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United States Department of Agriculture

Agricultural Research Service

Title: Purification and Characterization of An Oat Fructan Exohydrolase That Preferentially Hydrolyzes Beta-2,6-Fructans

Authors
item Henson, Cynthia
item Livingston, David

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 23, 1995
Publication Date: N/A

Interpretive Summary: An important part of a plant's ability to survive hostile environmental conditions depends on its capability to store excess food when it's available, for retrieval during periods of limited food supply. Most plants store excess food as large carbohydrates. Retrieval is dependent upon the breakdown of stored carbohydrates to small sugars that cells can easily use for energy. The objective of this work was to determine how oats breakdown, or retrieve, the carbohydrates stored in their stems and synthesize the small sugars that cells need for energy. This work successfully determined which protein is responsible for breaking down the most abundant carbohydrate stored in oat stems and showed that this protein produces fructose, a small sugar. This protein rapidly breaks down the carbohydrate that is known to accumulate during cold hardening/acclamation, and therefore plays a significant role by degrading the stored carbohydrate ein the spring allowing plants to regrow when the weather is favorable. Th biochemical characteristics of this protein that we determined indicate it functions under conditions known to exist inside oat cells. The protein identified and characterized in this work is considered a good candidate for manipulation with biotechnology procedures because it is a primary component of an oat plant's biochemical defense against hostile environmental conditions, such as low temperature or drought.

Technical Abstract: Fructans were isolated from oats, purified and their structures determined. These oat fructans were used as substrates for purification of a fructan hydrolase from oats and were also used to characterize the substrate specificity of the purified hydrolase. The purified protein had a mol. mass of 43 kD as determined by SDS-PAGE. The purified fructan hydrolase catalyzed hydrolysis of the terminal beta-2,6-bond of (6G,6)- kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal beta-2,6-bond of 6G-kestotriose and approximately 10 times faster than it hydrolyzed terminal beta-2,1-bonds of chicory inulin. Sucrose was not a substrate. The Km for avenose (beta-2,6-linked fructans with 7-14 degrees of polymerization) hydrolysis was 2.8% (w/v) and the Vmax was 0.041 umol/min/ml. The Km for hydrolysis of (6G,6)-kestotetraose was 5.6% (w/v) and the Vmax was 0.138 umol/min/ml. Catalysis was exolytic and by multiple chain attack. The enzyme was maximally active at pH 4.5-5.0. Chemical modification of oat fructan hydrolase with n-bromosuccinimide in the presence and absence of avenose identified tryptophan as an essential functional group at or near the active site. Chemical modification with diethylpyrocarbonate suggests that histidine may also have a significant role in enzyme function but is probably not at the active site.

Last Modified: 12/21/2014
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