Quaternary states of CBM64 and its interaction with Polisaccharide substrates

• Grant number: 16/23477-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: March 01, 2017
• End date: June 30, 2017
• Field of knowledge: Physical Sciences and Mathematics - Chemistry - Physical-Chemistry
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Munir Salomao Skaf
• Grantee: Bruna Medéia de Campos
• Host Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 13/08293-7 - CCES - Center for Computational Engineering and Sciences, AP.CEPID

Abstract

The production of biofuels and high added value chemical inputs from depolymerization of lignocellulosic biomass is still an expensive and low efficiency technology. One of the main factors that contribute for this is the absence of efficient and low cost enzymatic cocktails. The Carbohydrate-binding modules (CBMs) are an important part of these enzymes and have gained space in this field for being the main protein domains capable of orientating the catalytic modules of polisaccharidases, which are necessary for cellulosic biomass digestion into simple soluble sugars. Recently, cellulosome and CBMs studies are showing that CBMs might have a more important function in carbohydrate degradation than just simply link enzyme and substrate. CBM64 is a protein from CBM family 64 and is bound to a GH10, which has xyloglucanase function, and was first isolated in Spirochaeta thermophile, bacteria that has optimal growth temperature of 65 °C. This CBM has been studied by Dr. Fabio Squina group and has shown a very promising protein, as is a dimer in solution at 25 °C, binding to soluble and insoluble substrates with high affinity. Interestingly, at 65°C, CBM64 is a mixture of monomers and dimers and its capacity of binding to soluble substrates is diminished. The tridimensional structure of CBM64 was solved and many studies regarding the understanding of protein-ligand interaction was carried out. However, it was not possible to map the ligand-binding site of the protein and to elucidate how the dimer and temperature affect protein-ligand interaction or to understand the part of this dimer in biomass degradation.This Project has, therefore, the objective of elucidating the mechanism of interaction of CBM64 and its substrates, aiming to map the ligand-binding site of the dimeric protein to the ligand e how temperature and oligomeric state interfere with the interaction, being able to evaluate how it helps the carbohydrate degradation. The project also aims the study of the full protein, composed by enzyme, linker and CBM64, to clarify how CBM dimerization affects enzyme activity and how it can help to improve biomass degradation. This project is an important contribution to scientific collaboration between the groups from Prof. Munir Skaf and Prof. Fabio Squina, conducted respectively at Centro Cepid "Center for Computational Engineering & Sciences" from UNICAMP and CTBE/CNPEM, both supported by FAPESP. (AU)