1株烟曲霉产耐热纤维素酶发酵条件优化及酶学性质研究

Optimization of Fermentation Conditions and Enzymatic Characteristics of the Thermophilic Cellulase from Aspergillus fumigatus

【目的】优化1株产耐热纤维素酶烟曲霉的最适产酶条件,分析所产酶液对秸秆的纤维降解效果,为其在纤维素物质生物降解中的应用提供依据。【方法】利用3,5-二硝基水杨酸(DNS)法测定前期研究分离的烟曲霉WL002产纤维素酶的活性,并分析其酶学性质;采用单次单因子法和正交试验对其产酶的培养条件和发酵培养基进行优化;利用体外发酵法分析粗酶对不同粗饲料的纤维降解效果。【结果】酶学性质分析表明,菌株WL002所产纤维素酶的最适反应pH为7.0,pH 6.0~8.0保存0.5和1 h后,残余酶活为74%~95%和56%~90%;最适反应温度为60℃,70℃保温0.5~1.5 h后,残余酶活为70%~80%。产酶条件优化结果显示,孢子悬液以7%比例接种至含1.0%蛋白胨、2.0%小麦秸秆粉、pH为7.0的发酵培养基,50℃培养48 h,菌株WL002所产粗酶的内切葡聚糖酶活性最高,达3.11 U/mL。利用菌株WL002所产粗酶处理小麦秸秆和玉米秸秆60 h,其粗纤维降解率分别为6.1%和4.7%。【结论】烟曲霉WL002菌株所产的纤维素酶具有较宽泛的pH适应性和较强的热耐受性,对饲料粗纤维具有一定的降解能力。本研究初步获得了该菌的最优发酵条件,为其后续开发应用奠定了基础。

【Objective】The purpose of this experiment was to optimize the optimum enzyme production conditions of a strain of Aspergillus fumigatus producing heat-resistant cellulase,analyze the fiber degradation effect of the enzyme solution on straw,and provide the basis for its application in the biodegradation of cellulose.【Method】3,5-dinitrosalicylic acid(DNS)method was used to determine the activities of cellulase produced by Aspergillus fumigatus WL002 from the previous study,and its enzymatic properties were analyzed.The culture conditions and fermentation medium were optimized by the one factor at a time strategy combined with orthogonal test.The crude fiber degradation effects of the crude enzyme on roughage were analyzed by in vitro fermentation.【Result】Enzymatic characterization analysis revealed that the optimum reaction pH of cellulase produced by WL002 was 7.0,and the residual enzyme activity was 74%-95%and 56%-90%after preservation at pH 6.0-8.0 for 0.5 and 1 h,respectively.The optimal reaction temperature was 60℃,and after holding at 70℃for 0.5-1.5 h,the residual enzyme activity was 70%-80%.The maximum cellulase production by strain WL002 were achieved with peptone(1.0%)and wheat straw(2.0%)as the best carbon and nitrogen sources,respectively,at pH 7.0,7%initial inoculum,the incubation time of 48 h and a culture temperature of 50℃,the average cellulase activity reached 3.11 U/mL.The degradation rates of wheat straw and corn straw were 6.1%and 4.7%,respectively,when treated with the crude enzyme produced by strain WL002 for 60 h.【Conclusion】The cellulase produced by Aspergillus fumigatus WL002 had a wide pH range,strong thermostability and fiber degradability.The proper conditions for producing cellulase of strain WL002 were achieved,which laid a foundation for its follow-up exploitation and application.