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-sulfonic 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 microM 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 microgram/ml.
Mangos
T J
TJ
United States Department of Agriculture, Eastern Regional Research Center, Agricultural Research Service, Wyndmoor, Pennsylvania 19038, USA. tmangos@arserrc.gov
Haas
M J
MJ
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D052160:Benzothiazoles; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000379:methods; D005557:Formaldehyde / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007202:Indicators and Reagents; D010368:Pectins / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*; D013451:Sulfonic Acids
D052160:Benzothiazoles; D007202:Indicators and Reagents; D013451:Sulfonic Acids; D005557:Formaldehyde; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D010368:Pectins; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D009195:Peroxidase; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase