Viscometric Method for Assaying of Total Endodepolymerase Activity of Pectinases

A. V. Gusakov^*, A. V. Markov, S. G. Grishutin, M. V. Semenova, E. G. Kondratyeva, and A. P. Sinitsyn

School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 939-0997; E-mail: avgusakov@enzyme.chem.msu.ru

^* To whom correspondence should be addressed.

Received April 16, 2001

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An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40°C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.

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KEY WORDS: pectin, pectinase, polygalacturonase, polymethylgalacturonase, pectin lyase, pectate lyase, endodepolymerase activity, viscometry

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