Fusarium oxysporim M1菌株的rDNA ITS鉴定及其纤维素酶活性及纯化研究

以1株分离于北大仓白酒大曲的产纤维素酶真菌M1为材料,对其进行了形态及分子生物学鉴定;纯化并研究了其纤维素酶的酶学性质。以真菌ITS1/ITS4通用引物,扩增真菌M1的rDNA ITS序列,再与GenBank中其他菌株rDNA ITS序列进行比对,使用Mega5.0软件,采用最大似然法进行聚类分析。结果显示,该真菌同已经报道的Fusarium oxysporim strain Bt3聚为一类,一致率达99%,与形态学方法鉴定一致,命名为Fusarium oxysporum M1。该菌具有很高纤维素酶活力,FPA和CMCA分别高达16.84 IU/mL和35.31 IU/mL。经过发酵条件优化酶活性进一步提高。经硫酸铵分级分离、疏水和离子交换层析,纯化了该菌纤维素酶,纯化倍数高达17.97倍,得率为3.676%,SDS-PAGE分析表明,该纤维素酶分子量达60 k Da。本研究为进一步研究该酶高效催化机理及实际应用提供参考。

rDNA ITS Identification, Its Cellulase Activity and Purification of Fusarium oxysporum Strain M1

In this paper, a cellulase producing strain M1 isolated from Beidacang white spirit was taken as a material and carried out morphological and bio molecular characterization, purified and studied on rDNA ITS sequences of the strain M1 genome amplified using the eukaryotic universal primer pair ITS1/ITS4 were compared with similar sequences of other stains obtained in GenBank. Maximum likelihood tree was made using Mage5.0 analysis software. The Blast results of rDNA ITS sequences showed that strain M1 can be gathered into a class of Fusarium oxysporum strain Bt3 reported in GenBank. It belonged to the genus Fusarium, called as Fusarium oxysporum M1. FPA and CMCA from crude cellulase liquid reached 16.84 IU/mL and 35.31 IU/mL respectively. The cellulase activity further improved via the optimization of culture conditions. The crude cellulase was purified by using fractionation deposition of (NH₄)₂SO₄, hydrophobic interaction chromatography and ion exchange chromatography. The purification fold reached to 17.97 and the yield of enzyme was 3.676%. The enzyme molecular was determined about 60 KDa by the SDS-PAGE. It provided the basis for the further study on structure of the cellulose.