Isolation and screening of cellulolytic bacteria
The bacterial strains were isolated from cow dung by serial dilution on Omeliansky’s agar medium (Omeliansky 1902) [g/L (W/V), (NH4)2SO4 1.0; K2HPO4 1.0; MgSO4.7 H2O 0.5; NaCl traces; carboxymethyl cellulose (CMC) 1%, pH 7.0]. Preliminary screening of cellulase producing isolates was carried out on CMC agar plates as mentioned by Teather and Wood (Teather and Wood 1982). Twenty bacterial isolates were selected out of which one (designated C1) with highest CMCase activity was finally selected. The strain C1 was identified as Bacillus sp. by full 16 S rDNA sequence homology and deposited to Microbial Type Culture Collection Centre and Gene Bank with accession number MTCC 10046.
DNA extraction and molecular phylogenetic analyses using 16S rRNA gene sequence
Genomic DNA was extracted according to Marmur (Johnson 1994). PCR amplification of the 16S rRNA gene was carried out using universal primers 8–27f and 1492r and amplified PCR product was purified and sequenced as described by Saha and Chakrabarti (2006). A continuous stretch of 1514-nucleotide long gene sequences of 16S rRNA gene was used to search for similar sequences from RDP database Release 10 http://rdp.cme.msu.edu/) using various online tools (CLASSIFIER, SEQMATCH). After confirmation of generic affiliation, sequences from type strains of different species were retrieved from GenBank. All these sequences were aligned by CLUSTAL_X programme (Thompson et al. 1997) and edited manually. Similarity values were determined after pair wise alignment by CLUSTAL_X programme followed by manual calculation. A phylogenetic tree showing relationship between C1 and other reference strains was constructed by neighbor-joining (NJ) method with Jukes and Cantor correction using TREECON software as described by Saha and Chakrabarti (2006).
Suspension of active bacterial culture (Grown on TSA at 25 C° for 72 hours) of defined density (as per manufacture’s instruction) was inoculated into microtitre plate. An inoculum of 150 μl was dispensed into Biolog GN2 Microplates using a multichannel micropipette. The oxidation of various carbon sources as indicated by reduction of tetrazolium violet dye that results in production of purple color was monitored and recorded by using a micro plate reader that is coupled to a computer. The later has software for comparison of various patterns of oxidation as per Biolog database.
Preparation of agricultural and paper waste used as carbon source for CMCase production
The Banana agro waste (pseudo stem, leaves, etc.) used for saccharification were freshly collected. The waste was washed thoroughly with water and air dried. It was ground to powder using electric grinder and sieved. Orange fruit was washed thoroughly with water, peeled and sliced. The juice was removed with the aid of a squeezer and the pulp separated from the pericarp (albedo) and the three materials were sun-dried separately. They were later oven-dried at 70°C, still being handle separately and then pounded using a mortar and pestle. All waste substrates then ground using a blender. Raw rice straw (RS) was obtained from local farmers. It was cut to 1–2 cm length and washed thoroughly with tap water until the washings were clean and colorless and then dried in a oven at 65°C to constant weight. Oven dried RS was then ground with electric grinder and was used in the experiments. Sugarcane baggasse also prepared in the same method. Papers were cut to 1 × 6 cm length and were used in the experiment.
Optimum concentration of CMC
We find that among different carbon sources CMC was the best carbon source. So, to find out the suitable concentration of CMC, different concentrations of CMC was tested.
The isolate C1 was inoculated in 100 ml of Omeliansky medium (with 8% CMC as carbon source) and was grown at 50°C for 7 days. Cell separation was made by centrifugation of fermented broth at 10,000 × g for 20 min and the cell free broth used as crude enzyme.
Partial purification of endoglucanase
Crude enzyme preparation was obtained after cultivation of the bacteria under submerged fermentation in shake flask condition. After centrifugation of the fermented broth the supernatant was subjected to ammonium sulphate precipitation with continuous stirring at 4°C. The supernatant was precipitated with ammonium sulphate at 40-80% saturation for two hours with gentle stirring. The precipitated proteins were recovered by centrifugation at 8000 rpm for 20 min and were dialyzed against 0.02 M sodium acetate buffer (pH 5.2) for 24 h. The dialyzed solution was then applied to DEAE-Sepharose column.
The semi-purified enzyme solution was subjected to DEAE-Sepharose column. The 35 ml of the enzyme solution was applied to DEAE-Sepharose (Sigma-aldrich.) column which was previously equilibrated with 0.02 M Acetate buffer (pH 5.2). The entire purification was carried out at 4°C. The column was first washed with equilibration buffer, and then bound proteins were eluted using linear gradient of 0.05 -1.0 M NaCl (in acetate buffer) at a flow rate of 1 ml/minute. The elute fractions (3.0 ml) was by an automatic fraction collector and monitored for enzyme activity as well as for protein concentration at 280 nm. After step-wise eluted each fraction from DEAE-Sepharose column, the total activity and specific activity of endoglucanase was assayed by DNS method, the fraction showing high activity were pooled and used for SDS-PAGE analysis.
SDS-PAGE and determination of molecular weight of endoglucanase
The active eluted fraction (E5 = 5th eluted fraction) collected from DEAE-Sepharose chromatography was used for analysis by SDS-PAGE (Robyt and White 1990). Resolving gel consisted of 12% polyacrylamide in Tris–HCl (1.5 M, pH 8.8), while stacking gel consisted of 4.5% polyacrylamide in Tris–HCl (0.5 M, pH 6.8). Agarose slab gel method was used for congo red assay. An agarose slab gel (0.5% W/V) was prepared containing 0.2% (W/V) CMC as substrate for the enzyme assay. Four slots were punched using a gel puncher and the different dilutions (1:5, 1:10 and undiluted) of fractions E5 and negative control were loaded in it. The slab was placed overnight in a moist chamber at room temperature for diffusion, followed by flooding with 0.5% aqueous congo red solution and de-staining of slab with 2 M NaCl solution and recorded as photo image for further confirmation.
Determination of protein concentration
Concentration of protein was determined following the method of Lowry et al. (1951) using BSA as standard and the protein in column elute fraction was also monitored by spectrophotometrically at 280 nm.
Assay of endoglucanase activity
The activity of cellulase was assayed by incubating 1 ml of reaction mixture consisting of 0.5 ml of 1% CMC in 0.02 M Sodium acetate buffer, pH 5.2 and 0.5 ml of suitably diluted enzyme solution incubated at 40°C for 1 hr. Enzyme and reagent blanks were incubated maintaining the same condition simultaneously. The amount of reducing sugar released was determined by dinitrosalicylic acid (DNS) method (Miller 1959). One International Unit (IU) of enzyme activity for endoglucanase was defined as the amount of enzyme releasing 1 μmol of reducing sugar from CMC per minute. The specific activity determined as the number of units of enzyme activity per milligram of enzyme protein.
Determination of Km value
The Michaelis-Menten constant (Km) of purified extracellular cellulase of strain C1 was determined by varying the concentration of CMC in 0.02 M acetate buffer, pH 5.2. The kinetic parameters were determined from Lineweaver Burk double reciprocal plot (Lineweaver and Burk 1934). The initial velocity measured by quantitatively measuring the amount of one of the product at various time intervals (Robyt and White 1990).
Effect of various chemicals on endoglucanase activity
Enzyme activity measured in the presence of detergents (SDS, Tween-80), metal ions (MnCl2, ZnCl2) and EDTA at a concentration of 10 mM.
Effect of temperature on endoglucanase activity
Enzyme activity was measured by treating the enzyme mixture at various temperatures, ranging from 20 to 70°C.
Effect of pH on endoglucanase activity
The activity was measured at various pH (5.0-9.0), maintained using different buffering system.
Effect of pH and temperature on stability of the enzyme
The effect of pH on stability of the enzyme was determined by incubating 5 IU of enzyme for 1 h. at 50°C, in a buffer of desired pH before addition to the reaction mixture; similarly the effect of temperature was determined by treating the enzyme at varying temperatures before addition to the reaction mixture.
Stability of endoglucanase in commercial detergents
The stability of endoglucanase in the presence of the commercial detergents such as Ariel, Surf Excel and Tide was investigated by incubating the enzyme in the presence of the detergent (7 mg/ml) at 50°C. Aliquots of enzymes were removed at intervals of 10 min. and the residual activity of the enzyme was determined using standard assay conditions.
All the data generated in the study are the mean ± SEM of 3 replicates. All data were subjected to student’s t- test analysis with significance level of P < 0.01 using SPSS software package.