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Multiple Forms of Pectinesterase are Present in Tomato and Other Fruit

PE Assay development

A colorimetric method which measures PE activity using a plate reader was described by Cameron et al. (1992). The method determines PE activity by the measurement of H+ ions released from the PE mediated demethylation of pectin using a pH indicator. Our method further develops the one described by Randall et. al., using the more sensitive pH indicator phenol red, instead of bromothymol blue.

PE enzyme was extracted from tomato and a series of enzyme dilutions made. These dilutions were used to calibrate the plate reader by calculating PE activity from the titrator and comparing them to the rate colour change of the pH indicator on the plate reader. Bromothymol blue was found to have a 3x lower sensitivity relative to phenol red.

To provide the enzyme for isoform analysis, acetone insoluble solids were prepared from fruit tissues, extracted into aqueous buffer, and precipitated with ammonium sulphate. The protein was resuspended into buffer for analysis using high resolution liquid chromatography (HRLC). Protein was applied to two BioRad Econo-Pac CM-Sepharose cartridges connected in series and enzyme eluted by increasing the NaCl gradient with time. Buffer flow rate was a constant 2 ml per min., with 75 x 2 ml fractions collected. Using the precalibrated plate reader software, a PE assay was performed, with PE activity being calculated as µeqH⁺ min^-1 per 2 ml column fraction.

Further Reading

• Title Page
• Introduction
• Assay development
• Total Pectinesterase activity in fruit
• Pectinesterase in Tomato
• Pectinesterase in Strawberry
• Pectinesterase in citrus fruits
• Pectinesterase in Grapes
• Summary & Future work
• References