Structural and functional studies of glycoside hydrolases from thermophilic microorganisms

Author(s)

Leonardo Rodrigues de Almeida

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2022-04-29

Examining board members

João Renato Carvalho Muniz; João Alexandre Ribeiro Gonçalves Barbosa; Fernando Masarin; Ronaldo Alves Pinto Nagem

• Advisor: João Renato Carvalho Muniz

Abstract

The depletion of petroleum reserves, the growing energy demand from emerging countries, and the need to reduce the emission of carbon dioxide signal the importance of searching for new sources of renewable energy. However, in order for such sources to have a character of effective replacement and not only complementation, it is also mandatory to search for new strategies to obtain it. Thus, the production of bioethanol through the hydrolysis of lignocellulosic biomass, such as sugarcane bagasse, by cellulases has received great attention. Due to its potential for evolution and cost reduction, the enzymatic hydrolysis of cellulose can be a fundamental part for the production of sugars, 2G ethanol and carboxylic acids, with a competitive cost in the long term. Some thermophilic organisms, such as the bacteria Clostridium thermocellum and Lactobacillus gasseri, the fungus Thermothielavioides terrestris and the mesophilic bacterium Bacillus licheniformis, are examples known for their ability to secrete cellulases. C. thermocellum produces an extracellular multienzymatic protein complex called cellulosome, which demonstrates a high capacity to perform an efficient degradation of cellulosic biomass, especially in the crystalline portion of cellulose. The enzyme CtBgl3B, -glycosidases (BGls) cellulosomal from C. thermocellum from the GH3 family of glycoside hydrolases, was expressed in E. coli (BL21), purified by affinity and size-exclusion chromatography. It showed thermophilic characteristics, showing high activity at temperatures above 60 °C, ranging from pH 4.5 to 7.0, with greater activity at pH 5.5 to 70 °C. Tm in 70 °C, determined by differential scanning fluorimetry (DSF) and circular dichroism (CD). In addition, its enzymatic activity and kinetic parameters were evaluated with pNPG: Sp. act. = 124 U mg1, KM = 0.37 (mM), catalytic constant kcat = 173 (s1), and catalytic efficiency kcat/KM = 469 (mM1 s1). The enzyme also demonstrated the ability to act against synthetic substrates such as pNPX (40%) and CpNPG2 (25%). While in the additive test, it had its conversion rates increased 70% in the presence of Triton X-100, and 60% with Tween20. On the other hand, it showed a significant decrease in the reactions with the presence of divalent ions, DMSO and SDS, respectively, below 25-75% its relative activity to pNPG. The crystallographic structure of CtBGl3 was determined employing X-ray diffraction using molecular replacement, with anisotropic resolution limits in Å along their respective directions 3.47 (a), 2.22 (b) e 1.78 (c), demonstrating a dimer arrangement, indicators of refinement quality Rwork and Rfree, respectively 0.20 and 0.23. Multi-angle light scattering (MALS) and small-angle X-ray scattering (SAXS) experiments corroborate that in solution, CtBGl3 also adopts dimer arrangement. The mesophilic bacterium B. licheniformis produces a hypothetical protein BlYlmD, which was expressed in E. coli (Rosetta DE3) and had its structure determined at 2.66 Å resolution. This protein domain, whose alignment indicated the presence of a Cu-oxidase_4 domain (PF02578), also found in laccases, and widely distributed between prokaryotes and eukaryotes, or a putative CNF1/YfiH-like cysteine hydrolase from B. licheniformis BlYlmD, drew attention due to its sequential similarities with well-known laccase domains, and became the secondary object of study in this research. Its structure is classified as a / protein that forms four layers of /// with mixed antiparallel beta sheets and assembles into a dimer in the asymmetric unit. It did not have its function characterized for the suggested functions. It has recently been compared to a multifunctional purine nucleoside phosphorylase PNP FAMIN. However, also without detection of any enzymatic activity, the main structural indication for its lack of activity being centred on the conformation of His47, and the differences between the residues Tyr106 (Phe) and Glu77 (Asp), which can affect the volume of the catalytic cavity, preventing the entry of substrates. (AU)

• FAPESP's process: 16/09152-6 - Structural and functional studies of cellulosomal thermophilic glycoside hydrolases involved on lignocellulosic biomass hydrolysis
• Grantee: Leonardo Rodrigues de Almeida
• Support Opportunities: Scholarships in Brazil - Doctorate