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

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 (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. 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.

Discovery of a Novel β-xylosidase with Xylanase Activity and Its Application in the Production of Xylitol from Corncob Xylan

“…This finding was significantly different from those reported for β-xylosidases, which exhibited no activity toward xylan substrates [25]. Several β-xylosidases have been found to have additional α-arabinosidase activity [26,27], but β-xylosidases with xylanase activity have rarely been reported and, in such reports, the xylanase activity is very low. As shown in Table 1, β-xylosidases from Phanerochaete chrysosporium and Colletotrichum graminicola had the ability to hydrolyze birchwood xylan and beechwood xylan, but their activities were very low.…”

Simultaneous Improvement of Final Product-Tolerance and Thermostability of GH39 Xylosidase for Prebiotic Production by Directed Evolution

“…Notably, Xyl21 had better ethanol tolerance than the other reported tolerant xylosidases (Table 1). In terms of xylose tolerance, Xyl21 was better than most of the other xylosidases, but worse than Xln-DT from Dictyoglomus thermophilum and XylP81 isolated from high temperature horse manure compost [16,25]. Thus, considering its good product tolerance, xylosidase Xyl21 is an excellent starting enzyme for further enhancement.…”

“…The enzyme activities of purified proteins were measured spectrophotometrically on a microplate reader using pNPX (2 mM) as a substrate at 405 nm. Different concentrations of ethanol (5,10,15,20,25, 30% v/v) or xylose (0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1 M) were added to the reaction mixture and incubated at 45 • C for 10 min. Afterward, the reaction was blocked at 100 • C for 10 min.…”