耐热嗜酸β-甘露聚糖酶TaMan5A在毕赤酵母中高效表达及酶学性质研究

Efficient expression and enzymatic characterization of thermostable and acidophilicβ-mannanase TaMan5A in Komagataella phaffii

甘露聚糖酶是一类将甘露聚糖降解为短链甘露寡糖及甘露糖的水解酶。为了开发耐酸耐高温的工程酶,从棘孢木霉(Trichodema asperellum)ND-1中克隆甘露聚糖酶基因TaMan5A并在毕赤酵母(Komagataella phaffii)X-33中成功表达。结果显示,该酶的蛋白质分子质量约为67 kDa。最适pH值和温度分别为4.0和65℃,在pH 2.0~6.0具有较强的稳定性,孵育1 h后仍具有80%的活性,且在20~65℃时,残留酶活力均能达到90%以上。1 mmol/L Co^(2+)、脲和5 mmol/L SDS、Zn^(2+)、Fe^(2+)、Mn^(2+)、Ni^(2+)对TaMan5A的活力都有不同程度的激活作用。由于玉米秸秆富含纤维素与半纤维素,因此进一步确定了TaMan5A水解秸秆的最佳组合条件(玉米秸秆用量0.5 g,TaMan5A用量0.5 g,pH 4.0,水解2 d),并得到玉米秸秆用量和TaMan5A用量对玉米秸秆降解效率影响最大。该研究首次从棘孢木霉ND-1中克隆出TaMan5A基因,实验结果表明,TaMan5A是一种嗜酸、耐高温、热稳定性强的酶,具有较大的应用前景,值得进一步研究。

Mannanases are enzymes capable of degrading mannans into shorter mannooligosaccharides and mannose.In pursuit of developing acid-resistant and thermotolerant engineering enzymes,this experiment successfully cloned the mannanase gene TaMan5A from Trichoderma asperellum ND-1 and expressed it in Komagataella phaffii X-33.The recombinant TaMan5A exhibited a molecular weight of 67 kDa approximately.The optimum pH and temperature were determined to be 4.0 and 65℃,respectively.TaMan5A displayed robust stability within the pH range of 2.0 to 6.0,retaining 80%activity after incubation for 1 h.Furthermore,the TaMan5A retained over 90%activity between 20℃and 65℃.1 mmol/L Co^(2+),urea,and 5 mmol/L SDS,Zn^(2+),Fe^(2+),Mn^(2+),and Ni^(2+)all exhibited varying degrees of activation on the activity of TaMan5A.Due to the high content of cellulose and hemicellulose in corn stalks,the optimal conditions for the enzymatic hydrolysis of mannooligosaccharides in the stalks were further determined:corn stalk 0.5 g,TaMan5A 0.5 g,pH 4.0,and 2 d.It was found that the dosage of corn stalks and TaMan5A had the greatest impact on the degradation efficiency of corn stalks.Notably,the study innovatively cloned the TaMan5A gene from T.asperellum ND-1 for the first time.Our findings indicate that TaMan5A is an acidophilic enzyme with high thermal stability,offering promising applications and warranting further investigation.