Universidade Federal do Paraná - UFPR, Curitiba, PR, 81530-980, Brazil.
Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, 70770-917, Brazil.
Sci Rep. 2019 Dec 20;9(1):19580. doi: 10.1038/s41598-019-56070-1.
In the last years, the production of ethanol fuel has started to change with the introduction of second-generation ethanol (2 G Ethanol) in the energy sector. However, in Brazil, the process of obtaining 2 G ethanol did not reach a basic standard to achieve relevant and economically viable results. Several studies have currently been addressed to solve these issues. A critical stage in the bioethanol production is the deployment of efficient and stable enzymes to catalyze the saccharification step into the process of biomass conversion. The present study comprises a screening for genes coding for plant biomass degradation enzymes, followed by cloning a selected gene, addressing its heterologous expression, and characterizing enzymatic activity towards cellulose derived substrates, with a view to second-generation ethanol production. A cDNA database of the Cotton Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae), an insect that feeds on cotton plant biomass, was used as a source of plant biomass degradation enzyme genes. A larva and adult midgut-specific β-1,4-Endoglucanase-coding gene (AgraGH45-1) was cloned and expressed in the yeast Pichia pastoris. Its amino acid sequence, including the two catalytic domains, shares high identity with other Coleoptera Glycosyl Hydrolases from family 45 (GH45). AgraGH45-1 activity was detected in a Carboxymethylcellulose (CMC) and Hydroxyethylcellulose (HEC) degradation assay and the optimal conditions for enzymatic activity was pH 5.0 at 50 °C. When compared to commercial cellulase from Aspergillus niger, Agra GH45-1 was 1.3-fold more efficient to degrade HEC substrate. Together, these results show that AgraGH45-1 is a valid candidate to be engineered and be tested for 2 G ethanol production.
在过去几年中，随着第二代乙醇（2G 乙醇）在能源领域的引入，乙醇燃料的生产开始发生变化。然而，在巴西，获得 2G 乙醇的过程并未达到实现相关且经济可行结果的基本标准。目前已经有几项研究旨在解决这些问题。在生物乙醇生产中，一个关键阶段是部署高效且稳定的酶来催化糖化步骤，将生物质转化为过程。本研究包括筛选编码植物生物质降解酶的基因，然后克隆选定的基因，解决其异源表达，并对纤维素衍生底物的酶活性进行表征，以期用于第二代乙醇生产。棉铃象鼻虫（鞘翅目：象甲科）幼虫和成虫中肠特异性 β-1,4-内切葡聚糖酶编码基因（AgraGH45-1）的 cDNA 数据库被用作植物生物质降解酶基因的来源。该基因在巴斯德毕赤酵母中被克隆和表达。其氨基酸序列，包括两个催化结构域，与其他鞘翅目糖苷水解酶家族 45（GH45）具有高度的同源性。AgraGH45-1 的活性在羧甲基纤维素（CMC）和羟乙基纤维素（HEC）降解测定中被检测到，并且酶活性的最佳条件为 pH5.0 和 50°C。与黑曲霉的商业纤维素酶相比，Agra GH45-1 降解 HEC 底物的效率高 1.3 倍。总之，这些结果表明 AgraGH45-1 是一种有效的候选酶，可用于工程化并用于 2G 乙醇生产的测试。
