Department of Biotechnology, Lorena School of Engineering, University of São Paulo, Lorena, SP, Brazil.
Department of Biotechnology, Lorena School of Engineering, University of São Paulo, Lorena, SP, Brazil.
Biochim Biophys Acta Proteins Proteom. 2023 Jul 1;1871(4):140919. doi: 10.1016/j.bbapap.2023.140919. Epub 2023 May 8.
Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.
木质纤维素生物质是生产生物燃料的有前途的替代品，尽管它具有顽固性。自然界中有能够高效降解生物质的微生物，例如丝状真菌。其中，烟曲霉变种。雪白（AFUMN）具有各种各样的碳水化合物活性酶（CAZymes），特别是水解酶，但基因组中的氧化酶数量较少。为了确认该真菌的酶谱，本研究分析了以甘蔗渣为唯一碳源培养的 AFUMN 的分泌组。不出所料，与氧化活性相比，分泌组显示出水解酶的优势。然而，众所周知，水解酶与氧化蛋白协同作用以有效地降解纤维素聚合物，例如溶细胞多糖单加氧酶（LPMOs）。因此，从嗜热真菌Thermothelomyces thermophilus（TtLPMO9D、TtLPMO9H 和 TtLPMO9O）中选择了三种 LPMO，在 Aspergillus nidulans 中异源表达、纯化，并用于补充 AFUMN 分泌组，以评估它们对甘蔗渣糖化的影响。通过 Design-Expert 软件创建的统计设计进行了糖化分析，使用不同浓度的补充有重组 T. thermophilus LPMOs 的 AFUMN 分泌组，以及抗坏血酸作为氧化酶的还原剂。结果表明，一般来说，添加 TtLPMO9D 和抗坏血酸并没有在本研究中有利于转化过程，而与仅使用 AFUMN 分泌组相比，TtLPMO9O 在蔗渣糖化中具有高度显著的协同作用。
