嗜热毛壳菌多糖单加氧酶的氧化特性及协同作用

以嗜热毛壳菌Chaetomium thermophilum多糖单加氧酶CtPMO1为研究对象,利用薄层层析色谱法(TLC)、高效液相色谱-示差折光检测法(HPLC-RID)和飞行时间质谱(MALDI-TOF-MS)检测CtPMO1的酶活性,并根据定点突变的原理,将CtPMO1第1位的组氨酸(His1)和第166位的谷氨酰胺(Gln166)突变为H1A、Q166A和Q166E,研究两个突变位点是否参与CtPMO1的氧化作用;另外,采用3,5-二硝基水杨酸(DNS)法检测CtPMO1与纤维素酶(EGⅡ、BGLⅠ和CBHⅠ)的协同效应。研究发现CtPMO1在温度为50 ℃、pH为5.0的条件下降解磷酸膨胀纤维素(PASC),其酶解产物中不仅存在纤维二糖至纤维五糖,还存在C1氧化寡糖和C4氧化寡糖;此外,发现突变酶H1A完全丧失了酶活,Q166A丧失了C1和C4氧化活性,而Q166E保留了部分C1氧化活性;通过对CtPMO1与纤维素酶协同作用的探究,发现利用CtPMO1预处理磷酸膨胀纤维素(PASC),分别添加EGⅡ、BGLⅠ和CBHⅠ,使还原糖产量分别提高2.10倍、2.08倍和2.16倍,协同度分别是1.022、0.799和0.875。研究结果表明CtPMO1对底物具有C1和C4氧化的功能,其反应的最适温度为50 ℃、最适pH为5.0;CtPMO1活性中心氨基酸His1和平坦表面氨基酸Gln166均是关键性位点;CtPMO1预处理PASC,使纤维素酶的降解效率发生不同程度的提高。

The oxidation properties and synergism of polysaccharide monooxygenase from Chaetomium thermophilum

The enzyme activity of polysaccharide monooxygenase CtPMO1 from Chaetomium thermophilum was detected by a series of methods like thin-layer chromatography (TLC), high-performance liquid chromatography-refractive index detector (HPLC-RID) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). Two residues (His1 and Gln166) of wild-type CtPMO1 enzyme (WT) were mutated by site- directed mutagenesis to form three mutated enzymes (H1A, Q166A and Q166E), and whether the three mutants of CtPMO1 are involved in catalytic activity of C1 and C4 oxidation was clarified. The synergism of CtPMO1 with cellulase (EGⅡ, BGLⅠand CBHⅠ) was detected by 3,5-dinitrosalicylic acid (DNS). It was found that treatment of phosphoric acid-swollen cellulase (PASC) with CtPMO1 at 50 °C and pH 5.0 mainly produced cello-oligosaccharides with a degree of polymerization (DP) from DP2 to DP5, and C1- and C4-oxidized oligosaccharides in the enzymatic hydrolysis products. It was also found that H1A completely lost its activity, Q166A completely lost activity of C1 and C4 oxidation, and Q166E retained partial activity of C1 oxidation. According to the results of the synergism, the reducing sugar yield increased by 2.10, 2.08 and 2.16 times, respectively, when PASC was catalyzed by CtPMO1 and added EGⅡ, BGLⅠand CBHⅠ. The corresponding synergism degrees were 1.022, 0.799 and 0.875, respectively. We concluded that CtPMO1 could oxidized C1 and C4 position on cellulose, and when CtPMO1 was incubated with insoluble substrate phosphoric acid-swollen cellulase (PASC), the highest activity was exhibited at optimal reaction conditions of 50 °C and pH 5.0. CtPMO1 active center amino acid His1 and flat surface amino acid Gln166 are key sites. After CtPMO1 pretreated PASC, the degradation efficiency of cellulase was improved in different degrees.