# US 2014/0154721 A1 · Cellulase Assay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154721 A1 - Document ID: US20140154721A1 - Status: Published application - Kind: A1 - Application number: 14/092,545 - Filed: 2013-11-27 - Published: 2014-06-05 - Earliest claimed: 2012-12-04 - Applicants: Megazyme IP Limited - Inventors: Barry V. McCleary; David Peter Mangan - Web page: https://patentgrep.com/patent/US20140154721A1 ## CPC - C12Q 1/34: involving hydrolase - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) A reagent kit as claimed in claim 1 further comprising an exo-enzyme capable of cleaving the bonds between the non-blocked glucose residues and the label. ### Claim 3 (depends on 2) A kit as claimed in claim 2 further comprising a second exo-enzyme if the oligosaccharide is α-linked to the label. ### Claim 4 (depends on 1, 2, 3) A kit as claimed in claim 1, 2 or 3 wherein the measurable change upon cleavage of the bond is optically measurable. ### Claim 5 (depends on 1, 2, 3) A kit as claimed in any one of claims 1 to 3 wherein the label is a chromophore, a fluorophore, a chemiluminescent substituent, or a bioluminescent substituent. ### Claim 6 (depends on 5) A kit as claimed in claim 5 wherein the label is selected from 2-chloro-4-nitrophenol (C1PNP), p-nitrophenol (PNP), o-nitrophenol, coumarin derivatives such as 4-methylumbelliferone, and luciferin. ### Claim 7 (depends on 2, 3) A kit as claimed in claim 2 or 3 wherein the first exo-enzyme is β-glucosidase or a mixture of β-glucosidase and α-glucosidase. ### Claim 8 (depends on 3) A kit as claimed in claim 3 wherein the second exo-enzyme is α-glucosidase. ### Claim 9 (depends on 1, 2, 3) A kit as claimed in an one of claims 1 to 3 wherein the substrate has six or fewer glucose units. ### Claim 10 (depends on 9) A kit as claimed in claim 9 wherein the substrate has two, three or four glucose units. ### Claim 11 (depends on 1, 2, 3) A kit as claimed in any one of claims 1 to 3 wherein the chemical blocking substituent is selected from the group acetals, ketals, and benzylidene. ### Claim 12 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. ### Claim 13 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. ### Claim 14 (depends on 13) A process as claimed in claim 13 wherein the chaotropic solvent inhibits neither the cellulase being measured nor the enzymes β-glucosidase and α-glucosidase. ### Claim 15 (depends on 13) A process as claimed in claim 13 wherein the solvent is dimethyl sulphoxide or another chaotrophic solvent able to dissolve the substrate.