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United States Department of Agriculture

Agricultural Research Service

Title: Purification of Alternanase by Affinity Chromatography

Authors
item Ahlgren, Jeffrey
item Cote, Gregory

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 5, 2002
Publication Date: January 11, 2003
Citation: AHLGREN, J.A., COTE, G.L. PURIFICATION OF ALTERNANASE BY AFFINITY CHROMATOGRAPHY. JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY. 2003. V. 30. P. 114-117.

Interpretive Summary: Certain naturally occurring microorganisms have the ability to convert the sugar sucrose, an abundant agricultural commodity derived from both sugar beets as well as sugar cane, into a complex carbohydrate known as alternan. This value-added product may have important food and/or commercial applications, which are currently being studied. Some applications may require a degree of processing of the alternan to change its physical attributes to suit a particular application. Among the methods developed to date to process alternan, one of the most natural and environmentally friendly methods is to use a recently discovered enzyme, produced by a different microorganism than used to produce the alternan, to convert the alternan into smaller fragments than are produced naturally from sugar. A few years ago, NCAUR scientists developed a means for producing the needed enzyme in a highly purified form, but it used laboratory methods that would dlikely not be very useful in producing industrial amounts of the processin enzyme. The research reported here describes a new process for producing large amounts of the enzyme by methods that would be scalable to industrial processing, which is more likely to be adopted by commercial manufacturers interested in producing the modified alternan for their particular applications.

Technical Abstract: The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating alpha(1,6), alpha(1,3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been recently developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 107 kDa was observed when the product was analyzed by SDS-PAGE, and a molecular mass of 124 kDa was measured when the purified protein was analyzed by HPLC SEC-MALLS (size-exclusion chromatography-multi angle laser light scattering). A pI of 4.5 was determined by analytical isoelectric focusing. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high yield; 5.5 mg of purified alternanase was obtained from the cell-free supernatant of a 3 liter culture. The process produces enzyme of excellent purity which can be used to hydrolyze alternan to its various products for further investigation.

Last Modified: 4/16/2014
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