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Purification of alternanase by affinity chromatography
Jeffrey A Ahlgren1, Gregory L Côté
1Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N University Street, Peoria, IL 61604-3999, USA.
Journal of Industrial Microbiology & Biotechnology
|March 4, 2003
Summary
Researchers purified alternanase, an enzyme that breaks down the polysaccharide alternan. This improved method yields pure enzyme, producing a novel cyclic glucose tetrasaccharide.
Area of Science:
- Enzymology
- Carbohydrate Chemistry
- Biochemistry
Background:
- Alternan is a polysaccharide synthesized by Leuconostoc mesenteroides.
- Alternanase enzyme from Bacillus sp. NRRL B-21195 hydrolyzes alternan.
- Enzymatic hydrolysis yields a unique cyclic glucose tetrasaccharide.
Purpose of the Study:
- To develop an improved purification scheme for alternanase.
- To characterize the purified enzyme and its product.
- To enable large-scale purification of active alternanase.
Main Methods:
- Affinity chromatography using isomaltosyl-agarose for selective enzyme binding.
- Elution with 0.5 M sodium chloride.
- Purification confirmation via preparative isoelectric focusing and HPLC size-exclusion chromatography/multiangle light scattering.
Main Results:
- A highly efficient purification protocol for alternanase was established.
- The purified enzyme preparation was nearly homogeneous.
- Analysis confirmed a single protein band of 117 kDa.
- The overall yield of the purification procedure was 36%.
Conclusions:
- The developed purification method is effective for obtaining high-purity alternanase.
- This scalable procedure allows for the production of substantial quantities of the enzyme.
- The purified alternanase can be used for further studies on alternan hydrolysis and its products.