Floriculture Pathology Laboratory, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, Himachal Pradesh, India.
J Basic Microbiol. 2012 Jun;52(3):324-31. doi: 10.1002/jobm.201100145. Epub 2011 Sep 23.
A Trichoderma saturnisporum Hamill isolate GITX-Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek-Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE-Cellulose anion-exchange and Sephacryl S-200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4-methylumbelliferyl -N,N ' diacetyl-β-D-chitobioside (4-Mu-(GluNAc)(2) , the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn(2+) and Zn(2+) stimulated the enzyme activity by 63% and 41%, respectively. The K(m) and V(max) values of the purified enzyme for 4-Mu-(GluNAc)(2) were 338.9 μM ml(-1) and 0.119 μM ml(-1) min(-1) , respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum.
一株对康乃馨维管束枯萎病菌（腐皮镰刀菌，引起康乃馨维管束枯萎病的病原菌）具有强烈几丁质酶活性和抗真菌活性的土星轮枝孢（Hamill）分离株 GITX-Panog（C）被用于从添加真菌菌丝作为碳源的察氏培养基中纯化胞外壳二糖苷酶。该酶使用硫酸铵沉淀、DEAE-纤维素阴离子交换和 Sephacryl S-200 高分辨率凝胶过滤层析进行纯化。通过 SDS-PAGE 确定酶的纯度，估计其分子量为 24 kDa。在含有 4-甲基伞形酮-N,N '二乙酰基-β-D-壳二糖苷（4-Mu-（GluNAc）（2）的天然凝胶测定中，纯化的壳二糖苷酶显示为单条荧光带。该酶对短寡聚天然底物的活性表明，该酶具有外切壳聚糖酶的特性。该酶在 60°C 以下和 pH4.0 时具有活性，在 50°C 时显示出最大稳定性。Mn(2+)和 Zn(2+)分别使酶活性提高了 63%和 41%。该纯化酶对 4-Mu-（GluNAc）（2）的 K(m)和 V(max)值分别为 338.9 μM ml(-1)和 0.119 μM ml(-1) min(-1)。这似乎是首次报道拮抗的土星轮枝孢产生的壳二糖苷酶的特性。
