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Multiple Forms of Pectinesterase are Present in Tomato and Other Fruit

Introduction

Pectinesterase (PE; EC 3.1.1.11) is an enzyme involved in the degradation of plant cell walls. As well as its role in fruit ripening, PE often interferes with the subsequent processing of fruit products. An understanding of its action would, therefore, further basic research and bring commercial benefits to the food industry.

Most work on PE has been conducted on tomato, in which at least 3 isoforms have been identified. The dominant isoform (PE2) has been purified, and the entire protein sequenced in order to determine its primary structure. A cDNA clone corresponding to this isoform was isolated, and antisense technology used to suppress PE2 gene expression. Fruit from these plants contained less than 10% PE activity of normal fruit.

The residual PE activity in these plants was thought to be due to the activity of minor isoforms with insufficient homology to PE2 for antisense downregulation to suppress their gene expression. The downregulation of PE2 appeared to have little effect on the ripening and processing characteristics of the fruit. Consequently, recent work has focused on the minor isoforms, known as PE1 and PE3. Despite these being present in far smaller amounts than PE2, their proteins have also been purified and sequenced. Additionally, unlike PE2, these isoforms are not fruit specific, so could have other important roles in the plant.

Formerly, PE isoforms were separated using anion exchange on DEAE Sephadex A50. Our method uses cation exchange on CM-Sepharose, which was reported by Warrilow et al. (1994) to improve separation of the enzyme. Some work has also been conducted using affinity chromatography on heparin, which was shown by Rillo et al. (1992) to give excellent separation of PE.

Traditionally, a titrator had been used to measure PE activity. While satisfactory for the calculation of total PE activity in fruit, it was obvious that the method would be too time consuming for the analysis of column fractions, so a new technique was developed. Our method uses a plate reader which can simultaneously read 96 samples, so is ideal for the analysis of isoform profiles.

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