Purification and Characterization of Exo-β-d-Glucosaminidase from a Cellulolytic Fungus,Trichoderma reesei PC-3-7

ABSTRACT Chitosan-degrading activities induced by glucosamine (GlcN) orN-acetylglucosamine (GlcNAc) were found in a culture filtrate of Trichoderma reesei PC-3-7. One of the chitosan-degrading enzymes was purified to homogeneity by precipitation with ammonium sulfate followed by anion-exchange and hydrophobic-interaction chromatographies. The enzyme was monomeric, and its molecular mass was 93 kDa. The optimum pH and temperature of the enzyme were 4.0 and 50°C, respectively. The activity was stable in the pH range 6.0 to 9.0 and at a temperature below 50°C. Reaction product analysis from the viscosimetric assay and thin-layer chromatography and 1H nuclear magnetic resonance spectroscopy clearly indicated that the enzyme was an exo-type chitosanase, exo-β-d-glucosaminidase, that releases GlcN from the nonreducing end of the chitosan chain. 1H nuclear magnetic resonance spectroscopy also showed that the exo-β-d-glucosaminidase produced a β-form of GlcN, demonstrating that the enzyme is a retaining glycanase. Time-dependent liberation of the reducing sugar from partially acetylated chitosan with exo-β-d-glucosaminidase and the partially purified exo-β-d-N-acetylglucosaminidase from T. reesei PC-3-7 suggested that the exo-β-d-glucosaminidase cleaves the glycosidic link of either GlcN-β(1→4)-GlcN or GlcN-β(1→4)-GlcNAc.

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Metabolic Profiling of Saponin-Rich Ophiopogon japonicus Roots Based on 1H NMR and HPTLC Platforms

Planta Medica ◽

10.1055/a-0947-5797 ◽

2019 ◽

Vol 85 (11/12) ◽

pp. 917-924 ◽

Cited By ~ 3

Author(s):

Yanhui Ge ◽

Xiaojia Chen ◽

Dejan Gođevac ◽

Paula C. P. Bueno ◽

Luis F. Salomé Abarca ◽

...

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Magnetic Resonance Spectroscopy ◽

Thin Layer ◽

Thin Layer Chromatography ◽

Nuclear Magnetic Resonance Spectroscopy ◽

High Performance ◽

Resonance Spectroscopy ◽

Layer Chromatography ◽

Nuclear Magnetic

AbstractIdeally, metabolomics should deal with all the metabolites that are found within cells and biological systems. The most common technologies for metabolomics include mass spectrometry, and in most cases, hyphenated to chromatographic separations (liquid chromatography- or gas chromatography-mass spectrometry) and nuclear magnetic resonance spectroscopy. However, limitations such as low sensitivity and highly congested spectra in nuclear magnetic resonance spectroscopy and relatively low signal reproducibility in mass spectrometry impede the progression of these techniques from being universal metabolomics tools. These disadvantages are more notorious in studies of certain plant secondary metabolites, such as saponins, which are difficult to analyse, but have a great biological importance in organisms. In this study, high-performance thin-layer chromatography was used as a supplementary tool for metabolomics. A method consisting of coupling 1H nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography was applied to distinguish between Ophiopogon japonicus roots that were collected from two growth locations and were of different ages. The results allowed the root samples from the two growth locations to be clearly distinguished. The difficulties encountered in the identification of the marker compounds by 1H nuclear magnetic resonance spectroscopy was overcome using high-performance thin-layer chromatography to separate and isolate the compounds. The saponins, ophiojaponin C or ophiopogonin D, were found to be marker metabolites in the root samples and proved to be greatly influenced by plant growth location, but barely by age variation. The procedure used in this study is fully described with the purpose of making a valuable contribution to the quality control of saponin-rich herbal drugs using high-performance thin-layer chromatography as a supplementary analytical tool for metabolomics research.

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