一种几丁质裂解性多糖单加氧酶的活性评价及稳定性研究

The activity and stability analyses of chitin-activity lytic polysaccharide monooxygenase

摘要:

【目的】裂解性多糖单加氧酶（LPMO）是一类铜离子依赖型的单加氧酶，能够通过氧化的方式断裂糖苷键，进而显著提高多糖的降解效率，受到广泛的关注。但是LPMO单加氧酶的性质使其容易被自身氧化而失活，且底物的聚合性质和释放产物的多样性使得对LPMO催化过程活性的评估变得十分困难。【方法】本研究以2，6-二甲氧基苯酚（2，6-DMP）和H₂O₂为底物，建立了测定几丁质裂解性多糖单加氧酶（BtLPMO10A）活性的评价体系，并研究该酶在降解几丁质底物过程中的稳定性。【结果】研究发现，在测定BtLPMO10A活性的过程中，较高的酶浓度，过氧化氢浓度和2，6-DMP浓度均使得反应过程脱离了线性范围，而抗坏血酸的加入能够提高灵敏度，但是对活性测定过程有较大影响。BtLPMO10A对2，6-DMP和H₂O₂的K_m分别为0.53 mmol/L和5.31 mmol/L，亲和性高于纤维素裂解活性的NcLPMO9C。BtLPMO10A在还原剂抗坏血酸存在的条件下容易失活，但底物几丁质的加入能够一定程度上稳定LPMO的活性，但是其在降解几丁质过程中活性依然会下降。【结论】本研究以2，6-二甲氧基苯酚为底物检测BtLPMO10A对底物的亲和力，并研究BtLPMO10A的稳定性，为深入评价具有几丁质氧化裂解活性的LPMO的稳定性及其在几丁质降解过程中的应用提供了重要信息。

Abstract:

[Objective] Lytic polysaccharide monooxygenase (LPMO) is a recently discovered copper ion-dependent oxidase, which can break glycosidic bonds by oxidation, thus significantly improving the efficiency of polysaccharides degradation. However, it is easy to be inactivated and difficult to evaluate the activity of LPMO due to the properties of its substrates and the diversity of its released products.[Methods] In this study, we established a spectrophotometric activity assay to detect the chitin-active LPMO (BtLPMO10A) using 2,6-dimethoxyphenol (2,6-DMP) and H₂O₂ as substrates, and to evaluate the stability of LPMO during chitin degradation.[Results] The results suggested that high concentration of enzyme, 2,6-DMP or H₂O₂ would deviate the reaction from linear range. The K_m of BtLPMO10A toward 2,6-DMP and H₂O₂ was determined to be 0.53 mmol/L and 5.31 mmol/L respectively. It suggested BtLPMO10A possessed a higher affinity toward 2,6-DMP and H₂O₂ than NcLPMO9C. BtLPMO10A was easy to be inactivated in the presence of reducing agent ascorbic acid. The substrate chitin could stabilize the enzyme, but the activity still decreased during the degradation of chitin.[Conclusion] This work evaluated the factors that impacted on the assay for detecting the activity of BtLPMO10A using 2,6-DMP as substrate, and estimated the stability of BtLPMO10A during chitin degradation. It will provide important information for the investigation of chitin-active LPMOs.