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

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.

Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

“…According to the results, the concentration of xylooligosaccharides increase as a function of the reaction time. Previous studies have indicated that endoxylanase is the key enzyme for conversion of xylans into xylooligosaccharides [ 78 , 79 ]. The soft and green conditions used for enzymatic production of xylooligosaccharides and the specificity of the biocatalysts make enzymatic methods more convenient than chemical procedures [ 80 ], constituting a reasonable and reliable alternative to produce the large amounts required, for example, in aquaculture and poultry industry [ 81 ].…”

Discovery of a bifunctional xylanolytic enzyme with arabinoxylan arabinofuranohydrolase‐d3 and endo‐xylanase activities and its application in the hydrolysis of cereal arabinoxylans

“…However, their activities against double-Araf-substituted substrates could only achieve 0.5%-7.2% of that against mono (Dos Santos et al, 2018;Geng et al, 2019;Wang et al, 2018). That is to say, double-Araf-substituted xylans are particularly recalcitrant to enzymatic attack (Liu et al, 2021).…”

“…In addition, Araf substitution is also the base point for the covalent crosslink of xylan-xylan and xylan-lignin (Freeman et al, 2017). The reason is that its C(O)-5 position can be esterified with FA and these feruloyl substituents will form diferulates between xylans through dehydrodimerization or be bonded to lignin directly (Lin et al, 2023;Liu et al, 2021;Sumiyoshi et al, 2013). Therefore, efficient removal of Araf decoration is the key to decrease xylans recalcitrance, increase enzymatic accessibility and improve utilization rate of lignocellulose.…”

“…Indeed, researchers found that the yield of arabinose or reducing sugars increased by 16.7%-111% when AXHd3 was added in hydrolysis mixtures containing AXHm2,3 or endo-xylanase (Mroueh et al, 2019;Orita et al, 2017;Pouvreau et al, 2011;Sorensen et al, 2006). However, just a few AXHd3s have been reported to date (Liu et al, 2021;Mroueh et al, 2019;Orita et al, 2017;Pouvreau et al, 2011;Rogowski et al, 2015;Saito et al, 2020;Sorensen et al, 2006;van den Broek et al, 2005), which all belong to the GH43 family. In CAZy database, GH43 is one of the largest GH families and most reported multifunctional xylanolytic enzymes belong to this family (Limsakul et al, 2021;Mewis et al, 2016).…”

Biochemical and Molecular Dynamics Study of a Novel GH 43 α-l-Arabinofuranosidase/β-Xylosidase From Caldicellulosiruptor saccharolyticus DSM8903

“…Various arabinofuranosidases from both fungal and bacterial origins have recently been found, offering essential information for understanding the enzymes. Thermotolerant biomass-degrading enzymes often perform well due to their higher stability and potentiality, and thermophilic bacteria are one of the finest sources of arabinofuranosidases (Hu et al, 2018;Long et al, 2020).…”