Characterization of an alpha-L-rhamnosidase from Aspergillus kawachii and its gene.

An alpha-L-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on L-rhamnose as the sole carbon source. The alpha-L-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 degrees C. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 degrees C for 1 h. Its T (50) value was determined to be 72 degrees C. The enzyme was able to hydrolyze alpha-1,2- and alpha-1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii alpha-L-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the alpha-L-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins.

Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii.

The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61DeltaCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61DeltaCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61DeltaCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61DeltaCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl beta-D: -glucoside, p-nitrophenyl beta-D: -cellobioside, and p-nitrophenyl beta-D -cellotrioside.

The family 42 carbohydrate-binding module of family 54 alpha-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose.

Alpha-L-arabinofuranosidase catalyses the hydrolysis of the alpha-1,2-, alpha-1,3-, and alpha-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-alpha-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 10(3) M(-1). These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii alpha-L-arabinofuranosidase 54.

A role for N-linked oligosaccharides on the biochemical properties of recombinant alpha-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn(83)-Thr-Thr and Asn(202)-Ser-Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn(83), Asn(202), and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn(202) may contribute to thermostability and catalysis.