Submitted to: Meeting Abstract
Publication Type: Abstract only
Publication Acceptance Date: 9/16/2009
Publication Date: 9/16/2009
Citation: Rose, D.J., Inglett, G.E. 2009. A Simple Method for the Determination of Xylanase Activity on Insoluble Substrates. AACC International Annual Meeting. 00:000-000. Interpretive Summary:
Technical Abstract: The propensity for a xylanase to convert insoluble (arabino)xylan into soluble oligosaccharides is an important parameter in the baking, pulp and paper, prebiotics, and biofuel industries. 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, rapid method for the determination of xylanase activity on native substrates was developed. This method involves incubation of the enzyme preparation with a material containing insoluble (arabino)xylan under the proper conditions. Following the reaction, total (arabino)xylan released is quantified as total pentose sugars in solution spectrophotometrically using a modification of the phloroglucinol method of Douglas (Food Chemistry 1981, 7, 139-145). This method was tested using two commercial xylanase preparations and four 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. Stopping the reaction by pH adjustment resulted in a significant increase in the absorbance of the blank. Relative standard deviations were below 5% on samples assayed on different days. Enzyme 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 xylanase activity.