裂解性多糖单加氧酶HcLPMO与纤维素酶协同降解纤维素

Synergistic degradation of cellulose by lytic polysaccharides monooxygenase HcLPMO and cellulase

摘要:

[目的] 为研究HcLPMO的活性测定方法及其与纤维素酶的协同降解特性。[方法] 利用大肠杆菌表达系统进行HcLPMO异源表达，研究以Amplex^TM Ultra Red为荧光底物的LPMOs活性检测条件；研究HcLPMO与纤维素酶最优配比协同降解微晶纤维素及其他多种生物质底物的能力。[结果] 表达条件确定最适装液量为20%，最适诱导温度为20℃。活性测定研究结果表明HcLPMO需先与铜离子结合才具有活性，电子供体抗坏血酸钠（ASC）最适浓度为10^–4 mol/L，并发现Amplex^TM Ultra Red浓度以及辣根过氧化物酶浓度对酶活的检测影响较小。HcLPMO与纤维素酶协同降解微晶纤维素研究确定HcLPMO与纤维素酶最优配比为2：3，葡萄糖产量相较纤维素酶单独作用提高了99.48%。此外，针对多种生物质底物，发现该酶与纤维素酶的复配体系对汽爆玉米秸秆和微晶纤维素的协同降解效果较好，相较于单独用纤维素水解酶，葡萄糖产量分别提高了63.81%和59.43%，而对碱处理玉米芯和木薯渣降解效果次之，葡萄糖产量仅分别提高35.41%和11.06%。[结论] HcLPMO与纤维素酶复配能够有效提高酶法降解纤维素效率；而底物前处理如蒸汽爆破或碱处理对于HcLPMO与纤维素酶协同降解木质纤维素影响较大。

关键词:裂解性多糖单加氧酶;纤维素酶;纤维素;活性检测;协同降解

Abstract:

[Objective] To study the synergistic degradation of cellulose by lytic polysaccharide monooxygenase HcLPMO and cellulase. [Methods] We used E. coli for heterologous expression of HcLPMO and studied the influences of various conditions on the activity detection of LPMOs with Amplex^TM Ultra Red as fluorogenic substrate. Subsequently, we studied the synergistic degradation of avicel and other biomass substrates with different proportion of HcLPMO and cellulase.[Results] We found the optimal filling liquid volume was 20% and the optimal induction temperature was 20℃ for HcLPMO expression. The results of activity determination show that HcLPMO had activity only when it was combined with copper ions. The optimal concentration of sodium ascorbate was 10^-4 mol/L. We also found that the concentration of Amplex^TM Ultra Red and horseradish peroxidase had little effect on the detection of enzyme activity. For the synergistic degradation of avicel by HcLPMO and cellulase, we found the optimal ratio of HcLPMO to cellulase was 2:3, and the yield of glucose increased by 99.48% compared with cellulase alone. In addition, for a variety of biomass substrates, HcLPMO and cellulase had better synergistic degradation efficiency on steam exploded straw and microcrystalline cellulose from which the yield of glucose increased by 63.81% and 59.43%, respectively, compared with using cellulase alone. For alkali treated corn cob and cassava residue, the yield of glucose only increased by 35.41% and 11.06%, respectively.[Conclusion] The appropriate ratio of HcLPMO and cellulase can effectively improve the efficiency of enzymatic degradation of cellulose, and substrate pretreatment is very helpful for the synergistic degradation of lignocellulose by HcLPMO and cellulase.

Key words:lytic polysaccharide monooxygenases;cellulase;cellulose;activity detection;cooperative degradation