314.39U/mg）的145.04%.十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDSPAGE）结果表明,TvL的3条肽链相对分子质量为58100,45200和21600,而COS-TvL的3条肽链相对分子质量为62900,53300和39700,说明酶分子修饰成功,氨基修饰率为30.7%.TvL的最适pH值在4.0附近,而COS-TvL的最适pH值在3.5~4.0之间.TvL的最适温度为40℃,而COS-TvL的最适温度为45℃.动力学研究结果显示,TvL的Km=9.929mmol/L,vmax=714.29mmol/（mg·min）;而COS-TvL的Km=8.989mmol/L,vmax=1250mmol/（mg·min）.通过红外光谱、紫外吸收光谱、紫外差示光谱和荧光发射光谱对TvL和COS-TvL进行了结构表征.

Chemical modification is an important pathway to transform the catalytic activity and enzymological properties of enzymes. In order to improve the catalytic activity and enzymological stability of laccase from Trametes versicolor（ EC 1. 10. 3. 2,Tv L）,the enzyme was purified by DEAE 52 weak anion exchange resin chromatography. The pure enzyme was activated by Na IO4. The activated enzyme was chemically modified with chitooligosaccharides（ COS,average relative molecular weight 5000）. The optimal modification conditions by single factor and orthogonal experiments were： temperature 5 ℃,p H = 4. 0,100 mg Na IO4,20 mg COS. Under these conditions the activity of modified enzyme（ COS-Tv L,456. 00 U / mg） was 145. 04% of that of unmodified enzyme（ Tv L,314. 39 U / mg）. The results of SDS-PAGE experiments indicated that the relative molecular weights of 3 polypeptides of Tv L were 58100,45200 and 21600,respectively. But the relative molecular weights of 3 polypeptides of COS-Tv L were 62900,53300 and 39700,respectively. This meant that the modification experiments were successful. The amino modification rates changed from 0 of Tv L to 30. 7% of COS-Tv L. The optimal p H values of Tv L and COS-Tv L were 4. 0 and 3. 5—4. 0,respectively. The optimal temperatures of Tv L and COS-Tv L were 40 and 45 ℃,respectively. The results of kinetic study indicated that Kmand vmaxof Tv L were 9. 929 mmol / L and 714. 29 mmol /（ mg·min）. But the Kmand vmaxof COS-Tv L were8. 989 mmol / L and 1250 mmol /（ mg · min）,respectively. The infrared spectra,the ultraviolet absorption spectra,the ultraviolet differential spectra,and fluorescence emission spectra of Tv L and COS-Tv L were also studied and analyzed.