真菌发酵液中纤维素酶分离提取工艺研究

Isolation and extraction process of cellulase from fungal fermentation broth

该研究建立了两种分离提取纤维素酶的方法。二级盐析-G-75凝胶色谱法分离提取工艺为:纤维素酶发酵液经0.22μm微滤膜预处理后,依次采用饱和度为20%、80%的(NH_4)_2SO_4对其进行二级盐析,经G-75凝胶色谱层析,冷冻干燥后测得纤维素酶活性为13 675.76 U/g,总酶活收率为91.96%,总纯化倍数为31.41;二级超滤-G-75凝胶色谱法分离提取工艺为:纤维素酶发酵液经0.22μm微滤膜预处理后,以30.165 mL/(min·m^2)的膜通量分别经6×10^(4 )Da和1×10^(4 )Da的超滤膜分离,操作压力分别为0.050 MPa和0.037 MPa,经G-75凝胶色谱层析,冷冻干燥后测得纤维素酶活性为12 769.87 U/g,总酶活收率为92.91%,总纯化倍数为21.23。二级盐析-G75凝胶色谱法适合小规模间歇操作;二级超滤-G-75凝胶色谱法适合大规模连续操作。

Two methods were established to separate and extract cellulase.The separation and extraction process of secondary salting out-G-75 gel chromatography was as follows:after pretreatment with 0.22 pm microfiltration membrane,the cellulase fermentation broth was treated by the secondary salting-out with a saturation of 20%and 80%(NH4)2SO4 and then G-75 gel chromatographic chromatography.After freeze-drying,the cellulase activity was 13 675.76 U/g,the total enzyme activity yield was 91.96%and the total purification multiple was 31.41.The separation and extraction process of secondary ultrafiltration-G-75 gel chromatography was as follows:after pretreatment with 0.22 pm microfiltration membrane,the cellulase fermentation broth was separated by ultrafiltration membrane of 6x104 Da and 1x104 Da with membrane flux 30.165 ml/(min*m2).The operating pressure was 0.050 MPa and 0.037 MPa,respectively.Aftereeze-drying,the cellulase activity was 12 769.87 U/g,the total enzyme activity yield was 92.91%and the total purification multiple was 21.23.The secondary salting out-G-75 gel chromatography was suitable for batch operation on a small scale,and the secondary ultrafiltration-G-75 gel chromatography was suitable for large-scale continuous operation.