来源于拟轮枝镰孢菌的纤维素酶基因的异源表达、纯化及酶学性质表征OA

To screen for highly active cellulase resources,two cellulases were identified from Fusarium verticillioides(UniProt accession numbers:W7N3I2 and W7MQN2).To further investigate their enzymatic activities,this study employed the Pichia pastoris heterologous ex-pression system to achieve recombinant protein secretion and systematically analyzed the differences in expression levels between the two enzymes.The produced cellulase was isola-ted and purified using a Ni2+affinity chromatography column.Combined with enzyme activity determination,the enzymatic properties were characterized.The experimental results showed that both W7N312 and W7MQN2 could be effectively secreted and expressed in Pichia pasto-ris,and the expression level of W7N312 was significantly higher than that of W7MQN2.The enzyme activity of the fermentation broth from its optimal expression strain(designated as W7N3I2-5)reached 4.10±0.73 U/mL.Enzymatic property analysis indicated that the opti-mal reaction temperature of this recombinant enzyme was 50 ℃,and it exhibited good ther-mal stability within this temperature range.The optimal reaction pH was 5.0,and the en-zyme maintained high catalytic stability in an acidic environment.Metal ion regulation experi-ments revealed that Cu2+had a significant promoting effect on the enzyme activity.The re-sults of kinetic parameter determination showed that the Michaelis constant(Km)of the en-zyme for the substrate was 0.177 g/mL,and the maximum reaction rate(Vmax)was 1.390μmol·mg-1·min-1.This study provides theoretical support for the exploration and applica-tion of new cellulase resources with high activity,which has important reference value for promoting the efficient conversion and utilization of cellulosic biomass resources.