Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme

Author(s)

Pellegrini, Vanessa O. A. ^[1] ; Serpa, Viviane Isabel ^[1] ; Godoy, Andre S. ^[1] ; Camilo, Cesar M. ^[1] ; Bernardes, Amanda ^[1] ; Rezende, Camila A. ^[2] ; Pereira Junior, Nei ^[3] ; Cairo, Joao Paulo L. Franco ^[4] ; Squina, Fabio M. ^[4] ; Polikarpov, Igor ^[1]

Affiliation

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

^[2] Univ Estadual Campinas, Inst Quim, BR-13083970 Campinas, SP - Brazil

^[3] Univ Fed Rio de Janeiro, Dept Engn Bioquim, Escola Quim, BR-21949900 Rio De Janeiro, RJ - Brazil

^[4] CNPEM, Lab Nacl Ciencia & Tecnol Bioetanol CTB, BR-13083970 Campinas, SP - Brazil

• Document type: Journal article
• Source: Applied Microbiology and Biotechnology; v. 99, n. 22, p. 9591-9604, NOV 2015.
• Web of Science Citations: 11

Abstract

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 A degrees C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for beta-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl beta-d-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions. (AU)

• FAPESP's process: 09/52840-7 - Center of Biological and Industrial Processes for Biofuels - CeProBIO
• Grantee: Igor Polikarpov
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 10/18773-8 - Molecular cloning, expression, purification and structural characterization of endoglucanase from Trichoderma harzianum aiming the development of enzymatic blends for production of lignocellulosic ethanol
• Grantee: Vanessa de Oliveira Arnoldi Pellegrini
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis
• Grantee: Igor Polikarpov
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 11/20977-3 - Investigation of plant biomass conversion in termite Coptotermes gestroi complementary to glycosyl hydrolases aiming at bioproducts formation derived from lignocellulosic biomass.
• Grantee: João Paulo Lourenço Franco Cairo
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 09/05328-9 - Expression, purification and cristalization studies of the ligand-binding domain of nuclear receptors PPAR-alpha e PPAR-gamma
• Grantee: Andre Schutzer de Godoy
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 11/05712-3 - Structural and functional studies of beta-galactosidases from Xanthomonas campestris
• Grantee: Andre Schutzer de Godoy
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)