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Enzyme assay of fungal α-1,2-mannosidase
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Enzyme assay of fungal α-1,2-mannosidase

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Introduction Protocol References Credit lines
Category
Biosynthesis & Metabolism
Protocol Name

Enzyme assay of fungal α-1,2-mannosidase

Authors
Yoshida, Takashi
Faculty of Agriculture and Life Science, Hirosaki University
KeyWords
Reagents

Pyridylaminated high-mannose type oligosaccharide (Man9GlcNAc2-PA) 1)

(PA-Labeled mannooligosaccharides are commercially provided by Takara Bio Inc., Otsu, Japan)

Instruments

HPLC system with a fluorescent detector.

Methods
1.

Reaction

1) 

 Mix 5 μL of Man9GlcNAc2-PA (total 50 pmol), 5 μL of 200 mM sodium acetate buffer, pH 5.0, water, and the enzyme to make total 50 μL of solution.

Comment 0
2) 

 Incubate at 30°C for an appropriate period of time.

Comment 0
3) 

 Stop the reaction by chilling in an iced bath.

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2.

Analysis

1) 

 Put the reaction mixture into a centrifugal membrane-filter (0.2 μm), then spin down.

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2) 

 Analyze aliquots of the solution by HPLC with an amide column (TSKgel Amide 80, 4 × 250 mm) which is run in 3% acetic acid-triethylamine, pH 7.3/acetonitrile/water (8:55:37) at a flow rate of 0.4 mL/min.2)

Comment 0
3) 

 Monitor the fluorescence of the effluent at an excitation 315 nm and emission 380 nm (Fig. 1).

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Notes

The reaction buffer should be at neutral pH in determining mammalian enzymatic activity. 

Figure & Legends

Figure & Legends

 

Fig. 1. Trimming of high-mannose oligosaccharides by α-1,2-mannosidase.

Number in each panel is the reaction period (min).

This figure was originally published in Biosci. Biotechnol. Biochem. Akao F, Yoshida T et al. "Cloning and Expression of 1,2-α-Mannosidase Gene (fmanIB) from Filamentous Fungus Aspergillus oryzae: in Vivo visualization of the FmanIBp-GFP Fusion Protein" 2006, 70(2) 471–479.

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