Construction of a multifunctional enzyme feruloyl esterase/acetyl xylan esterase by rational design

Author(s)

Luana de Fátima Alves

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2016-02-26

Examining board members

Richard John Ward; Luiz Alberto Beraldo de Moraes; Arthur Henrique Cavalcante de Oliveira

• Advisor: Richard John Ward

Abstract

The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed (AU)

• FAPESP's process: 14/07468-0 - Construction of a multifunctional enzyme feruloyl esterase/acetyl xylan esterase by rational design
• Grantee: Luana de Fátima Alves
• Support Opportunities: Scholarships in Brazil - Master