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Title: DEVELOPMENT OF A COUPLED ENZYME ASSAY FOR THE MEASUREMENT OF ALTERNANASE ACTIVITY

Author
item Ahlgren, Jeffrey
item Cote, Gregory

Submitted to: Biotechnology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2002
Publication Date: 8/1/2002
Citation: AHLGREN, J.A., COTE, G.L. DEVELOPMENT OF A COUPLED ENZYME ASSAY FOR THE MEASUREMENT OF ALTERNANASE ACTIVITY. BIOTECHNOLOGY LETTERS. 2002. V. 24. P. 1277-1280.

Interpretive Summary: Alternan is a type of complex carbohydrate that is made by a microorganism called Leuconostoc mesenteroides. There are potential applications for this biologically produced material in its unmodified form, but we are hoping to expand the range of applications by processing the material obtained from fermentation using environmentally friendly technology. We have previously described and subsequently patented an enzyme called alternanase that can cleave a portion of the full-size carbohydrate and process it into smaller, cyclic pieces; we are currently exploring potential applications for this unique material. One problem that has persisted during our studies with this enzyme is the method used to evaluate its function or to measure the amount of enzyme contained in a variety of samples. This report describes the inherent problems with previously used methods and reports on the development of a new method designed to measure the amount of alternanase present in a sample using a reliable and convenient protocol that uses standardized, commercially available materials. This new method is quite simple and easy to reproduce and should provide for a means for reporting results in a way that will make it much easier to compare these findings between different laboratories.

Technical Abstract: Alternanase is an endoglucanase that hydrolyzes the bacterial exopolysaccharide alternan. It will also hydrolyze the trisaccharide panose to produce glucose and a disaccharide that will ultimately be formed into a novel, cyclic tetrasaccharide. The resulting glucose can then be selectively and quantitatively measured by enzyme-based reactions, which forms the basis of a coupled enzyme system to quantitate alternanase activity. A unit of alternanase is defined as the amount of enzyme that forms one micromole of glucose per minute at 45 deg C from panose. Using a hexokinase-based method to quantitate glucose, a preparation of alternanase purified by affinity chromatography on immobilized isomaltose, had a maximum reaction rate (Vmax) of 750 umoles/min and a Km of 34 mM for panose. Two competitive inhibitors of alternanase activity were also evaluated using this coupled enzyme assay: isomaltose had a Ki of 94 mm while the cyclic tetrasaccharide had a Ki of 66 mM.