Abstract
The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme’ active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.
Highlights
Feruloyl esterases (EC 3.1.1.73, FAEs) are a subclass of carbohydrate esterases that are considered a biotechnological key for the degradation of lignocellulosic biomass, catalyzing the hydrolysis of the ester bond between hydroxycinnamic acids, such as ferulic acid (FA), caffeic acid (CA), sinapic acid (SA), p-coumaric acid and sugars found in plant cell walls
The type C FAE from Fusarium oxysporum (FoFaeC) is a newly discovered enzyme with broad pH stability and good synergistic action [17][18]. It belongs to the SF2 subfamily of phylogenetic classification [12] which is closely related to tannases, showing high similarity with AoFaeB from A. oryzae of known structure [16][19]
methyl ferulate (MFA) and methyl caffeate (MCA) were purchased by Alfa Aesar (Karlsruhe, Germany) while methyl sinapate (MSA) and methyl p-coumarate (MpCA) from Apin Chemicals Ltd. (Abingdon, UK). p-Nitrophenyl ferulate and 4-nitrocatechol-1-yl ferulate [24] (4NTC-Fe) were provided by Taros Chemicals GmbH & Co
Summary
Feruloyl esterases (EC 3.1.1.73, FAEs) are a subclass of carbohydrate esterases that are considered a biotechnological key for the degradation of lignocellulosic biomass, catalyzing the hydrolysis of the ester bond between hydroxycinnamic acids, such as ferulic acid (FA), caffeic acid (CA), sinapic acid (SA), p-coumaric acid (pCA) and sugars found in plant cell walls. Others show a profile of type B FAEs with weak or no activity towards MSA, including AnFaeB from A. niger [6], AoFaeB from Aspergillus oryzae [16] and FoFaeC from Fusarium oxysporum [17] Despite their differences in specificity, the aforementioned enzymes were eventually categorized as Type C FAEs due to phylogenetic similarity with tannases [8] [9] [12]. The type C FAE from Fusarium oxysporum (FoFaeC) is a newly discovered enzyme with broad pH stability and good synergistic action [17][18] It belongs to the SF2 subfamily of phylogenetic classification [12] which is closely related to tannases, showing high similarity with AoFaeB from A. oryzae of known structure [16][19]. To the authors’ knowledge, this is the first report of applying rational protein redesign on a FAE, opening the pathway for understanding the mechanisms behind FAE specificity towards model substrates and tailoring this diverse class of enzymes towards desired bioconversions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
