Enzyme Activity Measurement for Alpha-D-Xyloside Xylohydrolase

Creative Enzymes provides a variety of enzyme assays, especially enzyme activity measurement. We work closely with customers to support their product development and process optimization needs. Having tested activities of a wide range of enzymes in the past few years, Creative Enzymes has accumulated extensive experiences, which enable rapid and high-quality activity assays for hydrolases such as alpha-D-xyloside xylohydrolase.

Alpha-D-xyloside xylohydrolase (EC 3.2.1.177; α-xylosidase) is an enzyme that catalyzes the release of α-xylose from the non-reducing terminal side ofα-xyloside. Some α-xylosidases from microorganisms and plants have been purified and characterized, but their amino acid sequences were never completely examined. There are few examples in enzymatic studies of α-xylosidase with the amino acid sequence elucidated. Among these, α-xylosidase from Sulfolobus solfataricus shows optimal activity at 90 °C and high hydrolytic activity on α-xyloside such as isoprimeverose, α-D-xylopyranosyl-(1,6)-D-glucopyranose, as well as oligosaccharides, such as maltose and maltotriose. The nasturtium, Arabidopsis, and pine α-xylosidases, which are established to be glycoside hydrolase (GH) family 31 enzymes, are considered to involve in the metabolism of xyloglucan. In Lactobacillus pentosus, it has been shown that xylQ, encoding α-xylosidase, is responsible for the metabolism of isoprimeverose. However, little is known about the enzymatic property of the α-xylosidase.

Representative α-xylosidases from GH-31 utilize a two-step mechanism. In the first step, the enzyme catalyzes the departure of the aglycon group from the substrate (donor) and the consequent formation of a glycosyl ester intermediate. In the second step, the enzyme is deglycosylated by a nucleophile that attacks the anomeric carbon of the donor and cleaves the covalent intermediate, leading to the overall retention of the anomeric configuration of the substrate. When a nucleophile different from water intercepts the glycosyl enzyme intermediate, transglycosylation occurs, producing glycosylated products.

Although α-xylosidases have been characterized enzymologically, very little is known about their structures and mechanisms. One of the reasons for the limited amount of mechanistic information is the lack of a convenient activity assay. Creative Enzymes is proud to offer the best-in-class assays of the enzymatic activity of α-xylosidases. The activities of α-xylosidases is determined by measuring the release of xylose spectrophotometrically. The amount of xylose released in reaction mixture is measured using the p-bromoaniline method.

With the most important resources, the skillful and experienced scientists of Creative Enzymes, we are happy to provide the advanced assay for enzyme activities. Overall, our state-of-the-art technology of enzymatic activity measurement is an ideal choice for any research and development activities involving α-xylosidases.

Figure: The crystal Structure of α-xylosidase from Escherichia coli.
PDB: 1WE5