陕西科技大学学报2026，Vol.44Issue(3)：71-79,9.
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
Heterologous expression,purification,and enzymatic characterization of cellulase gene from Fusarium verticillioides
摘要
Abstract
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.关键词
拟轮枝镰孢菌/纤维素酶/异源表达/分离纯化/酶学性质Key words
Fusarium verticillioides/cellulase/heterologous expression/isolation and purifi-cation/enzymatic characterization分类
轻工纺织引用本文复制引用
靖云阁,李萌,杨金初,黄珍珍,董盼盼,徐永明,杨宗灿,冯颖杰,魏明杰,王春,许亚龙..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[J].陕西科技大学学报,2026,44(3):71-79,9.基金项目
国家自然科学基金项目(31401810) （31401810）
河南省科技攻关计划项目(212102110257) （212102110257）
河南中烟工业有限责任公司科技项目(AW2023008) （AW2023008）
中国烟草总公司重点研发项目(110202402035,110202202038) （110202402035,110202202038）
