Preparation and Purification of Glucanase and Chitinase from Bean Leaves

Abstract

Glucanase (endo-0-1,3-glucan 3-glucanohydrolase, EC 3.2.1.6, laminarinase, callase) and chitinase (poly-0-1,4-[2-acetamido-2-deDxyl-D-glucoside glycanohydrolase, EC 3.2.1.14) were extracted from ethylene-treated bean (Phaseolus vulgaris L. cv. Red Kidney) leaves and purified on hiydroxvapatite and carboxymethyl Sephadex columns. The glucanase prepared was homogeneous as judged by anaIlvtical centrifugation data, electrophoresis, and antibodyantigen reactions. On the basis of gel filtration, antibodyantigen reactions, and amino acid analysis, the molecular weight was estimated to be between 11,300 and 12,300. However, ultracentrifugation gave a higher estimate of 34,000. The glucanase had an isoelectric point near pH 11 and was specific for B-1,3-linkages. The chitinase was only partially purified as judged by electrophoretic behavior.Enzyme systems capable of generating (13) and degrading (8) ,B-1 ,3-glucans have been described. The f-1,3-glucans themselves are widespread and have been associated with callose, leaf and stem hairs, root hairs, cystoliths, pollen mother cells, laticifers, pollen grains, pollen tube walls, wounded parenchyma cells (10), ovules (12), development of microspores from tetrads (15), and cell walls (17). fl-1,3-Glucans are also important components of carbohydrate reserves of cereals, algae, and fungi (5).A number of roles have been proposed for glucanase (fl-1,3-glucanase, laminarinase, callase) in plants. These include degradation of seed glucans (11), control of cell elongation (17, 20) (see however 9, 26), regulation of pollen tube growth (25), cell expansion of yeast (27), fertilization (12, 15), and removal of phloem callose (8).In an earlier paper (1), we described an increase in glucanase in ethylene-treated bean leaves that was associated with protein synthesis de novo and correlated with the removal of callose from the phloem. In order to characterize the enzyme more fully and to evaluate the role of the enzyme in the normal phys- iology of the plant, we have selected a purification procedure that resulted in the preparation of electrophoretically pure glucanase. MATERIALS AND METHODS Preparation of Glucanase. Bean (Phaseolus vulgaris L. cv.Red Kidney) leaves (1,300 g) treated for 3 days with 10 ,ul/liter of ethylene (2) were homogenized with 1,400 ml of water in a Waring Blendor. Except where noted, this and subsequent steps were performed at 5 C. The homogenate was filtered through cheesecloth and centrifuged at 10,000g for 10 min, and the precipitate was discarded (step 1). The supernatant was heated to 60 C for 10 min by placing the flask containing the homogenate in a boiling water bath, stirring rapidly, and monitoring the temperature rise until 60 C was reached. The flask was then removed from the bath, cooled slowly at room temperature, and, after 10 min, placed in an ice bath to lower the temperature to 5 C. The denatured protein was removed by centrifuging at 10,000g for 10 min (step 2).The 60 C supernatant was mixed with diethylaminoethyl cellulose (Cellex-D,...

Chitinase from Leaves of Beta vulgaris and other Higher Plants

“…Result of the screening experiments indicated that the enzymatic activity was varied not only with the species of the tested plants, but also according to the morphology of the examined parts. These results are to great extent in accordance to those found by Powning and lrzykiewicz (1965), Abeles et al (1971), Hirano et al (1990), Leah et al (1991) and Chang et al (1996). The increase in activity of chitinase during germination may be due to activation of the latent form and for increase in the rate of de novo synthesis of enzymes.…”

“…The differences in optimum conditions for extraction of chitinase from sugar beet, red beet and cabbage leaves may be due to relative solubility and stability of each enzyme. Abeles et al (1971) used distilled H 2 O as an extraction solution for isolation of chitinase from bean leaves, while Fink et al (1988) used sodium phosphate buffer (pH 6.0) to isolate this enzyme from oat leaves. In this study, sugar beet leaves proved to be the best source for chitinase.…”

Physical and Chemical Properties ofβ-1,3-Glucanase from Cultured Tobacco Cells

“…The isoelectric point of the enzyme was estimated to be 9.9 by this method. (3-l,3-Glucanases from kidney bean leaves 18 ) and germinated rye O ) were also found to be basic proteins.…”

Host-Pathogen Interactions

“…The correction factors (J) for the four fractions are: I, 1.03; II, 1.76; III, 1.51; and IV, 1.71. The mannose content of each fraction is accounted for by multi-plying the anthrone-determined glucose equivalents of a fraction by the computed correction (J) factor for that fraction.…”