高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (4): 704-712.doi: 10.7503/cjcu20140771

• 有机化学 • 上一篇    下一篇

壳寡糖化学修饰对云芝漆酶催化活性和酶学性质的影响

黎春怡1, 黄卓烈2(), 王春晓1   

  1. 1. 茂名职业技术学院, 茂名 525000
    2. 华南农业大学生命科学学院, 广州 510642
  • 收稿日期:2014-08-25 出版日期:2015-04-10 发布日期:2015-03-27
  • 作者简介:联系人简介: 黄卓烈, 男, 教授, 博士生导师, 主要从事酶学与酶工程研究. E-mail: zhuolieh@scau.edu.cn
  • 基金资助:
    国家自然科学基金(批准号: 10074016)资助

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

LI Chunyi1, HUANG Zhuolie2,*(), WANG Chunxiao1   

  1. 1. Maoming Polytechnic, Maoming 525000, China
    2. College of Life Science, South China Agricultural University, Guangzhou 510642, China
  • Received:2014-08-25 Online:2015-04-10 Published:2015-03-27
  • Contact: HUANG Zhuolie E-mail:zhuolieh@scau.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(No.10074016)

摘要:

利用壳寡糖(COS)对纯化后的云芝漆酶(EC 1.10.3.2)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, 100 mg NaIO4, 20 mg COS. 在此条件下得到的修饰酶(COS-TvL)的活性(456.00 U/mg)为天然酶(TvL)活性(314.39 U/mg)的145.04%. 十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)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=9.929 mmol/L, vmax=714.29 mmol/(mg·min); 而COS-TvL的Km=8.989 mmol/L, vmax = 1250 mmol/(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.

关键词: 漆酶, 化学修饰, 酶活性, 壳寡糖

Abstract:

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, TvL), the enzyme was purified by DEAE 52 weak anion exchange resin chromatography. The pure enzyme was activated by NaIO4. 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 ℃, pH=4.0, 100 mg NaIO4, 20 mg COS. Under these conditions the activity of modified enzyme(COS-TvL, 456.00 U/mg) was 145.04% of that of unmodified enzyme(TvL, 314.39 U/mg). The results of SDS-PAGE experiments indicated that the relative molecular weights of 3 polypeptides of TvL were 58100, 45200 and 21600, respectively. But the relative molecular weights of 3 polypeptides of COS-TvL were 62900, 53300 and 39700, respectively. This meant that the modification experiments were successful. The amino modification rates changed from 0 of TvL to 30.7% of COS-TvL. The optimal pH values of TvL and COS-TvL were 4.0 and 3.5—4.0, respectively. The optimal temperatures of TvL and COS-TvL were 40 and 45 ℃, respectively. The results of kinetic study indicated that Km and vmax of TvL were 9.929 mmol/L and 714.29 mmol/(mg·min). But the Km and vmax of COS-TvL were 8.989 mmol/L and 1250 mmol/(mg·min), respectively. The infrared spectra, the ultraviolet absorption spectra, the ultraviolet differential spectra, and fluorescence emission spectra of TvL and COS-TvL were also studied and analyzed.

Key words: Laccase, Chemical modification, Enzyme activity, Chitooligosaccharide

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