A spectrophotometric assay for the enzymatic demethoxylation of pectins and the determination of pectinesterase activity

A rapid spectrophotometric method for the determination of pectinesterase activity is presented. In this assay, methanol released from pectin 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 possible to couple the methanol assay directly to the action of pectinesterase for the real-time determination of this enzyme. The assay is reliable and sensitive, being capable of quantitating a minimum pectinesterase activity of 0.0625 unit (1 unit = 1 mu M methanol released per minute). It is also capable of detecting the enzymatic demethoxylation of galactopyranosyl uronic acid methyl esters of pectin down to a minimum concentration of 1.56 nM of methanol per milliliter using a pectin substrate with a methoxy content of 10% (w/w) at a concentration of 0.5 mu g/ml.