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.

A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

“…These results show that speci c activity of CmNAGase toward Nacetyl COSs decreased when the degree of polymerization increased, which is similar with other reports [59]. The catalytic e ciency of GH20 LeHex20A from Lentinula edodes for (GlcNAc) 6 was greater than for (GlcNAc) 2 [60]. The GH20 NAGase VhNag2 from V. harveyi showed the highest activity against (GlcNAc) 4 , while the lowest activity was observed with (GlcNAc) 2 [61].…”

“…Furthermore, CmNAGase showed some activity (0.02 U/mg) toward colloidal chitin. The result is similar with the GH20 NAGases from V. harveyi [61] and the fungal NAGases from Myceliopthora thermophila [62], L. edodes [60], and M. anisopliae [63], which were reported to degrade chitin to some extent without the cooperation of chitinase.…”

“…Finally, (GlcNAc) 2 −(GlcNAc) 6 were both hydrolyzed to GlcNAc as the nal product with the increase of hydrolysis time. Similar results were reported for GH20 NAGase (VhNag2) from V. harveyi [61] and LeHex20A from L. edodes [60]. However, the hydrolysis rates of VhNag2 and LeHex20A toward N-acetyl COSs were in the order: (GlcNAc) 4 >(GlcNAc) 3 >(GlcNAc) 5 >(GlcNAc) 6 >(GlcNAc) 2 .…”

“…4a, hydrolysis of colloidal chitin resulted in GlcNAc as the only product, and its concentration increased with the increase of hydrolysis time. Konno et al [60] reported that the NAGase LeHex20A from L. edodes hydrolyzes colloidal chitin to various N-acetyl COSs at the start of the reaction, and these N-acetyl COSs convert to GlcNAc after 3 h, which is different from CmNAGase.…”

A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

“…This phenomenon shows that SYBC-H1 cells rupture during a long period of fermentation, which leads to the release of CmNAGase. To date, most reported NAGases are secretory proteins [21][22][23]. The results showed that the transport of N-acetyl COSs into the intracellular compartments of strain SYBC-H1 could be achieved by specific membrane transporters, then further hydrolyzed to GlcNAc by NAGase for cell growth, which is similar with other reports [24,25].…”