Monteiro Valdirene Neves, Ulhoa Cirano José
Universidade Federal de Goiás, Laboratório de Enzimologia, 74.001-940, Goiânia, GO, Brazil.
Curr Microbiol. 2006 Feb;52(2):92-6. doi: 10.1007/s00284-005-0090-2. Epub 2006 Jan 31.
Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of beta-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of beta-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The Km and Vmax values for beta-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL-1 and 0.159 U.min-1, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50 degrees C. Hg2+ inhibited the purified enzyme.
通过常规方法很容易从巴西塞拉多土壤中分离出木霉菌种，其中一些被鉴定为康氏木霉。研究了碳源对特定康氏木霉菌株（ALL 13）培养滤液中β-1,3-葡聚糖酶产生的影响。在所有测试的碳源中均检测到酶活性，并且在非变性聚丙烯酰胺凝胶电泳中仅检测到一条β-1,3-葡聚糖酶条带。该酶通过Sephacryl S-200凝胶过滤和苯基琼脂糖CL 4B色谱法纯化。一个典型的步骤可实现105倍的纯化，产率为13.4%。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，纯化酶的分子量为75 kDa。该酶以内切酶样方式水解海带多糖，形成小寡糖和葡萄糖。以海带多糖为底物时，β-1,3-葡聚糖酶的米氏常数（Km）和最大反应速度（Vmax）值分别为0.148 mg·mL-1和0.159 U·min-1。该酶的最适pH为4.6，在50℃时获得最大活性。汞离子抑制纯化后的酶。
