Functional Characterization and Low-Resolution Structure of an Endoglucanase Cel45A from the Filamentous Fungus Neurospora crassa OR74A: Thermostable Enzyme with High Activity Toward Lichenan and beta-Glucan

Autor(es)

Seiki Kadowaki, Marco Antonio ^[1] ; Camilo, Cesar Moises ^[1] ; Muniz, Amanda Bernardes ^[1] ; Polikarpov, Igor ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Fis Sao Carlos, BR-13566590 Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: MOLECULAR BIOTECHNOLOGY; v. 57, n. 6, p. 574-588, JUN 2015.
• Citações Web of Science: 7

Resumo

Biomass is the most abundant and short-term renewable natural resource on Earth whose recalcitrance toward enzymatic degradation represents significant challenge for a number of biotechnological applications. The not so abundant but critically necessary class of GH45 endoglucanases constitutes an essential component of tailored industrial enzyme cocktails because they randomly and internally cleave cellulose molecules. Moreover, GH45 glucanases are core constituents of major-brand detergent formulations as well as enzymatic aid components in the cotton processing industry, clipping unwanted cellulosic fibers from cotton (cellulosic)-based tissues. Here we report on a recombinant high-yield Neurospora crassa OR74A NcCel45A production system, a single-band GH45 endoglucanase purification, and a complete enzyme functional characterization. NcCel45A is a bimodular endoglucanase showing maximum activity at pH 6.0 and 60 A degrees C, while most active against lichenan and beta-glucans and lesser active toward filter paper, carboxymethylcellulose, and phosphoric acid-swollen cellulose. Gluco-oligosaccharide degradation fingerprinting experiments suggest cellopentaose as the minimal length substrate and ThermalFluor studies indicate that NcCel45A displays excellent stability at elevated temperatures up to 70 A degrees C and pHs ranging from 5 to 9. Remarkably, we show that NcCel45A is uniquely resistant to a wide-range of organic solvents and small-angle X-ray scattering show a monkey-wrench molecular shape structure in solution, which indicates, unlike to other known cellulases, a non-fully extended conformation, thus conferring solvent protection. These NcCel45A unique enzymatic properties maybe key for specific industrial applications such as cotton fiber processing and detergent formulations. (AU)

• Processo FAPESP: 09/54035-4 - EMU: facility para estudos avançados de materiais nanoestruturados e biossistemas / FAMA
• Beneficiário: Igor Polikarpov
• Linha de fomento: Auxílio à Pesquisa - Programa Equipamentos Multiusuários
• Processo FAPESP: 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis
• Beneficiário: Igor Polikarpov
• Linha de fomento: Auxílio à Pesquisa - Programa BIOEN - Temático
• Processo FAPESP: 10/08680-2 - Aspectos moleculares da degradação de biomassa lignocelulósica: dinâmica de enzimas e nanoarquitetura de paredes celulares de plantas
• Beneficiário: Rodrigo Leandro Silveira
• Linha de fomento: Bolsas no Brasil - Doutorado
• Processo FAPESP: 11/20505-4 - Duas classes importantes de glicosil hidrolases: estudos funcionais e análise estrutural
• Beneficiário: Marco Antonio Seiki Kadowaki
• Linha de fomento: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 09/11536-3 - Estudos bioquímicos e estruturais dos sistemas celulolíticos de Trichoderma reesei e Phanerochaete chrysosporium aplicados à melhoria da eficiência de degradação de biomassa
• Beneficiário: Cesar Moises Camilo
• Linha de fomento: Bolsas no Brasil - Pós-Doutorado