Pectic polysaccharides solubilized in vivo during ripening, were isolated using phenol, acetic acid, and water (PAW) from the outer pericarp of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang and A.R. Ferguson var deliciosa 'Hayward') before and after postharvest ethylene treatment. Insoluble polysaccharides of the cell wall materials (CWMs) were solubilized in vitro by chemical extraction with 0.05 molar cyclohexane-trans-1,2-diamine tetraacetate (CDTA), 0.05 molar Na2CO3, 6 molar guanidinium thiocyanate, and 4 molar KOH. The Na2CO3-soluble fraction decreased by 26%, and the CDTA-soluble fraction increased by 54% 1 day after ethylene treatment. Concomitantly, an increase in the pectic polymer content of the PAW-soluble fraction occurred without loss of galactose from the cell wall. The molecular weight of the PAW-soluble pectic fraction 1 day after ethylene treatment was similar to that of the Na2CO3-soluble fraction before ethylene treatment. Four days after ethylene treatment, 60% of cell wall polyuronide was solubilized, and 50% of the galactose was lost from the CWM, but the degree of galactosylation and molecular weight of pectic polymers remaining in the CWMs did not decrease. The exception was the CDTA-soluble fraction which showed an apparent decrease in molecular weight during ripening. Concurrently, the PAW-soluble pectic fraction showed a 20-fold reduction in molecular weight. The results suggest that considerable solubilization of the pectic polymers occurred during ripening without changes to their primary structure or degree of polymerization. Following solubilization, the polymers then became susceptible to depolymerization and degalactosidation.Pectolytic enzymes such as endopolygalacturonase and #-galactosidase were therefore implicated in the degradation of solubilized cell wall pectic polymers but not the initial solubilization of the bulk of the pectic polymers in vivo.Studies detailing the involvement of pectolytic enzymes such as PG2 in cell wall degradation have been prompted by ' Supported in part by a grant from the New Zealand Kiwifruit Board.2 Abbreviations: PG, endopolygalacturonase; CWM, cell wall material; CDTA, cyclohexane-trans-1 ,2-diamine tetra-acetate; GTC, guanidinium thiocyanate; PAW, phenol:acetic acid:water (2:1:1, w/ v/v); SSC, soluble solids concentration; CTAB, hexadecyltrimethylammonium bromide; d.e., degree of esterification.the observation that the cell wall pectin content of many fruit decreases or changes its form during ripening. Attempts have been made to relate an increase in the activity of a particular cell wall-degrading enzyme to the onset and development of fruit softening. This approach has provided apparent correlations, but most are inconclusive, and one, polygalacturonase involvement in tomato softening, has largely been discredited by results of genetic experiments. PG activity in tomato fruit increases dramatically during ripening because of de novo synthesis of the enzyme (3, 5). However, when the PG gene was introduced into a nonripenin...

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The exo-b-1,3-glucanase of Candida albicans (Exg) has a marked specificity for b-1,3-glucosidic linkages as judged by the kinetic constants for p-nitophenyl b-glucoside, b-linked disaccharides of glucose (laminaribiose, gentiobiose, and cellobiose), oligosaccharides of the laminari series, laminarin and pustulan. The k cat /K m ratios for a series of laminari oligosaccharides from -biose to -heptaose showed that Exg has an extended substratebinding site which contains at least five binding sites for sugar residues. Binding at position +2 (the third sugar residue) increases the k cat twofold while positions +3 and +4 lower the K m value further and thereby increase the catalytic efficiency. Exg catalyses an efficient transglucosylation reaction with high concentrations of laminarioligosaccharides which specifically form b-1,3 linkages and with yields up to 50%. The rate of the transglucosylation is concentration-dependent and can be more than 10 times faster than the hydrolytic reaction with excess donor substrates such as laminaritriose and laminarihexaose. The kinetics of Exg and the predicted substrate-binding site for up to five sugar residues are consistent with a recent structural analysis of the enzymebinding site.Keywords: Candida albicans; exo-b-1, 3-glucanase; glycosidase; transglucosylation.b2Glucan, a homopolymer of glucose containing b-1,3, b-1,6 and b-1,3,6 linkages is the main structural component of the cell walls of many yeasts and some filamentous fungi (for reviews, see [1,2]). Linear b-1,3-glucan is produced by a UDP-glucose-dependent synthetase located in the plasma membrane and delivered to the nascent cell wall in a vectorial process [3]. Other reactions involved in the assembly of b-glucan have not been identified but must include: the formation of b-1,6 linkages, b-1,3,6 branch points and crosslinkages to other wall constituents [2,4].Enzymes implicated in glucan catabolism have been studied in some detail. Endoglucanases and exoglucanases are secreted into the cell wall by many organisms and the putative roles for these include localized breakdown of b-glucan for wall expansion, mobilization of glucan for use as a fuel and the hydrolysis of exogenous material for uptake as a nutrient (for reviews see [5,6]). It has also been suggested that some glucanases may, within the wall, catalyze transglycosylation rather than hydrolytic reactions and thereby contribute to rearrangement and assembly of wall glucan [7]. For example, the BGL2 gene product of Candida albicans, Saccharomyces cerevisiae and Aspergillus fumigatus previously described as an endo-b-1,3-glucanase [8] preferentially catalyzes a glucanosyltransferase reaction in which new 1,6 linkages are introduced into 1,3 glucan [9±12].Exo-b-1,3-glucanase (Exg) has been purified from several sources. The genes for EXG1 of S. cerevisiae [13] and EXG of C. albicans [14] share 58% identity, and closely related genes have been detected in other Candida species [15]. The enzymes from S. cerevisiae and C. albicans are similar in many respects al...

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