α-L-Arabinofuranosidase (Aspergillus nidulans)

The combination of hydrothermal pretreatment (autohydrolysis) and enzymatic hydrolysis was comprehensively evaluated for the efficient release of monosaccharides and ferulic acid from corn bran. Arabinan was depolymerized and solubilized more easily during autohydrolysis compared to xylan, esterified ferulic acid, and the acetyl group. Also, the enzymatic xylose yield showed strong linear correlation with arabinan, ferulic acid, and acetic acid content in autohydrolysis residues while correlations between enzymatic glucose yield and hemicellulose contents were separated into two stages with different slopes. The addition of a few debranching enzymes to commercial cellulase and xylanase only slightly enhanced enzymatic hydrolysis of autohydrolysis residues, whereas an enzyme blend from Aspergillus oryzae and Eupenicillium parvum showed a significant synergistic effect. Desirable combined hydrolysis yields of glucose (72.26%), xylose (75.87%), arabinose (76.95%), and ferulic acid (74.13%) were obtained after autohydrolysis at 165°C for 40 min and subsequent hydrolysis by an equal mixture blend produced by A. oryzae and E. parvum at an enzyme loading dosage of 14.1 mg protein/g dry destarched corn bran.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k_cat = 178/s, K_m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3–5 (37–80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu¹⁸⁸) and base (Asp²⁸) catalysts, and the general acid pK_a modulator (Asp¹³⁶) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp²³ and Tyr⁴⁴, situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with Ki of WAX substrate inhibition increasing 4–7-folds, but lost 77–96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp²³ is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp ²³ and Tyr⁴⁴ belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.