Electrophoresis zyxwvutsrqponm 1988, zyxwvutsrqponmlk 9, zyxwvutsrqponm 327-330 Purification of cellulases by electroendosmotic preparative electrophoresis 327 z Andrea Curioni Angelo Dal Belin Peruffo Marco P. Nuti Dipartimento di Biotecnologie Agrarie, Universita di Padova 1 Introduction Purification of cellulases from Streptomyces strain A20 by electroendosmotic preparative electrophoresis Five cellulase components were identified and purified in one step from Streptomyces strain A20 using electroendosmotic preparative electrophoresis. By this procedure up to 18 mg of protein mixture could be loaded on the column, with an estimated recovery of 60-70 % of total activity; activity and protein recovery could be es- timated 32 % and 47 %, respectively, if only activity peaks were considered. In com- parison to other purification methods, this technique results in high protein recovery and resolution of applied samples. Several methods have been described for the separation and purification of microbial cellulolytic enzymes: gel filtration [ 1, 21, ionic-exchange chromatography [3, 41, affinity chroma- tography 15, 61, preparative isoelectric focusing [4, 71, high performance liquid chromatography [81, hydrophobic chro- matography [91, or separation on hydroxylapatite [ 11. These methods are often considered expensive and/or time-consum- ing. Recently a preparative gel electrophoresis system based on electroendosmosis, which is the backward flow occurring between the electrodes during electrophoresis, has been proposed [lo]. In this system a dialysis membrane is tightly held against the terminal end of a cylindrical gel. The surface between the membrane and the gel forms a near-zero volume elution chamber, the latter being connected by a capillary, situated in the middle of the gel, with the exterior. When an electrical current is applied between the electrodes, the electroendosmotic flow of the buffer crosses the membrane rinsing the terminal end of the gel. Any emerging molecular species is therefore eluted through the outlet capillary. This method may offer the advantage of rapid isolation of large quantities of pure protein. The aim of the present study was to assess the usefulness of this electrophoretic method in op- timizing the separation conditions for microbial cellulases. 2 Materials and methods 2.1 Materials Acrylamide, N,N'-methylenebisacrylamide, N,N,N',N' tet- ramethylethylenediamine (TEMED), ammonium persulfate and Bio-Gel P-30 were purchased from Bio-Rad, tris-(hydrox- ymethyl) aminomethane (Tris), CM (carboxymethy1)-cellu- lose were purchased from Sigma, Avicel was from Merck. All other chemicals were of analytical grade. 2.2 Microorganism and culture conditions A wild-type Streptomyces sp., strain A20, isolated from the gut ofhigher termites [ 111was used throughout this study. The actinomycete was maintained as stock in 20 % glycerol at + 4 OC. For cellulase production, the strain was grown in the presence of 2 % Avicel as a sole carbon source, for 72 h at 37 "C, on a rotatory shaker at 150 rpm. Correspondence: Angelo Dal Belin Peruffo, Dipartimento di Biotecnologie Agrarie, Universita, Via Gradenigo, 6, zyxwvutsrqp 1-35 13 1 Padova, Italy Abbreviations: CM, carboxymethyl 2.3 CM-cellulaseactivity CM-cellulase activity was determined by measuring the release of reducing equivalents (with glucose as standard) in a solution of CM-cellulose by the dinitrosalicilic acid method 1121. The assay conditions were 0.95 mL of 2 % w/v CM- cellulose in 0.1 M citric/phosphate buffer, pH 6, and 0.05 mL of enzyme solution incubated at 37 OC. One international unit of activity (I. U.) is equivalent to one mole of reducing sugar produced/min. 2.4 Preparation of cellulases for electrophoresis Cells and undegraded Avicel were removed from cultures by centrifugation for 20 min at 10 000 g and 4 "C. (NH&SO4 was added to the supernatant (3 L) at 4 OC to give 80 % satura- tion. After stirring for 30 min the precipitated protein was collected by filtration on Whatman No. 1 paper and resus- pended in distilled water. This solution, after centrifugation for 15 min at 10 000 g and 4 "C, was subjected to chromato- graphy on a Bio-Gel P-30 column (4 x 85 cm), equilibrated with water at a flow rate of 50 mL/h. CM-cellulase activity was eluted as a single symmetrical peak. The appropriate fractions were pooled, concentrated and dialyzed with H,O by ultrafiltration through Spectrum C-5K membranes. The resulting solution was used for subsequent electroendosmotic preparative electrophoresis. The protein content and the specific activity of the concentrated solution were 20.5 mg/ mL and 21 I. U., respectively. 2.5 Preparative gel eleetrophoresis The apparatus consisted of a) an electroendosmotic prepara- tive electrophoresis unit (ELFE, supplied by M-Medical, Firenze, Italy) (Fig. I), b) a detector (HM-Holochrome, Gilson with a 70 pL flow cell), set at 280 nm, c) a fraction collector (Model 202, Gilson), d) a double channel recorder (Model 22 10, LKB), e) a power supply (Model 500/200 Bio- Rad). The method of Laemmli I1 31 was used for the prepara- tion of both the running gel (T = 7.5 %, C = 2.5 %) and the stacking gel (T = 4 %, C = 4 %). The detergent sodium dodecyl sulfate was omitted. The dimensions of the running gel were 2.00 (diameter) x 3.5 cm long and those ofthe stack- ing gel 2.00 x 1.6 cm. After polymerization, the base of the gel was released from the solid support and subsequently covered with a glass-wool filter (Whatman GF/C) and a dialysis membrane (Millipore type VS 0.025 pm) moistened with running buffer; both filter and membrane were previously deaerated for 10 rnin with a vacuum pump. To obtain a con- tinuous flow ofrunning buffer through the capillary tube it was 0 173-0835/880707-0327$02.50/0 OVCH Verlagsgesellschaft zyxwvutsrqp mbH, D-6940 Weinheim, 1988