Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

Kanyisa Ndata; Walter Nevondo; Bongi Cekuse; Leonardo Joaquim van Zyl; Marla Trindade

doi:10.1186/s12896-021-00722-6

Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

BMC Biotechnol. 2021 Oct 24;21(1):61. doi: 10.1186/s12896-021-00722-6.

Authors

Kanyisa Ndata¹, Walter Nevondo^{1

2}, Bongi Cekuse¹, Leonardo Joaquim van Zyl³, Marla Trindade¹

Affiliations

¹ Institute for Microbial Biotechnology and Metagenomics, Department of Biotechnology, University of the Western Cape, Bellville, South Africa.
² Division of Medical Virology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
³ Institute for Microbial Biotechnology and Metagenomics, Department of Biotechnology, University of the Western Cape, Bellville, South Africa. lvanzyl@uwc.ac.za.

Abstract

Background: There 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.

Results: Screening 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 (K_M-5.3 mM; V_max-122 U/mg; k_cat-107; T_opt-50 °C; pH_opt-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 K_i of 1.33 M, a feature that is highly desirable for commercial applications.

Conclusions: Here 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.

Keywords: Glycoside hydrolase 39; Lignocellulose; Metagenomics; β-xylosidase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Composting*
Horses
Hydrogen-Ion Concentration
Manure
Substrate Specificity
Temperature
Xylose
Xylosidases* / genetics
Xylosidases* / metabolism

Substances

Manure
Xylose
Xylosidases
exo-1,4-beta-D-xylosidase