Enzymes polygalacturonase

The objective of this research was to follow the course of growth of H.annuus 1805 cell suspension in parallel with the changes in the amount of cell walls, as well as the course of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase and the... 

H.annuus 1805 cell suspension was cultivated on a shaker (11.6 rad/s) at 26-28 °C in the dark in 1/5 net volume flasks. Duration of growth was different from 5 days for obtaining the inoculum to 10 days for studying the course of growth, the course of changes in the cell walls and the courses of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase as well. For inoculation 20 % (v/v) five-day cell suspension, containing 9 g/L dry cell biomass, was used. 

Enzyme.—Polygalacturonase (PG) was obtained from a culture of Rhizopus nigricans using Citrus pectin as carbon source [11,12], The enzyme used for oligouronides obtention was the residual activity after a thermal treatment (100°C, 60 sec) of the native Rh. nigricans endo and exoPG, since the endo-enzyme was thermoresistant while the exo-enzyme was thermolabile [13]. 

There are several groups of pectic enzymes, including pectinesterase, the enzyme that hydrolyzes methoxyl groups, and the depolymerizing enzymes polygalacturonase and pectate lyase. 

In liquid medium, the thiobarbuturic acid test was used to determine polygalacturonase and pectate lyase activity (Sherwood, 1965). 1 ml of the crude enzyme preparation was added to 2 ml of 0.5 N HCl in a test tube. 4 ml of 0.01 M thiobarbuturic acid, dissolved in distilled water, were added. The tubes were heated in a boiling water for Ih and centrifuged. The absorption of the supernatant was determined in the spectrophotometer over the range 480-580 nm. Reaction mixtures without enzyme, which showed no reaction with thiobarbuturic acid, were used as controls. 

In order to characterize the pectinolytic enzymes encoded by these clones, the culture supernatants of all these clones were tested for pectate lyase and polygalacturonase activity, using thiobarbutiric acid as described in materials and methods. Absorption at 550 nm indicates the activity of pectate lyase whereas absorption at 510 nm indicates the activity of polygalacturonase. 

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