ELSEVIER 0308-8146(95)00221-9 Food Chemistry, Vol. 56, No. 4, pp. 433437, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0308-8146196 $15.00 + .OO Use of crosslinked mucilage prepared from ruredzo (Dicerocaryum zanguebarium) in the purification of polygalacturonase extracted from tomato Mudadi A. N. Benhura & Isabella Mavhudzi Department of Biochemistry, University of Zimbabwe, P. 0. Box MP 167, Mt Pleasant, Harare, Zimbabwe The mucilage from ruredzo (Dicerocaryum zanguebarium) was crosslinked in alkaline conditions with epichlorohydrin. Polygalacturonase (PG) was extracted from ripe tomatoes by first macerating at pH 3 at low ionic strength. After washing off sugars, the enzyme was extracted in buffer containing 1.7 M NaCI. PG was bound to crosslinked mucilage (CLM) at pH 4 and released by eluting with buffer containing 1 M NaCI after washing off unbound proteins. A six-fold purification of PG was achieved. The purified enzyme showed two main bands of molecular weights 30 000 and 44 000. Used CLM was regenerated by treatment in 1 M NaCl and 1 M HCI. Treatment of PG with regenerated CLM produced results that were similar to the purification with fresh CLM. Copyright G 1996 Elsevier Science Ltd INTRODUCTION There is an increased trend towards the use of renew- able resources including plant materials. We have been trying to find applications for the mucilage that is isolated from ruredzo. If suitable uses can be iden- tified, we believe that a plentiful supply of the material would be available even when relying on the naturally growing plant as the only source of the mucilage. Ruredzo is a plant that grows widely in the sandy soils of many parts of Southern Africa. The creeping plant has traditionally been used for a variety of pur- poses that depend on the presence of a slimy material in the leaves (Benhura & Marume, 1993). The mucilage from ruredzo is a pectic material with a molecular weight of about 500000 and has been shown to contain 8% uranic acid groups. Crosslinked pectins have been used in the purification of polygalacturonase (PG), a cell wall enzyme that is responsible for the softening of vegetable material such as tomatoes (Inoue et al., 1984). The purification meth- ods are based on the ability of crosslinked pectate to act as an affinity adsorbent for PG. After the enzyme is bound to the matrix, environmental conditions such as pH and ionic strength can be changed in order to elute the enzyme (Kohn et al., 1976). At present, the purification of pectic enzymes involves several steps in order to produce a preparation of acceptable activity. One attraction of affinity chroma- tography is that substantial purification of proteins may be effected in a single step. Reduction in the number of steps that are involved in the purification of a given enzyme would be expected to reduce the cost of preparation of that enzyme. The purpose of this study was to investigate the use of crosslinked ruredzo mucilage (CSM) as an affinity adsorbent in the purification of PG from tomatoes. The procedure would not only provide an application for ruredzo mucilage but it would also effect purification of the enzyme in a single step. MATERIALS AND METHODS Extraction of PC Soft ripe tomatoes were purchased from a local super- market and stored frozen at -20°C. Tomatoes, chilled and sliced (200 g) were homogenized in a Waring Blen- der in 200 g of ice until all the ice had melted. The pH of the homogenate was adjusted to 3 with HCI and the mixture centrifuged at 8000 rpm in an ICE centrifuge for 20 min. The pellet was suspended in 180 ml of cold 433