Abstract：
In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 μmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.
摘要：
在这项工作中,嗜热真菌嗜热酵母的多聚半乳糖醛酸酶（TL-PG1）首次在酵母Komagataellaphafii中异源产生。TL-PG1在AOX1启动子的控制下成功表达,并通过His-标签亲和力顺序纯化。纯化的重组果胶酶在最佳条件（pH6和55℃）下对聚半乳糖醛酸的活性为462.6U/mL，Km和Vmax为2.83mg/mL和0.063μmol/分钟，分别。当用作生物质水解的补充时，TL-PG1表现出与酶促混合物Ctec3的协同作用，以解聚橙色柑橘果肉，释放1.43mg/mL的还原糖。此外,TL-PG1在织物生物精练中表现出效率，显示出在纺织工业中的潜在用途。应用7mg/mL的蛋白质剂量，织物吸水的时间为19.77秒(比对照快10倍)。向处理中加入表面活性剂Triton允许酶剂量减少50%,并且吸水时间减少至6.38秒。总之,这项工作描述了一种来自T.lanuginosus的新型多功能聚半乳糖醛酸酶，具有用于木质纤维素生物质水解和生物精练的潜力。
