A dyed substrate for the assay of endo-1,4-b-glucanases

RESCIGNO, ANTONIO;RINALDI, ANDREA;CURRELI, NICOLETTA;OLIANAS, ALESSANDRA;SANJUST, ENRICO

1994-01-01

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Abstract

A new dyed substrate was prepared for the rapid determination of endo-1,4-beta-glucanases. Carboxymethylcellulose was coupled with Ruthenium red to obtain a violet powder, which was stable enough to allow for reproducible assays. The spontaneous reaction is based on ionic interactions between the negatively charged carboxymethylcellulose and the complex cation of the dye. The enzymic assay is based on spectrophotometric measurement at 535 nm of the enzyme-released dyed fragments with low-molecular-weight filterable through a 0.22 mu m filter. The release of coloured fragments from this dyed substrate was proportional to its solubilization. The absorbance was directly proportional to the enzyme amount in the range 0.5-5.5 mU enzyme (A(535) = 0.1-0.95). This enzymic assay is advantageous for both rapid time of analysis and sensitivity.