Bifunctional xylanase-esterase enzyme obtained from the ruminal liquid metagenome

Author(s)

Gabriella Cavazzini Pavarina

• Document type: Master's Dissertation
• Press: Jaboticabal. 2020-08-18.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Agrárias e Veterinárias. Jaboticabal
• Defense date: 2020-07-28
• Advisor: João Martins Pizauro Junior; Eliana Gertrudes de Macedo Lemos

Abstract

Lignocellulosic biomass, an important and strategic source of renewable energy, is complex and heterogeneous. For the enzymatic hydrolysis of this raw material to occur, it needs the synergistic action of cellulose, hemicellulose and lignin degrading enzymes. The endo-β-xylanases, enzymes capable of degrading the main chain of xylans, the most abundant hemicellulose, have their efficiency reduced due to the presence of branches in their structure, such as ester bonds with ferulic and cumaric acid, which can be degraded by esterases. An alternative to increase efficiency would be the simultaneous hydrolysis of these branches and the main chain, which is possible using bifunctional enzymes. The aim of this work was to prospect and express a xylanase-esterase achieved by metagenome from the rumen fluid. The xylanase-esterase sequence was obtained based on the inference of function by similarity in the rumen metagenome. The amplification of the metagenomic DNA sequence by PCR was realized, insertion into vector pET28a and transformation into Escherichia coli BL21 (DE3) were made. The purification of the recombinant enzyme was performed using affinity chromatography for immobilized metal ions and gel filtration in the AKTA system. The endo-β-xylanase activity was verified by zymogram containing xylan from beechwood and esterase activity by the hydrolysis of the substrate of p-nitrophenyl ester. The results demonstrate the obtaining of a bifunctional enzyme (xylanase-esterase), active for both substrates, resistant to NaCl and organic solvents (ethanol, methanol, propanol and DMSO), with potential for application in the degradation of lignocellulosic biomass. (AU)

• FAPESP's process: 18/12885-0 - Prospecting, heterologous expression, purification and characterization of a xylanase
• Grantee: Gabriella Cavazzini Pavarina
• Support Opportunities: Scholarships in Brazil - Master