Biotechnology and Biomedicine, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.
Sino-Danish Center for Education and Research, Beijing, 100190, China.
Appl Microbiol Biotechnol. 2019 Jan;103(2):777-791. doi: 10.1007/s00253-018-9431-5. Epub 2018 Nov 5.
The early-lineage, aerobic, zoosporic fungi from the Chytridiomycota constitute less than 1% of the described fungi and can use diverse sources of nutrition from plant or animal products. One of the ancestral sources of fungal nutrition could be products following enzymatic degradation of plant material. However, carbohydrate-active enzymes from these ancient fungi have been less studied. A GH11 xylanase (RrXyn11A) (EC 3.2.1.8) and a GH43 xylosidase (RrXyl43A) (EC 3.2.1.37) were identified from an early-lineage aerobic zoosporic fungus, Rhizophlyctis rosea NBRC 105426. Both genes were heterologously expressed in Pichia pastoris and the recombinant enzymes were purified and characterized. The optimal pH for recombinant RrXyn11A and RrXyl43A was pH 7. RrXyn11A had high stability over a wide range of pH (4-8) and temperature (25-70 °C). RrXyn11A also showed high substrate specificity on both azurine-cross-linked (AZCL) arabinoxylan and AZCL xylan. RrXyl43A had β-xylosidase and minor α-L-arabinofuranosidase activity. This enzyme showed low product inhibition and retained 51% activity in the presence of 100 mM xylose. A combination of RrXyn11A and RrXyl43A exhibited significantly higher hydrolytic and polymer degradation capability and xylose release on wheat bran and beechwood xylan compared to treatment with commercial enzymes. This study was the first to heterologously express and characterize the GH11 xylanase (RrXyn11A) and GH43 xylosidase (RrXyl43A) from the ancient fungus, R. rosea. Meanwhile, this study also demonstrated that the enzymes from the ancient fungus R. rosea can be easily handled and heterologously expressed in Pichia, which presents a promising path to a new source of enzymes for biomass degradation.
从接合菌门的早期谱系需氧游动孢子真菌中获取的真菌，其利用的营养物质来自植物或动物产品，不到已描述真菌的 1%。真菌营养的一个古老来源可能是植物材料经酶解后的产物。然而，对于这些古老真菌的碳水化合物活性酶的研究较少。从早期谱系需氧游动孢子真菌罗氏散囊菌 NBRC 105426 中鉴定出一种 GH11 木聚糖酶（RrXyn11A）（EC 3.2.1.8）和一种 GH43 木糖苷酶（RrXyl43A）（EC 3.2.1.37）。这两个基因在巴斯德毕赤酵母中异源表达，并对重组酶进行了纯化和特性分析。重组 RrXyn11A 和 RrXyl43A 的最适 pH 值均为 7。RrXyn11A 在较宽的 pH（4-8）和温度（25-70°C）范围内具有高稳定性。RrXyn11A 对 AZCL 阿拉伯木聚糖和 AZCL 木聚糖均具有较高的底物特异性。RrXyl43A 具有β-木糖苷酶和少量的α-L-阿拉伯呋喃糖苷酶活性。该酶对产物抑制的抑制作用较低，在 100 mM 木糖存在的情况下保留 51%的活性。与使用商业酶处理相比，RrXyn11A 和 RrXyl43A 的组合在麦麸和山毛榉木聚糖上具有更高的水解和聚合物降解能力和释放木糖的能力。本研究首次异源表达和鉴定了古老真菌罗氏散囊菌的 GH11 木聚糖酶（RrXyn11A）和 GH43 木糖苷酶（RrXyl43A）。同时，本研究还表明，古老真菌罗氏散囊菌的酶可以在毕赤酵母中轻松处理和异源表达，这为生物量降解的新酶源提供了一个很有前景的途径。
