Isolation of bacteria from a paddy field for CMCel- lulase secretion. A small amount of soil from paddy

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37°C and 150 rpm of shaking. #is cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. #e cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. #e enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. #e enzyme exhibited its optimal activity at 50°C and pH 6.0. #e enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50°C and its activity got inhibited by Hg, Cu, Zn, Mg, Na, and Ca. K e y w o r d s: Bacillus sp., biological degradation, carboxymethyl cellulase, paddy field, cellulase purification * Corresponding author: P. Vijayaraghavan, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam-629 502, Kanyakumari District, Tamilnadu, India; e-mail: [email protected] Vijayaraghavan P. and Vincent P. 1 52 agar, 15, CMCellulose, 10) and incubated at 37°C for 48 h. A%er incubation, bacterial growth was seen as a single line on the plate. To visualize the hydrolysis zone, the agar medium was 'ooded with an aqueous solution of Gram’s iodine. Gram’s iodine forms a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone within 3–5 min (Kasana et al., 2008). Bacterial identification was conducted based on ‘Bergey’s Manual of Systematic Bacteriology’ (Jones and Collins, 1984). Microorganism and submerged fermentation. #e Bacillus sp. was cultured in the nutrient medium composed of in (g/l): peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; sodium chloride, 5.0 and CMCellulose, 10 (pH 7.0), and sterilized at 121°C for 20 min. A loopful culture of 18 h Bacillus sp. was inoculated into the nutrient medium with rotary shaking at 150 rpm at 37°C for 36 h. #e culture medium was centrifuged at 6000×g for 20 min and passed through a 0.22-μm membrane (Rankam, NY0213SF) to remove the cells. #e resulting supernatant was used for further CMCase purification. Enzyme activity assays: a) Endo-β-1,4-glucanase. Endo-β-1,4-glucanase (EG) activity was determined by incubation of 1.0 ml of 0.2% (w/v) CMC in 0.025 M phosphate bu"er (pH 7.0) with 1.0 ml of appropriate concentration of enzyme at 37°C. A%er 30 min of reaction, 1 ml of dinitrosalicylic acid (DNS) was added and boiled in a water bath for 5 min to stop the reaction. #e resulting samples were then cooled to room temperature and the absorbance measured at 540 nm (A 540 ). One unit of EG activity was defined as the amount of enzyme that could hydrolyze CMC and release 1 μg of glucose within 1 min at 37°C (Nelson, 1944). b) Avicelase. Avicelase activity was determined by incubation of 1.0 ml of 0.2% avicel (w/v) in 0.025 M phosphate bu"er (pH 7.0) with 1.0 ml of appropriate concentration of enzyme at 37°C. A%er 30 min of reaction, the activity was measured. One unit of avicelase activity was defined as the amount of enzyme that could hydrolyze avicel and release 1 μg of glucose within 1 min at 37°C (Nelson, 1944). c) Filter paperase. In this assay Whatman No. 1 filter paper was used as the substrate. Fi%y milligrams of filter paper was dissolved in 0.025 M phosphate bu"er (pH 7.0) and 1.0 ml of appropriate concentration of enzyme. #e mixture was incubated at 37°C for 30 min and the reaction was terminated by adding 2 ml of DNS. One unit of filter paperase activity was defined as the amount of enzyme that could hydrolyze filter paper and release 1 μg of glucose within 1 min at 37°C (Nelson, 1944). Growth and fermentation. A loopful culture of the bacterial isolate was inoculated into the minimal medium (g/l) (peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; and sodium chloride, 5.0 (pH 6.5)), and incubated at 37°C in an orbital shaker at 150 rpm. Five millilitres of the culture medium was withdrawn at regular 12 h intervals and the cell density determined at 600 nm (Henriette et al., 1993) up to 72 h. To determine the optimum fermentation period for enzyme production the culture medium was centrifuged at 6000×g for 10 min. #e culture supernatant was assayed for determining enzyme activity (Nelson, 1944). Determination of protein concentration. #e protein concentration of the crude enzyme as well as that of the purified enzyme was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as the standard. Substrate specificity. #e hydrolytic ability of the crude enzyme sample against 0.2% CMC, 0.2% avicel, 0.2% cellobiose, and filter paper (20 mg) in 0.025 M phosphate bu"er (pH 7.0) was determined to evaluate its substrate specificity. CMCase purification Ammonium sulphate precipitation. #e proteins in the crude preparation were precipitated by the addition of solid ammonium sulphate at 40% to 80% saturation. #e precipitate was allowed to form at 4°C for 24 h, and was collected by centrifugation at 5000 ×g in a cold centrifuge for 20 min. #e precipitate was redissolved in 10 ml of bu"er A (0.025 M sodium phosphate bu"er, pH 7.0 containing 0.001 M ethylene diaminetetraacetic acid (EDTA) and 0.001 M 2-mercaptoethanol). Purification by ion exchange chromatography on diethylaminoethyl (DEAE) cellulose. DEAE cellulose was packed into a vertically mounted column (1.5×15 cm) and the 'ow rate of the column was set at 0.5 ml/min. #e column was equilibrated with 4 bed volumes of bu"er A. #e enzyme concentrate obtained from ammonium sulphate precipitation was redissolved in a minimal amount of bu"er A and dialysed overnight against the same bu"er. #e column was washed to remove all unbound proteins and a linear gradient of 0 to 0.5 M NaCl-added bu"er A was used to elute the bound proteins. Fractions (2 ml) were collected at a 'ow rate of 0.5 ml/min. Fractions exhibiting cellulase activity were pooled and concentrated with ammonium sulphate. #e precipitate was collected by centrifugation at 5000 × g in a refrigerated centrifuge at 4°C for 20 min, redissolved in bu"er A and dialysed against the same bu"er for 4 h. Gel filtration on Sephadex G-75. Sephadex G-75 slurry was packed into a column (0.6 × 50 cm) and equilibrated with bu"er A. #e fractions with the highest cellulase activity from the ion exchange chromatography step were applied to the column. Fractions (2 ml) were collected at a 'ow rate of 0.25 ml/min. #e eluate was monitored for protein concentration at 280 nm and Carboxymethyl cellulase from Bacillus sp. 1 53 fied organism was Gram-positive, motile, rod-shaped, starch-hydrolyzing, and nitrate-, catalaseand ureasepositive. #e organism was seen to produce acid when fructose, galactose, mannitol, sucrose, trehalose, and cellobiose were added to it. Based on morphological and biochemical characteristics, the organism was tentatively identified as Bacillus sp. according to Bergey’s Manual of Systematic Bacteriology. Growth curve and fermentation. #e Bacillus sp. attained maximum growth a%er 36 h of fermentation and the absorbance was 1.69 at 600 nm. #e absorbance declined to 1.38, 1.14, and 0.836 at 48, 60, and 72 h of incubation, respectively. #e enzyme secretion increased to its maximum of 13 U/mg protein at 36 h of fermentation. Enzyme production was found to be 3.8, 7.1, 7, 3.1 and 0.7 U/mg protein at 12, 24, 48, 60 and 72 h of fermentation, respectively (Fig. 1). E"ect of pH on enzyme production. E"ect of pH on enzyme production was studied by culturing the organism at various pH levels (5.5–8.0) using 1 N HCl/ NaOH. Enzyme production was 3.6, 5.1, 9.6, 11.1, and 1.4 U/mg protein at pH 5.5, 6.0, 7.0, 7.5, and 8.0, respectively. Enzyme activity was found to be high at pH 6.5 (14.4 U/mg protein) (Fig. 2). Purification of cellulase from the Bacillus sp. #e enzyme was purified through 3 steps including ammonium sulphate fractionation, DEAE cellulose and gel filtration column chromatography. #e recovery and purification were 24% and 14.5-fold, respectively, a%er Fig. 1. E"ect of fermentation period on CMCellulase by Bacillus sp. in submerged fermentation Fig. 2. E"ect of pH on CMCellulase from Bacillus sp. was also assayed for enzyme activity. Fractions with high enzyme activity were pooled and precipitated with solid ammonium sulphate. Again, the precipitate was collected by centrifugation at 5000 × g at 4°C for 30 min, redissolved in a minimal amount of bu"er A and then dialysed against the same bu"er overnight. #e purified enzyme thus obtained was stored at –20°C. SDS-PAGE and molecular mass determination. Denaturing sodium dodecyl sulphate/polyacrylamide gel electrophoresis SDS/PAGE (10%) was performed to determine the molecular mass of the cellulase following the methods of Laemmli (1970). #e molecular weight of the subunit of the enzyme was estimated with phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa) markers. #e sample obtained from the gel filtration column was loaded into the gel. Following electrophoresis, the gel was documented using a gel documentation system (Syngene, UK) and the molecular weight of the purified cellulase determined. E"ect of pH on enzyme activity and stability. #e optimum pH for the activity of the purified enzyme was determined by using the following bu"ers (0.1 M): citrate bu"er (pH 4.0); succinate bu"er (pH 5.0–6.0); sodium phosphate bu"er (pH 7.0); tris bu"er (pH 8.0); glycine-NaOH bu"er (pH 9.0). #e stability of the enzyme at various pH was examined by incubating the enzyme solution with the above-mentioned bu"ers at 37°C for 30 min prior to incubation with substrate. Enzyme activity was assayed as described by Nelson (1944). E"ect of temperature on enzyme activity and stability. #e temperature profile of the purified cellulase enzyme was determined by performing the routine enzyme assay at di"erent temperatures: 30, 40, 50, 60, 70 and 80°C. To determine thermal stability, the crude enzyme was incubated (without substrate at increasing temperatures, from 30 to 80°C) for 30 min and cooled. Enzyme activity was assayed as described by Nelson (1944). E"ect of divalent ions on enzyme activity. To study the e"ect of divalent ions on enzyme activity, the enzyme sample was incubated with various divalent ions namely, Ca, Mg , Zn , Cu , Na , Hg at 0.005 M concentration for 30 min. Enzyme activity was assayed as described by Nelson (1944).