Bacillus subtilis as an Alternative Source of Beta-glucosidase

Article International Journal of Modern Cellular and Molecular Biology, 2014, 3(1): 1-9 International Journal of Modern Cellular and Molecular Biology Journal homepage: www.modernscientificpress.com/journals/ijcellbio.aspx ISSN: 2169-0995 Florida, USA Bacillus subtilis as an Alternative Source of Beta-glucosidase Bagudo, A. I 1*, A. U. Argungu 2, Aliero, A. A 1., S 1., Suleiman, N., 3 Kalpana, S. 1 1 Microbiology Unit, Department of Biological Sciences, Kebbi State University of Science and Technology, Aliero, Nigeria. 2 Department of Biochemistry, Kebbi State University of Science and Technology, Aliero, Nigeria. 3 Department of Veterinary Biochemistry Usmanu Danfodiyo University Sokoto, Nigeria. * Author to whom correspondence should be addressed; E-mail: aamadee96@gmail.com, +2347060975682 Article history: Received 22 November 2013, Received in revised form 27 December 2013, Accepted 16 January 2014, Published 21 2014. Abstract: Bacillus species of soil origin are known to produce a wide variety of extracellular enzymes which are of vast agricultural and industrial uses. One gram (1g) each of soil obtained from a local farm lands in Aliero, Kebbi State was tenfold serially diluted then 0.1ml of 10 5 and 10 6 dilutions was inoculated into primary media followed by inoculation in to production media. The bacterial isolate was identified as Bacillus subtilis which was used in this study for the production of Beta-glucosidase. The enzyme activity was determined using Dinitrosalicylic (DNS) method. Highest production by B. subtilis was obtained following ten(10) hours of incubation. The optimum temperature for the activity of Betaglucosidase produced was obtained at 60 C with a concentration of 0.55 mg/ml. Optimum ph was obtained at ph 7.0 with a concentration of 0.67 mg/ml. The results of this study revealed that B. subtilis is a potential source of extracellular Beta-glucosidase at high temperatures suitable for industrial uses. Keywords: Farm lands, Bacillus subtilis, Beta-glucosidase, Industrial applications

2 1. Introduction Huge amount of agricultural, domestics and industrial cellulosic wastes have been accumulating and are continuously deposited into our environment. Celluloses are considered as the most important renewable resource for bioconversion (Kapdan et al., 2006). Many Cellulosic substances were hydrolyzed to simple sugars for making Single Cell Protein, sweeteners etc. It has been become the economic interest to develop an effective method to hydrolyze the cellulosic biomass (Peng et al., 2011). One of the important sources for a number of extracellular enzymes is soil microorganisms. Soils, rice straw, wheat straw and naturally decaying organic matters that contain cellulose materials are the best sources to isolate these microorganisms. A number of bacteria have been reported for their cellulolytic activities. The type of extracellular enzyme formed may depend on the composition of the medium and culture conditions; therefore selection of appropriate organism for a particular enzyme plays a key role in high productivity of desirable enzyme (Christakopoulos et al., 1994). The genus Bacillus contains a number of industrially important species and at present approximately half of the industrial productions of the enzymes are obtained from the strains of Bacillus sp. These strains are specific producers of extracellular cellulases and can be cultivated under high temperature and at various ph conditions to give rise to products that are in turn, stable in a wide range of harsh environments (Chen et. al., 2006). Cellulase is composed of several enzymes complex which functions together to hydrolyze cellulose to its sub-units component i.e. glucose. Cellulases produced by some bacteria tend to be cell associated and form tight multi enzyme complexes called cellulosome, on the surface of the cell. Cellulosome exhibits a cellulose binding function that allows the bacterial cell to bind closely to its substrate. Cellulosome is microcellular machine, whose component interacts in a synergistic manner to catalyze the efficient degradation of cellulose (Bayer, et al., (2007).). The enzyme has great economic significance. An efficient cellobiose hydrolysis requires a large amount of Beta-glucosidase for the utilization of cellulose residues, on an industrial scale. Beta-glucosidases are heterogeneous enzymes that have aroused considerable interest primarily because of their involvement in the biological saccharification of cellulosic material. For industrial use, isolation and characterization of new high yielding strains using less costly carbon source still remains a challenge (Zhang et. al., 2006). The aim of this work is therefore to identify bacterial as a source of Beta-glucosidase for industrial applications. 2. Materials and Methods 2.1. Sample Collection and Processing Soil samples collected from different cultivations farms lands in Aliero local Government, Kebbi State, Nigeria for screening of Bacillus species producing Beta-glucosidase were brought to

3 Microbiology laboratory, Kebbi State University of Sciences and Technology, Aliero. Soil samples were heat-shocked briefly at 80 o C for 15 minutes to kill vegetative cells and non-spore former (Saraswati et al., 2012). Serial dilutions of each soil sample were made in sterilized distilled water and 0.1 ml of 10-5 and 10 6 dilutions of each sample was spread on the surface of modified Han s medium containing (Yeast extract 2g/L, Cellulose 5g/L, Peptone 2g/L and Agar 15g/L). The plates were flooded with Congo red to see the cellulolytic activity of isolated strain after incubated at 35 o C for 48 h Cellulase producing microorganism showed a zone of clearance on this agar (Christakopoulos,et al., 1994), figures 1 and 2. Figure 1. showing plate 10 5 dilutions Figure 2. showing plate 10 6 dilutions 2.2. Identification of Bacillus The selected isolates were identified to the genus level using morphological and biochemical methods. Morphological examination was observed by a light microscope and Gram strain. The conventional biochemical tests used for identification of Bacillus are motility, Voges-Proskauer, Nitrate reduction, acid and/gas production from glucose, Mannitol fermentation, Hydrolysis of (Starch, Gelatin or Tyrosine), Casein Hydrolysis, Catalase test and Citrate utilization test and Propionate utilization (Bergey s Manual, (2000). 2.3. Identification of Cellulase Producing Bacillus The colonies showing clear zone were sub-cultured and re-streaked on the first medium again to have pure cultures. The purified isolates were preserved at - 80 o C in glycerol solution and kept on slants at 4 o C for further study. 100 ml of autoclaved production medium (Second medium) with following composition: MgSO4.7H2O 0.3g/L, NH4SO4 1.0g/L, K2HPO4, 3.6g/L, Na2HPO4. 12H2O 18.8g/L, NaCl 0.4g/L and Sucrose 10.0g/L was inoculated with 1 ml of test culture and incubated in rotary shaker at 20, 000 rpm at 37 C for 72 hours (Kalaichelvan, et al., (2012). 2.4. Extraction of Enzyme from Bacteria

4 The selected isolates were incubated at 37 C for 24 h in 50 ml of 8% (w/v) starch medium in 250 ml conical flask and placed in a shaker incubator operated at 120 rpm at 30 C. The extracellular enzyme solutions were obtained by centrifugation at 5000 rpm for 20 min using a high speed centrifuge. The supernatant obtained was collected and used as enzyme source and used in enzyme assays (Singh et al., 1998). 2.5. Characterization of the Enzyme 2.5.1. Measurement of optimum ph for enzyme activity ph stability of the enzyme was determined by the incubation of enzyme for 30 minutes at room temperature with buffer solutions of varying ph range. The ph range of the phosphate buffers used was of ph 5.5-9.5. The residual activity was then determined at 600C under standard assay conditions (Liu, et al., 2008; and Qu, et al., 2008). 2.5.2. Measurement of optimum period for enzyme production The effect of time course on cells growth and Beta-glucosidase production were determined by measuring the absorbance of cells growth and determining the cellulase activity at different time intervals. The results indicated that maximum Beta-glucosidase activity was observed after 10 hours of fermentation with sucrose as a carbon source. After 10 hours a decline in growth and Beta-glucosidase activity was observed (Maki et al., 2009). 2.5.3. Measurement of optimum temperature for enzyme activity The optimum temperature stability of the enzyme was measured by the incubation of enzyme in 0.05M phosphate buffer of ph 7.0 for 2 hour at temperature ranging from 40-65 o C. The residual activity was then determined at 60 O C using standard assay conditions (Lo et al., 2009; and Bai et al., 2009). 2.5.4. Measurement of Beta-glucosidase-activity Measurement of the amount of reducing sugars released by dinitro-salicyclic acid (DNS) was used to assay the activity of beta-glucosidase. 0.5 ml, each of salicyclic acid and diluted enzymes respectively and 0.05 M phosphate buffer at ph 7.0 was mixed in a test tube and incubated at 60 o C for 10 minutes and this is followed by the addition of 3 ml of DNS reagent to the enzyme mixture and heated in water bath at 100 o C for 15 minutes and 0.5 ml of salicin, 3.0 ml DNS and 0.5 ml of the enzyme of the same dilution was used as a reference blank to estimate the reducing sugars. The mixture was then placed in boiling water bath for 15 minutes and cooled to room temperature. The absorbance was then measured at 600 nm. The absorbance of the sample corrected by subtraction of enzyme blank was used to calculate glucose concentration from the glucose standard curve (Mallier 1959).

5 3. Results The appearance of white-creamy and dry colonies was indicative of the growth of Bacillus-like bacteria and number of colonies ranged from 3 5 10 5 to 2 6 10 6 CFU g 1 and on gram staining, gram positive, motile, Voges-poskrauer negative, Nitrate reduction positive, acid and gas production from Glucose,, Starch Hydrolysis and Gelatin Hydrolysis positive rods were observed. The effect of temperature on the cell growth and Beta-glucosidase production was studied by growing the organism at different temperature. As fermentation temperature was increased from 40ºC to 65ºC, cell growth and Beta-glucosidase activity in the culture supernatant gradually increased from 0.50 U/mL to 1.72 U/mL (table 1). After 60ºC, the cell growth and Beta-glucosidase activity gradually decrease. Since the maximum Beta-glucosidase activity was observed at 60ºC, this temperature was used in the subsequent experiments to determine the effect of other parameters for growth and Beta-glucosidase production. Table 1: Measurement of optimum temperature for Cells growth and enzyme production Temperature (ºC) 40 45 50 55 60 65 Cells growth (O/D) 0.810 0.805 1.221 1.321 0.846 0.625 Beta-glucosidase activity u/ml 0.50 0.55 1.52 1.70 1.72 0.68 The effect of initial ph of the culture medium on the cell growth and Beta-glucosidase production was studied by growing the organism at different ph values ranging from 4.5 to 8.5 at 60ºC. As the ph of the culture medium increased from 4.5 to 7.0, Beta-glucosidase activity increased from 0.580 to 1.750 U/mL. The maximum cell growth was measured 1.750 at ph 7.0. Increasing the ph to 8.5, the Betaglucosidase activity was reduced to 0.425 U/mL. Since maximum Beta-glucosidase activity, again this ph 7.0 was chose, so the ph of the medium was selected as 7.0 (table 2). Different carbon sources on cell growth and Beta-glucosidase production was studied, a number of carbon sources at a concentration of 0.5% were used. The maximum yield of Beta-glucosidase activity was recorded after 10 hours of fermentation when sucrose was used as a carbon source. Sucrose was the best amongst the carbon sources i.e. glucose, lactose, cellobiose and avicel. The maximum Betaglucosidase activity was 1.750 U/mL, when sucrose was used as the carbon source (table 3). The

6 minimum cell growth and Beta-glucosidase activity was observed when avicel was used as carbon source. Therefore, sucrose was selected as the carbon source for the production of Beta-glucosidase. Table 2: Measurement of optimum ph for Cells growth and enzyme production Medium ph Cells growth (O/D) Beta-glucosidase activity U/mL 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 1.911 1.926 1.125 1.231 1.382 1.583 1.425 1.135 1.025 0.580 0.550 0.825 0.910 1.200 1.750 1.550 0.785 0.425 Table 3: Effects of different carbon source for cells growth and enzyme production Carbon source Cells growth (O/D) Beta-glucosidase activity U/mL Glucose Sucrose Lactose Cellulobiose Avicel 0.024 1.414 0.342 0.345 0.406 0.23 1.72 0.25 0.51 0.20 4. Discussion Production and partial characterization of Beta-glucosidase by Bacillus subtilis isolated from a local farm lands was investigated. Bacillus spp forms one of the major soil saprophytes as reported by Olajuyigbe et al. (2005) who identified twenty five bacterial isolates from soil of which nine were identified as Bacillus species. The occurrence of cellulolytic organisms in soil agrees with earlier reports of (Rehena et al., 1989) that soil is known to be a reserve of Bacillu sp producing Beta-glucosidase. The effect of incubation period on growth and Beta-glucosidase production by B. subtilis revealed that

7 production begins to increase from six hours (6hrs) to peak at ten hours (10hrs) at optical density of 0.760 to 1.636 and with enzyme activity of 0.580 to 1.750 u/ml. After 10hrs of incubation in the shaker incubator these parameters tend to decrease (Table-4). Similar reports were observed by Rehena et al., 1989. Increase in incubation period resulted in decrease in the production of Beta-glucosidase by B. subtilis. This may be due to the fact that after maximum production of Beta-glucosidase enzyme (maximum incubation time), there was production of other byproducts and a depletion of nutrients. These by-products inhibited the growth of the organisms and hence, enzyme formation (Christakopoulos, 1994). Table 4: Measurement of optimum time for Cells growth and enzyme production Period (hr) Cells growth (O/D) Beta-glucosidase activity u/ml 6 8 10 12 14 16 0.760 1.414 1.636 1.702 1.381 1.287 0.45 1.23 1.70 1.10 0.95 0.86 Temperature and ph are the most important factors, which markedly influence enzyme activity. B. sutilis had an enzyme activity of 0.50 mg/ml at 40 C and at optical density of 0.810. As the temperature increased to 60 C, the enzyme activity increased (1.72 u/ml). This was followed by a sharp decrease in enzyme activity at 650 C. Further increase in temperature was followed by a decrease in enzyme activity. The bacterium showed high level of cell growth and Beta-glucosidase production when grown at ph 7.0 in the presence of sucrose for 10 hours. Above this ph the production was decreased (Table-3). Maximum Beta-glucosidase yield (0.67 u/ml/min) was achieved at ph 7 by B. subtilis although, ph 4-10 supported amylase production (Figure 3). The results suggest that maximum Betaglucosidase activity was observed at neutral ph. But considerable amount of activity (0.46 0.31 u/ml/min) was obtained at alkaline ph showing the wide application nature of the amylase enzyme identified. This result is in agreement with Oyeleke and Oduwole (2009) and Daniel et al., (2010). Who stated in their report that most bacterial enzymes function between a ph range of 6 and 8. The results suggest that there is stimulation of enzymes at a neutral ph. Similar observations were made by Olajuyigbe and Ajele (2005) who recorded optimum ph of 8.0 for Bacillus species. The study revealed the potential of agricultural wastes capability to produce Beta-glucosidase by B. subtilis.

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