Isolation and Identification of Trehalase from Pullularia pullulans

Abstract

Trehalase has been isolated from Pullularia pullulans. The enzyme, which is specific for trehalose, was purified approximately 800-fold. The optimal pH was found to be 4.0 and the Michaelis dissociation constant, Km. was determined to be 3.2 x 10-3 M. Trehalose has been found in microorganisms, vertebrates, invertebrates, and phanerogams (15). In fungi, trehalose functions as a reserve carbohydrate sustaining respiration and accelerating germination (6,7,14). Trehalose has recently been isolated from the yeastlike fungus Pullularia pullulans in which its accumulation parallels the increase of pigment formation in the cells (12,13). P. pullulans is exceedingly common and found on fruits, vegetables, meat products, and cheese and is involved in the deterioration of paint and discoloration of lumber. Thus, it seemed of interest to undertake the isolation and characterization of trehalase from these cells. P. pullulans (strain NRRL YB-4515) was grown in a medium consisting of (Difco) 0.3% malt and yeast extracts, 1.5% glucose, 0.0135% K2HPO4, 0.01 % MgSO4-7H20, 0.01% KH2PO4, and 0.02% NH,Cl. After 5 days of shaking on a rotary shaker (220 rev/min and 2.5-inch thrust) at 28 C, the cells were harvested by centrifugation and washed first with 1% NaCI solution and then with distilled water until free from chloride. The cells were broken by adding to each gram of cells 3 g of 0.1-mm glass beads and 3 ml of 0.1 M sodium acetate buffer (pH 5.5) in a Sorvall Omni-mix. The rupturing was performed at 5 C at a rheostat setting of 100 for I hr, at which point microscopic examination indicated 80 to 90% breakage. The supernatant fluids were then centrifuged at 1,370 x g for 10 min, and the resulting residue was washed several times with 0.1 M acetate buffer (pH 5.5), hereafter referred to as "standard buffer." The washings were combined with the supernatant fluids, and the combined mixture was centrifuged at 6,590 x g for 10 min followed by centrifugation of the supernatant at 105,400 x g for 60 min. Enzyme activity was measured by employing samples of the fractions mixed with I ml of sugar solution containing 10 ,umoles of trehalose dissolved in standard buffer. After 15, 30, 45, and 60 min of incubation at 37 C, the amount of reducing sugar was determined by the Folin-Wu method (3). A unit of enzyme activity is defined as that amount of enzyme which produced 1.0 ,umole of glucose per min per mg of protein at 37 C. Protein content of the enzyme solutions was determined by the procedure of Lowry et al. (1 1).Ammonium sulfate was added to the supernatant liquid containing the enzyme with stirring at 5 C to a concentration of 40% saturation. After removal of the precipitate by centrifugation, the addition of ammonium sulfate was continued until a 70% saturation was obtained. After separation of the precipitate by centrifugation (20,000 x g), the supernatant was dialyzed against distilled water until free from ammonium sulfate. A small portion of this solution was dialyzed against standard buffer, whereas the remaining portion wa...

Regulation of trehalose mobilization in fungi.

“…Another characteristic of the regulatory type of trehalase might be a higher Km than that for the nonregulatory trehalases. The Km values of the nonregulatory trehalases fall in the range of about 0.5 to 3 mM (70,74,81,82,108,109,128,134,139,208,211). Km values reported for regulatory trehalases are 4.5 (88), 10 (6), and 55 (198) mM.…”

Regulation of trehalose mobilization in fungi

“…These data were taken as additional evidence for the existence of compartmentation between trehalose and trehalase. Other fungi in which trehalase was found only in the soluble fraction of cell extracts include Dictyostelium discoideum (82), Pullularia pullulans (109), and ascospores of Pichia pastoris (Thevelein, unpublished results). It was suggested that in spores of Dictyostelium discoideum, trehalose was localized in vesicles (62,114), as opposed to the localization of trehalase in the cytosol.…”

“…Another characteristic of the regulatory type of trehalase might be a higher Km than that for the nonregulatory trehalases. The Km values of the nonregulatory trehalases fall in the range of about 0.5 to 3 mM (70,74,81,82,108,109,128,134,139,208,211). Km values reported for regulatory trehalases are 4.5 (88), 10 (6), and 55 (198) mM.…”

A neutral trehalase gene from Candida albicans: molecular cloning, characterization and disruption

“…Trehalases, trehalose-hydrolysing enzymes, have been isolated from many organisms (Hill & Sussmann, 1963; Killick, 1985; Merdinger et al, 1971; Retief & Hewitt, 1973; Arguelles & Gacto, 1985). Fungal trehalases are divided into acid trehalases and neutral trehalases (Thevelein, 1984).…”