嗜热革节孢多糖单加氧酶氧化方式

Oxidation mode of polysaccharide monooxygenases from Scytalidium thermophilum

多糖单加氧酶(polysaccharidemonooxygenase,PMO)是一种铜离子依赖的氧化酶,属于辅助活性酶类第九家族(auxiliary activity 9,AA9),在存在电子供体维生素C(vitamin C,Vc)的情况下,可以氧化裂解纤维素的多糖链,显著提高纤维素的酶解效率。本文克隆了嗜热革节孢Scytalidium thermophilumAA9家族的一个编码基因pmo7651,并在毕赤酵母GS115进行诱导表达,通过His标签获得了重组蛋白PMO7651-His。以磷酸膨胀纤维素(PASC)为底物进行酶促反应,薄层层析法(TLC)结果显示PMO7651酶解产物主要为纤维二糖至纤维五糖;飞行时间质谱法(MALDI-TOF-MS)和溴氧化法确定PMO7651具有C1、C4、C6位的氧化活性;底物结合平面的3个芳香族氨基酸位点突变对酶的活性具有不同程度的影响;在PMO7651帮助下,纤维素酶的降解效率均具有不同程度的提高。

Polysaccharide monooxygenase(PMO) is a copper ion-dependent oxidase and belongs to the auxiliary activity family 9(AA9). PMO can oxidize and cleave the polysaccharide chains of cellulose with vitamin C, and significantly increase hydrolysis efficiency. In this study, a gene of auxiliary activity family 9(AA9) from Scytalidium thermophilum named pmo7651 was cloned and the enzyme PMO7651 was successfully expressed in Pichia pastoris. The recombinant protein PMO7651 was obtained by nickel affinity chromatography. PMO7651 with substrate phosphoric acid-swollen cellulose(PASC) was incubated in pH 5.0,50℃ for 48 hours. Thin layer chromatography(TLC) analysis showed that the hydrolysis products were mainly cello-oligosaccharides with a degree of polymerization(DP) from DP2 to DP5. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and bromine oxidation analyses showed that PMO7651 possessed the function of C1, C4 and C6 oxidation to cellulose. The mutation of the three aromatic amino acid sites exhibited different hydrolysis activities and final products.In addition, the enzymatic hydrolysis efficiency was improved at different degrees with the enzyme of PMO7651.