Characterization of a modular enzyme of exo-1,5-alpha-L-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893

Abstract

A gene encoding an alpha-L-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45 degrees C and was stable over the pH range of 5.0-6.5 at 30 degrees C. The enzyme hydrolyzed p-nitrophenol (PNP)-alpha-L-arabinofuranoside but did not hydrolyze PNP-alpha-L-arabinopyranoside, PNP-beta-D-xylopyranoside, or PNP-beta-D-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside and methyl 3-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside were not. These data suggested that the enzyme only cleaves alpha-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-alpha-L-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.

Fig. 1

a Multiple alignment of the catalytic domains of SaAraf43A and several enzymes belonging to GH43. The alignment was achieved with ClustalW. Identical amino acid residues are enclosed in black boxes. The asterisks indicate the putative catalytic amino acids in CjArb43A and BsArb43A. Boxes L1 to L9 are insertion or deletion loops between exo-1,5-α-l-arabinofuranosidases and α-l-arabinanases. ScAraf43A, exo-1,5-α-l-arabinofuranosidase from S. chartreusis (accession number, BAA90772); CjArb43A, arabinanase from C. japonicus (CAA71485); BsArb43A, arabinanase from B. subtilis (CAA99586). b Multiple alignments of SaCBM42 with other known members of CBM42. The alignment was achieved using ClustalW. Conserved amino acids are shown in black boxes, and asterisks indicate the amino acids related to the substrate binding. AkCBM42, CBM42 of α-l-arabinofuranosidase from A. kawachii (BAB96816); HjCBM42, CBM42 of α-l-arabinofuranosidase from Hypocrea jecorina (CAA93243)

Fig. 2

SDS-PAGE of purified SaAraf43A. M Molecular mass standard; lane 1 recombinant SaAraf43A; lane 2 CBM-deficient mutant of SaAraf43A. Approximately 1 μg of each sample was separated on 12% polyacrylamide gel

Fig. 3

Course of the hydrolysis of regioisomeric arabinofuranobiosides by SaAraf43A. Closed square Methyl 2-O-α-l-arabinofuranosyl-α-l-arabinofuranoside; closed triangle methyl 3-O-α-l-arabinofuranosyl-α-l-arabinofuranoside; closed circle methyl 5-O-α-l-arabinofuranosyl-α-l-arabinofuranoside. The enzyme was incubated with 0.5% (w/v) arabinofuranobioside at 30°C for the appropriate time. The hydrolysis rate of the substrate was estimated by the amount of released l-arabinose

Fig. 4

HPAEC-PAD analysis of the hydrolysis products of α-1,5-arabinopentaose by SaAraf43A. The enzyme was incubated with 0.5% (w/v) arabinopentaose at 30°C for the appropriate time; the samples were subjected to HPAEC-PAD analysis. A1l-arabinose; A2 α-1,5-l-arabinobiose; A3 α-1,5-l-arabinotriose; A4 α-1,5-l-arabinotetraose; A5, α-1,5-l-arabinopentaose

Fig. 5

HPAEC-PAD analysis of the hydrolysis products of debranched arabinan by SaAraf43A. The enzyme was incubated with 0.25% (w/v) debranched arabinan at 30°C for the appropriate time; the samples were analyzed by HPAEC-PAD. A1 indicates l-arabinose

Fig. 6

Plots of the affinity gel electrophoresis data used to determine the affinity of SaAraf43A for wheat arabinoxylan and arabinan. Closed circle wheat arabinoxylan; closed square arabinan; R relative mobility of SaAraf43A compared with the standard in the absence of the sugars; r relative mobility of SaAraf43A compared with the standard in the presence of the sugars

Fig. 7

Effect of SaCBM42 on hydrolysis rate of SaAraf43A. a Hydrolysis of PNP-α-l-arabinofuranoside; b hydrolysis of debranched arabinan. Closed circle SaAraf43A; closed square the catalytic domain of SaAraf43A. The enzyme (1 μM) was incubated with 1 mM PNP-α-l-Araf or 0.25% (w/v) debranched arabinan at 20°C for up to 20 min. The hydrolysis rate of the substrate was estimated by the amount of released PNP or l-arabinose