Trichoderma reesei纤维素水解酶的糖苷合成性质  

Glycoside Synthesis Ability of a Cellulase From Trichoderma reesei

作　　者：张玢[1] 余多慰[1] 

机构地区：[1]南京师范大学生命科学学院,江苏南京210097

出　　处：《南京师大学报（自然科学版）》2007年第2期69-76,共8页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：江苏省高校自然科学基金(03KJBI80064)资助项目

摘　　要：通过非变性聚丙烯酰胺凝胶电泳(从(Trichoderma reesei天然纤维素水解酶中分离纯化得到了一种相对分子质量约为65 000的蛋白组分.分别以pNP-Glu、pNP-Gal、oNP-Gal和Avicel作为底物,均检出该组分的水解活性.此外该酶对CMC-Na有可被测出的弱活性.以pNP-Glu为底物,45℃时,其水解酶性质的米氏常数Km=3.04 mg/mL,Vmax=3.77μmol/min.用DNS法和对硝基苯酚法(仅对含有对硝基酚结构的底物pNP-Glu、pNP-Gal和oNP-Gal)分别检测了在180 min反应时段内产物还原糖和对硝基苯酚的含量变化,结果显示在封闭的反应体系中有明显的可逆反应存在,并出现了周期性振荡的特征,有糖苷合成活性的表现,水解产生还原糖和对硝基苯酚的量,呈现出分别为25.43±8.34 min和36.25±2.50 min的含量增减振荡周期.与此同时,为了证实该酶糖苷合成活性的存在,以葡萄糖作为底物与酶反应,得到了7.00±4.83 min的葡萄糖含量周期性波状振荡结果.对底物为pNP-Glu反应15 min后的产物作乙酰化处理GC/MS分析,结果产物中有含二糖结构的产类型,揭示了这个水解酶的糖苷合成的特点.这是首次从Trichoderma reesei纤维素水解酶中分离得到了具有糖苷合成活性的酶.By PAGE, an enzyme was purified from commercial Cellulase, which was extracted by Trichoderma reesei. The molecular mass of the purified enzyme was 65 000 estimated by SDS - PAGE. The purified enzyme had hydrolytic activies correlating with some substrates containing p - Nitrophanyl - β - D - Gluopyranoside （ pNP - Glu）, p - Nitrophanyl - β - D - Galactopyranoside （ pNP - Gal）, o - Nitrophanyl - β - D - Galactopyranoside （ oNP - Gal） and Avicel. The enzyme had weak activity which can be detected using CMC - Na as substrate. Its Km was 3. 04 mg/ml and Vmax was 3. 77 μmol/min, using pNP - Glu as substrate, at 45℃. DNS method and p - nitrophenol method （ only for pNP - GIu,pNP - Gal and oNP - Gal as substrate） were used to analyze changes of quantity of reducing sugar and p - nitrophenol in 180 min. The results confirmed that the reversible reactions characteristic of period oscillation have displayed in the close reaction system. The quantity change period time of reducing suger and p - nitrophenol were 25. 429 ± 8. 343 rain and 36. 25 ± 2.5 min, respectively. Using glucose as substrate, period undulate oscillation was detected also, and the period time of which was 17 ± 4. 830 mln. The acetylated products were detected by GC/MS, using pNP - Glu as substrate, with reaction time 15 min. The result confirmed that the component existed containing disaccharide structure in the all reaction productions. It confirmed that the enzyme had characteristic of glycoside synthesis. It was firstly discovered that the enzyme possessed glycoside synthesis activity of Cellulose hydrolase from Trichoderma reesei.

关 键 词：TRICHODERMA REESEI 纤维素酶 酶活 糖苷合成 可逆反应 

分 类 号：O657.3[理学—分析化学] Q55[理学—化学]