Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced beta-glucanase activity monitored by electron paramagnetic resonance

Author(s)

Fonseca-Maldonado, Raquel ^{[1, 2]} ; Meleiro, Luana P. ^[1] ; Mendes, Luis F. S. ^[3] ; Alves, Luana F. ^[4] ; Carli, Sibeli ^[1] ; Morero, Lucas D. ^[3] ; Basso, Luis G. M. ^[3] ; Costa-Filho, Antonio J. ^[3] ; Ward, Richard J. ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Ave Bandeirantes 3900, Ribeirao Preto, SP - Brazil

^[2] Inst Fed Educ Ciencia & Tecnol Sao Paulo IFSP, Dept Gestao, Campus Jacarei, Jacarei, SP - Brazil

^[3] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Fis, Ribeirao Preto, SP - Brazil

^[4] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim, Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: BIOTECHNOLOGY FOR BIOFUELS; v. 10, NOV 14 2017.
• Web of Science Citations: 4

Abstract

Background: The Bacillus subtilis endo-beta-1,4-glucanase (BsCel5A) hydrolyzes beta-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds beta-1,4-linked glucan. Methods: Here we investigate the BsCel5A beta-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having beta-1,3-1,4 glucan affinity. Results: The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley beta-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with beta-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with beta-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan-or lignin-free lignocellulose was correlated with a 4.5-to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the beta-1,3-1,4 glucan. Conclusions: These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates. (AU)

• FAPESP's process: 10/18850-2 - Identification, characterization and engineering of plant cell wall degrading enzymes
• Grantee: Richard John Ward
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 14/50884-5 - INCT 2014: National Institute of Science and Technology of Bioethanol
• Grantee: Marcos Silveira Buckeridge
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 15/18149-6 - Study of conformational and functional changes of the catalytic domain and of the CBM (Carbohydrate Binding Module) of a GH5 endoglucanase during catalysis
• Grantee: Raquel Fonseca Maldonado
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/18390-5 - Electron magnetic resonance in molecular biophysics: new and old looks to new and old problems
• Grantee: Antonio José da Costa Filho
• Support Opportunities: Regular Research Grants