A method for the determination of soluble arabinoxylan released from insoluble substrates by xylanases

Authors: Rose, Devin; Inglett, George

Submitted to: Journal of Food Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2009
Publication Date: 2/14/2011
Citation: Rose, D.J., Inglett, G.E. 2011. A method for the determination of soluble arabinoxylan released from insoluble substrates by xylanases. Food Analytical Methods. 4(1):66-72.

Interpretive Summary: Arabinoylans are dietary fiber components in the cell walls of cereal grains and other plants. These arabinoxylans can be either water-soluble or water-insoluble depending on structure. Xylanases break down arabinoxylans into smaller, soluble molecules. The conversion of insoluble arabinoxylan to soluble compounds is an important parameter in the baking, pulp and paper, prebiotics, and fuel ethanol industries. Current methods for determining xylanase activity on insoluble substrates are labor intensive, non-specific, or utilize artificial substrates which may provide inaccurate results. Therefore, accurate and rapid methods for the determination of xylanase activity on insoluble substrates are needed. This manuscript describes the development of such a method, which was found to be applicable to multiple xylanases and insoluble arabinoxylan-containing substrates. This method will be useful for food, paper, and fuel ethanol manufacturers in the selection of more efficient xylanases in their applications, thus reducing processing costs.

Technical Abstract: The propensity for xylanase to convert insoluble arabinoxylan to soluble oligosaccharides is an important parameter in many applications. Current methods for determining xylanase activity on insoluble substrates are labor intensive, non-specific, or utilize artificial substrates which may provide much different results from native substrates. Therefore, a new method for the determination of xylanase activity on insoluble substrates was developed. This method involved incubation of the enzyme with a material containing insoluble arabinoxylan under the proper conditions. Following the reaction, total arabinoxylan released was quantified spectrophotometrically as xylose equivalents. Because this method measured total pentose sugars released into solution by the enzyme, (arabino)xylanase activity, rather than xylanase activity, was recommended for expressing results. The method was tested using two xylanases and six insoluble arabinoxylan-containing substrates. Proper buffer, reaction time, and stopping procedure were determined. Acetate and citrate buffers were suitable for the reaction; phosphate buffer substantially interfered with quantification of reaction products by reducing chromagen formation. The reaction was linear for at least 5 min under all conditions tested, after which the reaction was stopped by boiling for 2 min. Contaminating amylase and cellulase activity did not influence the results, despite the presence of starch and cellulose in many reaction slurries. Relative standard deviations were below 5% between reactions assayed on different days. Activity on different insoluble arabinoxylan-containing substrates differed by up to 100-fold, emphasizing the need for the use of application-specific substrates to obtain accurate estimations of enzyme activity.