Novel β-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans

Motoyuki Shimizu,Yuhei Kaneko,Saaya Ishihara,Mai Mochizuki,Kiyota Sakai,Miyuki Yamada,Shunsuke Murata,Eriko Itoh,Tatsuya Yamamoto,Yu Sugimura,Tatsuya Hirano,Naoki Takaya,Tetsuo Kobayashi,Masashi Kato

doi:10.1074/jbc.m115.661645

Motoyuki Shimizu, Yuhei Kaneko + Show 12 more

Open Access

https://doi.org/10.1074/jbc.m115.661645

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Abstract

Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-β-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. The action of Man5C toward β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans were the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.

Highlights

Filamentous fungi produce various mannanolytic enzymes including ␤-1,4-mannanases for ␤-mannan degradation
Identification of Extracellular Proteins Induced by ␤Mannans—The extracellular proteins of A. nidulans produced using various sole carbon sources were analyzed by SDS-PAGE (Fig. 1A)
The extracellular proteins of A. nidulans grown in MM medium containing glucomannan as the sole carbon source were resolved by twodimensional gel electrophoresis (Fig. 1B), which identified the same proteins as those found by SDS-PAGE

Summary

Introduction

Filamentous fungi produce various mannanolytic enzymes including ␤-1,4-mannanases for ␤-mannan degradation. Many filamentous fungi produce ␤-mannan-degrading ␤-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. We identified a novel ␤-1,4mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. The growth phenotype of a Man134A disruptant was poor when ␤-mannans were the sole carbon source, indicating that Man134A is involved in ␤-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of ␤-mannan degradation that is enhanced by the novel ␤-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-␤-1,4-mannanases

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