嗜热毁丝菌裂解性多糖单加氧酶TtLPMO9I的酶学性质及其功能研究

Characterization and Functional Analysis of Lytic Polysaccharide Monooxygenase TtLPMO9I from Thermothelomyces thermophilus

【目的】为挖掘新型裂解性多糖单加氧酶(LPMO)酶资源,探究LPMO在辅助降解纤维素过程中起到的重要作用。【方法】从Thermothelomyces thermophilus基因组中克隆表达了一个新型LPMO酶TtLPMO9I,系统地分析了其序列及结构的进化特征;采用DNS法表征了TtLPMO9I的酶学性质;在反应体系中添加不同浓度的抗坏血酸探究外部电子供体对TtLPMO9I活性的影响;以玉米秸秆和微晶纤维素为底物,通过检测还原糖的生成量计算获得TtLPMO9I与纤维素酶的协同作用效果。【结果】TtLPMO9I在60℃,pH 5.0时表现出最佳酶活力。在60℃孵育12 h后,仍能剩余54%的活性。经pH 6.0-8.0处理12 h后,酶活无损失。添加外部电子供体抗坏血酸使TtLPMO9I的活性提高至184%。在玉米秸秆和微晶纤维素降解过程中,TtLPMO9I与纤维素酶表现出良好的协同作用效果。将50-200μg的TtLPMO9I添加至降解体系中,还原糖产量分别提高了34%-142%和6%-46%。【结论】TtLPMO9I不仅具有良好的温度稳定性和pH稳定性,在木质纤维素的降解过程中也具有突出的作用效果,为工业生产应用提供了潜在的优质酶资源。

【Objective】To explore a novel lytic polysaccharide monooxygenase(LPMO)enzyme and to elucidate its crucial role in cellulose degradation.【Method】A new LPMO enzyme TtLPMO9I was cloned from Thermothelomyces thermophilus genome,and its sequence and structure were systematically analyzed to determine its evolutionary characteristics.The enzymatic properties of TtLPMO9I were characterized using the DNS method;the impact of different concentrations of ascorbic acid on the activity of TtLPMO9I was investigated by adding external electron donors to the reaction system.By detecting the production of reducing sugars,the synergistic effect between TtLPMO9I and cellulase was calculated when pretreated corn straw and Avicel were used as substrates.【Result】TtLPMO9I presented the highest activity at 60℃and pH 5.0.After 12 h of incubation at 60℃,approximately 54%of its activity remained.TtLPMO9I still maintained initial enzyme activity at pH 6.0-8.0 for up to 12 h.Adding an external electron donor,ascorbic acid,to the reaction system,the activity of TtLPMO9I increased to 184%.In addition,TtLPMO9I has shown promising synergistic effects with cellulase in the degradation of pretreated corn straw and Avicel.Adding 50-200μg of TtLPMO9I into the degradation system increased reducing sugar yields by 34%-142%and 6%-46%respectively.【Conclusion】TtLPMO9I not only has excellent thermal and pH stability,but also has outstanding performance in degrading lignocellulose,providing potential high-quality enzyme resources for industrial production.