Abstract
BackgroundThere is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood.ResultsScreening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications.ConclusionsHere we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.
Highlights
There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood
The three main components of plant biomass are cellulose, hemicellulose, and lignin of which hemicellulose and lignin can take a variety of chemical forms depending on the plant species
A total of 26 positive hits were identified, and of these, the insert sequences for 18 clones that showed highest β-xylosidase activity were determined
Summary
There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Enzymatic degradation, following physicochemical disruption (steam explosion, high/ low pH treatment or solubilization with ionic liquids), could offer a more efficient, environmentally friendly approach to assist in the degradation of plant biomass. The complete breakdown of lignocellulose requires the consortium of microorganisms which produce cellulases, hemicellulases and ligninases. In industrial processes, this is achieved through enzymatic cocktails which contain different enzymes that act on different components of lignocellulosic substrates [6]. Metagenomics is the direct interrogation of total DNA from an environmental sample without the need for culturing of the host organism This tool provides a powerful approach for functional screening and identification of novel biocatalysts from unculturable or uncultured microorganisms from any environment [8]
Sign-in/Register to view highlights & summary 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.
