Purification and characterization of extracellular α‐amylase and glucoamylase from the yeast Candida antarctica CBS 6678
An α‐amylase and a glucoamylase were purified to homogeneity from the culture fluid of β‐cyclodextringrown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G‐75 sf, Ultrogel AcA 54), DEAE‐Sephacel chromatography, hydroxyapatite chro...
| Published in: | European Journal of Biochemistry |
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| Main Authors: | , |
| Format: | Article in Journal/Newspaper |
| Language: | English |
| Published: |
Wiley
1987
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| Subjects: | |
| Online Access: | http://dx.doi.org/10.1111/j.1432-1033.1987.tb11175.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1432-1033.1987.tb11175.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1987.tb11175.x |
| Summary: | An α‐amylase and a glucoamylase were purified to homogeneity from the culture fluid of β‐cyclodextringrown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G‐75 sf, Ultrogel AcA 54), DEAE‐Sephacel chromatography, hydroxyapatite chromatography and affinity chomatography on acarbose—AH‐Sepharose 4B. Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient ( S ° 20, W ), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48 500, 4.7 S, 10.1, 1.74 cm 2 mg −1 , 4.2 and 57°C, respectively, for glucoamylase and as 50 000, 4.9 S, 10.3, 1.53 cm 2 mg −1 , 4.2 and 62°C, respectively, for α‐amylase. Kinetic analyses indicated that both enzymes preferentially hydrolyzed high‐molecular‐mass substrates, including some raw starches. α‐Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both α‐amylase ( K i < 1 μM) and glucoamylase ( K i < 0.1 μM), being more effective than Bay e 4609 ( K i < 10 μM). Glucoamylase was selectivity and strongly inhibited by acarbose ( K i < 0.1 μM). Activity of the latter enzyme was also affected by 1‐deoxynojirimycin ( K i < 1 mM), maltitol and amino alcohols ( K i < 10 mM). Unlike α‐amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non‐identical with the active site. |
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