Characterization and functional analysis of two novel thermotolerant \u03b1-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

Liangkun Long,Shaojun Ding,Franz J St John,Qunying Lin,Lu Sun

doi:10.1007/s00253-020-10867-7

Abstract

Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-l-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-l-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone.Key points• Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability.• The thermal stability of GH62 family EpABF62C was dependent on calcium.• Buffer pH affects the thermal stability of the two ABFs.• Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

Highlights

Hemicellulose is the second most abundant polysaccharide in the biosphere and is of interest for bioconversion to green chemicals, fuels, biomaterials, functional foods, and pharmaceuticals (Liu et al 2016; Zhou et al 2017)
Members of the GH43 family are active on α-1,5linked L-Araf oligosaccharides or are specific for α-1,2- and or α-1,3-linked Araf from xylan (Mewis et al 2016), those from GH51 or 54 hydrolyze mono- and di-substituted Araf side chains on arabinoxylan or arabinan, and GH62 family ABFs seem to be specialized in removing mono-substituted Araf residues from arabinoxylans (Wilkens et al 2017)
The constructed phylogenetic trees indicated that TtABF51A and EpABF62C were affiliated with the ABFs belonging to GH51 or 62 family (62_2 subfamily), respectively (Fig. 1a, b)

Summary

Introduction

Hemicellulose is the second most abundant polysaccharide in the biosphere and is of interest for bioconversion to green chemicals, fuels, biomaterials, functional foods, and pharmaceuticals (Liu et al 2016; Zhou et al 2017). The presence of Araf substitutions enhanced the resistance of xylan to enzymatic hydrolysis (Thakur et al 2019). Exo-α-L-arabinofuranosidases (ABFs, EC 3.2.1.55) catalyze the hydrolysis of terminal non-reducing α-1,2-, α-1,3-, or α-1,5-linked Araf residues from arabinose-substituted polysaccharides or shorter oligosaccharides (Thakur et al 2019; Yang et al 2015). Members of the GH43 family are active on α-1,5linked L-Araf oligosaccharides or are specific for α-1,2- and or α-1,3-linked Araf from xylan (Mewis et al 2016), those from GH51 or 54 hydrolyze mono- and di-substituted Araf side chains on arabinoxylan or arabinan (dos Santos et al 2018), and GH62 family ABFs seem to be specialized in removing mono-substituted Araf residues from arabinoxylans (Wilkens et al 2017). ABFs of families GH2, 3, 51, and 54 act on the glycosidic linkage by a retaining mechanism, and members of GH43 and 62 families (clan GH-F) display a five-bladed propeller arrangement with an inverting mechanism of hydrolysis (Maehara et al 2014; Numan and Bhosle 2006; Wang et al 2014)

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