A SPECTROPHOTOMETRIC ASSAY FOR THE ENZYMATIC DEMETHOXYLATION OF PECTINS AND THE DETERMINATION OF PECTINESTERASE ACTIVITY

A rapid spectrophotometric method for the determination of pectinester ase activity is presented. In this assay, methanol released from pecti n by pectinesterase is oxidized with alcohol oxidase to form hydrogen peroxide and formaldehyde. Hydrogen peroxide is then quantitated with peroxidase and the chromogen 2,2'-azino-bis(3-ethylbenzthiazoline-6-s acid). Since both reactions exhibit the same pH optimum it was possibl e to couple the methanol assay directly to the action of pectinesteras e for the real-time determination of this enzyme. The assay is reliabl e and sensitive, being capable of quantitating a minimum pectinesteras e activity of 0.0625 unit (1 unit = 1 mu M methanol released per minut e). It is also capable of detecting the enzymatic demethoxylation of g alactopyranosyl uronic acid methyl esters of pectin down to a minimum concentration of 1.56 nM of methanol per milliliter using a pectin sub strate with a methoxy content of 10% (w/w) at a concentration of 0.5 m u g/ml.