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US 2014/0154721 A1Published application
Cellulase Assay
OwnerBarry V. McCleary, David Peter Manganinventors
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OwnerBarry V. McCleary, David Peter Manganinventors
Estimating the term…
The invention involves the development of a substrate and assay procedure for the scientific measurement of cellulase in admixture with other enzymes active on cellulose or 1,4-β-D-cello-oligosaccharides, or in various levels of purity of the cellulase. This substrate being characterised by the fact that it contains at least 2 glucose units linked β-1,4-, with the reducing end glucose unit linked β- or α- to a compound which exhibits an optically measurable change on cleavage of the bond between it and the adjacent D-glucosyl residue and the non-reducing terminal D-glucosyl unit is covalently linked to a blocking substituent which inhibits cleavage by exo-acting enzymes of the bond between the terminal and non-reducing end, penultimate D-glucose unit.
A reagent kit for measurement of cellulase comprising, an oligosaccharide substrate for assay of cellulase, the said substrate containing at least 2 glucose units linked by a 1,4-β-linkage, with the reducing-end glucose of the oligosaccharide linked via a bond cleavable by β- or α-glucosidase, to a label which exhibits a measurable change upon cleavage of the bond, and the non-reducing glucose unit being linked to a chemical blocking substituent which inhibits cleavage by exo-enzymes of the bond between the non-reducing end glucose unit and the adjacent glucose unit.
A method for the measurement of cellulase in a sample comprising incubating the sample with an oligosaccharide substrate and with or without an exo-enzyme, the substrate containing at least 2 glucose units linked by a 1,4-β-linkage, with the reducing-end glucose of the oligosaccharide linked via a bond cleavable by β- or α-glucosidase, to a label which exhibits a measurable change upon cleavage of the bond, the exo-enzyme if present being capable of cleaving the bond between the reducing-end glucose unit and the label, and measuring a change between the sample and a control, on cleavage of the bond, the change indicating the amount of cellulase in the sample.
A process for the dissolution of the end-blocked p-nitrophenyl cello-oligosaccharide or end-blocked 2-Cl-4-nitrophenyl cello-oligosaccharide, either of which are sparingly soluble in water or buffer solutions, comprising use of a chaotropic solvent.
Dependencies are inferred from the claim text.