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ARS Home » Midwest Area » Madison, Wisconsin » Cereal Crops Research » Research » Publications at this Location » Publication #102373
Title: OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF BARLEY ALPHA-GLUCOSIDASE SECRETED BY PICHIA PASTORIS

Author
item MUSLIN, E - UNIVERSITY OF WISCONSIN
item KANIKULA, A - UNIVERSITY OF WISCONSIN
item CLARK, S - UNIVERSITY OF WISCONSIN
item Henson, Cynthia

Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/21/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: The two primary uses of barley seeds, in both domestic and international markets, are as animal feed and for malting to produce alcohol. When barley seeds are used for malting, special characteristics are required that are not necessary for barley used as animal feed. One of these special characteristics is that the malt must be able to rapidly degrade starch and produce high amounts of fermentable sugars. Furthermore, the malt must be able to do this at the relatively high temperatures used by industries that convert starch to fermentable sugars. Therefore, we have studied the effects of temperature on one of the key components of malt responsible for starch conversion to fermentable sugars. The component we studied is the enzyme alpha-glucosidase (AGL). We established that AGL by itself has greatly reduced activity at temperatures used by industry, but that its activity can be stabilized significantly by the addition of sugars. This information can be used to develop protocols for addition of adjuncts during starch conversion at high temperature. Future studies should address the optimal composition and concentration of an adjunct and the optimal timing of its addition.

Technical Abstract: Alpha-glucosidases are recognized as important in starch degradation during cereal seed germination, yet no studies have been published of the purified protein's characteristics that demonstrate its suitability for use as an industrial enzyme. A barley alpha-glucosidase (AGL) expressed in Pichia pastoris was obtained from shake-flask culture, however the yield was low necessitating the use of multiple batches. Problems arose because of significant batch-to-batch variation. We solved these problems by switching to a fermentation system producing sufficient quantity of a uniform sample. Methyl-hydrophobic interaction chromatography was used to purify the enzyme to a single band when analyzed by denaturing electrophoresis. We examined the enzyme's thermal stability at pHs that occur during seed germination and at pHs used by industry during high temperature starch conversions. We demonstrated that AGL was more sensitive to high temperature at acidic pHs and that additions of sugars significantly stabilize the activity at acidic pH. We clearly showed that the enzyme hydrolyzed the glucosidic bonds present in maltose and nigerose, but, in contrast to earlier reports using impure enzyme, hydrolyzes trehalose at rates only marginally above the detection limits.