Osiro Karen O, de Camargo Brenda R, Satomi Rachel, Hamann Pedro Ricardo V, Silva Jéssica Pinheiro, de Sousa Marcelo Valle, Quirino Betania F, Aquino Elaine N, Felix Carlos R, Murad André Melro, Noronha Eliane F
Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
Biochemistry and Protein Chemistry Laboratory, Department of Cellular Biology, University of Brasília, Brasília, DF, Brazil.
Enzyme Microb Technol. 2017 Feb;97:43-54. doi: 10.1016/j.enzmictec.2016.11.002. Epub 2016 Nov 8.
The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren't detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.
本研究的主要目标是对从残留底物结合蛋白（RSBP）以及嗜热栖热菌B8在以微晶纤维素作为碳源生长期间分泌的纤维小体中洗脱的总蛋白进行完整的蛋白质组学表征。第二个目标是评估它们作为酶混合物用于水解农业工业残留物以生产可发酵糖的潜在用途。通过液相色谱-串联质谱（LC-MS/MS）进行蛋白质鉴定表明，RSBP样品除了纤维小体蛋白外，还包含多种蛋白质，包括那些在植物细胞壁降解中作用尚未明确的蛋白质。定义为纯化纤维小体（PC）的RSBP子样品主要由糖苷水解酶组成，这些酶分属于第5、8、9、10和48家族。对PC进行动态光散射（DLS）分析产生了两个蛋白质峰（pi1和pi2），其分子量与先前描述的纤维小体和多纤维小体的分子量一致。在用1.0%吐温处理PC后未检测到这些峰。PC和RSBP在60°至70°C的温度范围和pH 5.0时表现出最大活性。RSBP在50、60和70°C孵育后几乎保留了其所有活性，而PC在50和60°C时表现出显著的热稳定性。RSBP的全纤维素分解活性受到酚类化合物的抑制，而PC则表现出活性增加或抑制程度较小。RSBP和PC能水解甘蔗秸秆、棉花废料和微晶纤维素，释放出多种糖类；然而，以PC作为酶混合物并在50°C下反应十天后进行的测定获得了最高浓度的释放糖。
