Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom)

Abstract

We purified and cloned a β-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of β-N-acetylgalactosaminides and β-N-acetylglucosaminides, indicating that LeHex20A is a β-N-acetylhexosaminidase classified into EC 3.2.1.52. The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 β-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.

Figure 1

SDS-PAGE of purified LeHex20A. Approximately 1 μg of sample was separated on a 10% (w/v) polyacrylamide gel. Lane 1, molecular mass standards (kDa); lane 2, purified LeHex20A.

Figure 2

HPLC analysis of LeHex20A action on colloidal chitin. Colloidal chitin (2%, w/v) was incubated with LeHex20A (0.4 nM) in 20 mM sodium acetate buffer (pH 4.2), at 30°C. Eluted products were detected by monitoring UV absorption at 205 nm. Standards (Std.) were GlcNAc and chitooligosaccharides (GlcNAc_2-6). DP; degree of polymerization.

Figure 3

Multiple sequence alignment of a region surrounding the catalytic residue of GH family 20 members (β-N-acetylhexosaminidases) fromLentinula edodes(LeHex20A),Hypocrea lixii(AAB50829),Aspergillus oryzae(XP_001817681),Ostrinia furnacalis(ABI81756),Homo sapiens(AAB00965),Arabidopsis thaliana(BAE99290) andStreptomyces plicatus(AAC38798). Protein sequences were aligned with the MAFFT program (version 6; http://align.bmr.kyushu-u.ac.jp/mafft/online/server/) using the E-INS-I algorithm. A consensus motif preceding the catalytic residue is boxed. The catalytic glutamate is bold.