Molecular cloning, characterization, and expression analysis of a β-N-acetylhexosaminidase (LeHex20B) from the shiitake mushroom, Lentinula edodes
We previously reported on a β-N-acetylhexosaminidase, LeHex20A, belonging to glycoside hydrolase family 20 (GH20), from the fruiting body of Lentinula edodes (shiitake mushroom). In this study, we purified, cloned, and characterized another β-N-acetylhexosaminidase, LeHex20B, from L. edodes fruiting bodies. The cDNA of LeHex20B includes an open reading frame of 1,686 bp encoding a 20 amino acid signal peptide and a 541 amino acid mature protein. The amino acid sequence identity of LeHex20A and LeHex20B was 57 %, and LeHex20B had high sequence identity to GH20 proteins; thus, LeHex20B belongs to GH family 20. LeHex20B showed β-N-acetylhexosaminidase activity and catalyzed degradation of chitooligosaccharides (GlcNAc2-6) exolytically with N-acetylglucosamine (GlcNAc) production. The maximum LeHex20B activity was observed at pH 5.0 and at 60 °C. LeHex20B had highest catalytic efficiency (k cat/K m) for GlcNAc3 and showed high affinity for GlcNAc3-6. The transcript level of LeHex20A was significantly increased in fruiting bodies after harvest, suggesting that LeHex20A is mainly involved in fruiting body autolysis. On the other hand, LeHex20B was highly expressed in young fruiting bodies and mycelia. Therefore, LeHex20B seems to be mainly involved in elongation of fruiting bodies and mycelia.
KeywordsShitake mushroom Basidiomycete Chitin β-N-acetylhexosaminidase Glycoside hydrolase family 20
We thank Ms. Akiko Uchidate, Ms. Miyuki Ito, Ms. Junko Kawaguchi, and Ms. Shiho Sato for their help with experiments. We are grateful to Dr. Arend F. van Peer for his suggestion and comments. This research was supported by a Grant-in-Aid for Scientific Research to N. K. (no. 2510648) from the Japan Society for the Promotion of Science (JSPS), by grants for project research (Development of fundamental technology for analysis and evaluation of functional agricultural products and functional foods).
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