Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates

Abstract

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K i values for D-glucose (83.3-357 mM) and D-xylose (15.6-70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t (0.5) ∼ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s(-1), 138 s(-1) mM(-1)), xylotriose (235 s(-1), 80.8 s(-1) mM(-1)), and xylotetraose (146 s(-1), 32.6 s(-1) mM(-1)).

Secretory Expression and Characterization of Two Hemicellulases, Xylanase, and β-Xylosidase, Isolated from Bacillus Subtilis M015

“…Therefore, XynB’s optimum temperature falls within the range of closely-related β-xylosidases. Similar to its temperature profile, XynB’s optimum pH of 7.0 agrees with the pH profile of most GH-43 β-xylosidases [ 22 – 24 ]. XynB showed highest activity in sodium phosphate buffer at pH 7.0–7.5 but dropped to a relative activity of around 20 % at pH 6.0.…”

“…; 25 °C for B. subtilis subsp. subtilis 168 [ 22 ], 30 °C for B. subtilis PAP115 [ 23 ], and 50 °C for B. subtilis (subspecies not reported) [ 24 ]. Therefore, XynB’s optimum temperature falls within the range of closely-related β-xylosidases.…”

Characterization of a furan aldehyde-tolerantβ-xylosidase/α-arabinosidase obtained through a synthetic metagenomics approach

“…In addition, XylM1989 was predicted to belong to the GH43 family, which contains mostly β ‐xylosidases (EC 3.2.1.37), α ‐arabinosidases (EC 3.2.1.55), galactan 1,3‐ β ‐galactosidases (EC 3.2.145) and endo‐ α ‐arabinases (EC 3.2.1.99) (Jordan et al . ; Lagaert et al . ).…”

“…The protein XylM1989 has a calculated isoelectric point (pI) of 6Á16 and a molecular weight of 37Á5 kDa. In addition, XylM1989 was predicted to belong to the GH43 family, which contains mostly b-xylosidases (EC 3.2.1.37), a-arabinosidases (EC 3.2.1.55), galactan 1,3-bgalactosidases (EC 3.2.145) and endo-a-arabinases (EC 3.2.1.99) (Jordan et al 2013;Lagaert et al 2014). Based on the BLASTP analysis, the amino acid sequence of XylM1989 showed 95% identity (100% coverage) with an uncharacterized GH43 family protein (ACX30651) encoded by a chromosomal segment of Sphingobacterium sp.…”

Golgi-localized exo-β1,3-galactosidases involved in AGP modification and root cell expansion in Arabidopsis

“…The family is divided into α-L-arabinofuranosidase, β-D-xylosidase, α-L-arabinanase, and β-D-galactosidase groups (Mewis et al, 2016). Several GH43 enzymes from prokaryotes and eukaryotes have recently been characterized because of their potential in biomass degradation and other biotechnological applications (Jordan et al, 2013; McCleary et al, 2015). Based on their diverse identified enzymatic activities and amino acid motifs, the GH43s have been further subdivided into 37 subclades (Mewis et al, 2016).…”