Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography
A highly purified extracellular α-amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.
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- 1.Bernfeld P (1951) Enzymes of starch degradation and synthesis.Adv Enzymol 12:379–428Google Scholar
- 2.Davis BJ (1964) Disc gel electrophoresis. II. Method and application to human serum proteins. Annu Rev NY Acad Sci 121:404–427Google Scholar
- 3.Deploey JJ, Nasta M, Adams PR (1982) Quantitative determinations of the temperature and pH stability of extracellular amylase obtained fromMucor pusillus. Mycologia 74(5):847–850Google Scholar
- 4.Fergus CC (1969) The production of amylase by some thermophilic fungi. Mycologia 61:1171–1175Google Scholar
- 5.Lowry DH, Rosebrough NH, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275Google Scholar
- 6.Parikh Z, Cautrecasas P (1985) Affinity chromatography. Chem Eng News 26:17–32Google Scholar
- 7.Somkuti GA, Steinburg DH (1980) Thermoacidophilic extracellular amylase ofMucor pusillus. Dev Indust Microbiol 21:327–337Google Scholar
- 8.Sundburg L, Porath J (1974) Preparation of adsorbants for biospecific affinity chromatography. J Chromatog 9:87–98Google Scholar
- 9.Uy R, Wold F (1977) Use of carbohydrates as ligands for biospecific adsorbants. Anal Biochem 81:98–107Google Scholar
- 10.Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412Google Scholar