纤维素降解真菌A25-2的分离、鉴定及其产纤维素酶的酶学特性

本研究以Avicel-刚果红选择培养基为初筛培养基,从云南哀牢山国家级自然保护区和广西猫儿山国家级自然保护区的土壤样品中分离筛选得到4200株真菌,从中筛选出透明圈与菌落直径比较大、透明程度较为清晰的12个菌株。通过液体培养发酵,测定其上清液中的羧甲基纤维素酶活力、滤纸酶活力和Avicel酶活力,最终筛选出一株产该三种酶且其活力均最高的真菌菌株A25-2。通过对菌株A25-2形态学观察和其内转录间隔区（internal transcribed spacer,ITS）序列同源性比对分析,将菌株A25-2鉴定为哈茨木霉（Hypocrea lixii）。酶活测定结果表明菌株A25-2产纤维素酶的酶活力较高,在最适作用pH4.5和最适作用温度55℃下,其羧甲基纤维素酶活力为2.26IU/mL,滤纸酶活力为0.58IU/mL,Avicel酶活力为0.39IU/mL。薄层层析实验表明A25-2具有完整的纤维素酶系统。因此,真菌A25-2可作为饲料加工等生产和纤维素酶相关研究的备选菌株。

Isolation and Identification of Cellulose-Degrading Fungal Strain A25-2 and Enzymology Characterization of Its Produced Cellulases

Using agar medium plate containing Avicel and congo red as primary screening medium,4 200 fungal strains were isolated from soil samples collected from AiLao mountain National Nature Reserve of Yunnan and MaoEr mountain National Nature Reserve of Guangxi Zhuang Autonomous Region.Twelve strains which showed larger and clearer hydrolyzing zone around the colony were selected for further screening.After flask liquid fermentation,enzyme activities of supernatant of the 12 strains were measured towards substrates carboxymethylcellulose（CMC）,filter paper and Avicel,respectively.A fungal stain A25-2 showing the highest activity towards the three substrates was selected for further study.The strain A25-2 was identified as Hypocrea lixii based on its morphological characteristics and the internal transcribed spacer（ITS） sequence analysis.Under optimum pH 4.5 and optimum temperature 55℃,the crude cellulase activities degrading carboxymethylcellulose,filter paper and Avicel were 2.26 IU/mL,0.58 IU/mL and 0.39 IU/mL,respectively.Thin layer chromatography demonstrated that A25-2 produces a complete cellulase system.In this work,we would like to recommend that fungal strain A25-2 as a candidate for feed production and for further studying cellulase.