Functional characterization and comparative analysis of two heterologous endoglucanases from diverging subfamilies of glycosyl hydrolase family 45

Author(s)

Berto, Gabriela Leila ^[1] ; Velasco, Josman ^[1] ; Cabos Ribeiro, Caio Tasso ^[1] ; Zanphorlin, Leticia Maria ^[2] ; Domingues, Mariane Noronha ^[2] ; Murakami, Mario Tyago ^[2] ; Polikarpov, Igor ^[3] ; de Oliveira, Leandro Cristante ^[4] ; Ferraz, Andre ^[1] ; Segato, Fernando ^[1]

Affiliation

^[1] Univ Sao Paulo, Escola Engn Lorena, Dept Biotecnol, Estr Municipal Campinho, S-N, BR-12602810 Lorena, SP - Brazil

^[2] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Ciencia & Tecnol Bioetanol, Campinas, SP - Brazil

^[3] Univ Sao Paulo, Inst Fis Sao Carlos, Dept Fis & Ciencias Aplicadas, Sao Carlos, SP - Brazil

^[4] Univ Estadual Paulista UNESP, Inst Biociencias Letras & Ciencias Exatas, Sao Jose Do Rio Preto, SP - Brazil

• Document type: Journal article
• Source: Enzyme and Microbial Technology; v. 120, p. 23-35, JAN 2019.
• Web of Science Citations: 1

Abstract

Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study retorts cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0 degrees C and 80.9 degrees C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70 degrees C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis. (AU)

• FAPESP's process: 14/18714-2 - Enzymatic oxidation of sugarcane bagasse: discovery, characterization and new application of oxidative enzymes active in carbohydrates, applied to the enhancement of a fungal cell factory
• Grantee: Fernando Segato
• Support type: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants
• FAPESP's process: 15/13684-0 - Structural and functional studies of enzymes that participate in complex carbohydrates synthesis and degradation
• Grantee: Igor Polikarpov
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 15/26982-0 - Exploring novel strategies for depolymerization of plant cell-wall polysaccharides: from structure, function and rational design of glycosyl hydrolases to biological implications and potential biotechnological applications
• Grantee: Mário Tyago Murakami
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 14/06923-6 - Sugar cane biomass recalcitrance: basic knowledge related to the cell wall construction, pretreatment and enzymatic digestion, applied for the development of innovative biorefinery models
• Grantee: Andre Luis Ferraz
• Support type: Program for Research on Bioenergy (BIOEN) - Thematic Grants