Production and Immobilization of α-amylase from Bacillus subtilis

MATERIALS AND METHODS

Present study was conducted throughout the years 2005 and 2006 and carried out in the laboratories of Botany Department, Faculty of Science, New Damietta, Mansoura University, Egypt.

Organism: Bacillus subtilis was isolated from a fertile soil sample collected from Egypt and identified according to Bergey’s manual of systematic bacteriology (Sneath, 1986).

Cultivation: The organism was grown in 250 mL Erlenmeyer flasks, each containing 50 mL of starch nutrient broth medium that has the following composition (g/L): beef extract, 3.0; peptone, 10.0; NaCl, 5.0; starch, 10.0; distilled water, 1000 mL. The pH was adjusted to 7.0. The flasks were inoculated with 1 mL of the spore suspension obtained from nutrient agar cultures and incubated at 40°C with shaking at 150 rev min^-1 for 48 h. The filtrate was then separated by centrifugation at 10,000 for 10 min.

Estimation of α-amylase: α-amylase was assayed by measuring the amount of reducing sugar released from 1% starch. The amount of reducing sugar was estimated according to Miller (1959). The reaction mixture contained 0.4 mL of (1%) (w/v) soluble starch plus 0.5 mL each of diluted culture supernatant or partial purified enzyme preparation and 1.1 phosphate buffer (0.2 M, pH 7). The mixture was incubated at 50°C for 20 min. centrifuged at 10,000 rpm for 10 min and aliquots of the supernatants were assayed for reducing sugars. One unit of the enzyme activity was defined as the amount of enzyme that released 1 μmole of reducing sugar (expressed as glucose equivalents) per milliliter per minute under assay conditions. Enzyme and substrate controls were included routinely.

Growth estimation: Growth was determined spectrophotometrically by measuring optical density of the culture at 600 nm. The final pH was also recorded at the end of each experiment.

Factors affecting α-amylase production by Bacillus subtilis

Incubation periods: The basal medium containing 1% soluble starch was used; 25 mL medium in 250 mL conical flasks was inoculated with 1 mL of an overnight culture in triplicate manner. The culture was incubated in an incubator shaker at a shake rate of 150 rpm. Samples were taken at 12 h intervals (12, 24, 36, 48, 60 and 72 h).

Effect of pH: Different flasks of basal amylase production media of different initial pH values (4, 5, 6, 7, 8 and 9). The pH was adjusted by using 0.1 N NaOH and 0.1 N HCl. The inoculated flasks were incubated at 40°C for 48 h.

Effect of different temperatures: Temperature effect was carried out by inoculating the flasks containing α-amylase production medium at pH 7 with the tested strain and incubated at different temperatures, notably, 30, 35, 40, 45 and 50°C for 48 h.

Effect of different carbon sources: Different carbon sources including; D-glucose, fructose, lactose, xylose, galactose, mannitol, sucrose, potato starch, rice starch, flower starch) were incorporated into flasks containing the production medium to the final concentration of 1% (w/v). The flasks were inoculated and incubated at 40°C for 48 h.

Effect of different concentration of soluble starch: Different concentrations of soluble starch solution (0.5, 1.0, 1.5 and 2.0%) in 25 mL basal medium and the flasks were inoculated with the bacterial culture and after incubation at 40°C for 48 h.

Effect of different nitrogen sources: Different nitrogen sources were added to the medium according to the nitrogen content of peptone (16.16% nitrogen) and beef extract (12% nitrogen). These nitrogen sources included peptone, tryptone, beef extract, yeast extract, NH₄Cl, (NH₄)₂SO₄, NH₄NO₃ and KNO₃) were inoculated and incubated at 40°C for 48 h.

Physicochemical properties of the α-amylase

The pH: To study the effect of pH on the activity of α-amylase, citrate phosphate buffer with pH's (from 3 to 5), phosphate buffer with different pH's (from 6 to 8) and carbonate buffer with pH’s (from 9.5 to 10.5) were used in the standard assays. Aliquots of the enzyme preparations were mixed with the different buffers at each pH and the enzyme activity was assayed as previously described. Activities were expressed as a percentage of the maximal activity.

Temperature: Reaction mixtures at different temperature (30, 40, 50, 60, 70, 80°C), while the assay was carried out under the standard assay methods. Activity was expressed as % of the maximal activity. The effect of temperature on the stability of α-amylase was determined by incubating the enzyme solution in the absence of substrate for different time at 60°C at pH. The residual activity was assayed as described before.

Effect of metals: 0.5 mL of the dialyzed precipitated α-amylase was mixed with 0.5 mL of calcium chloride, sodium chloride, cobalt chloride, nickel chloride and EDTA solutions, 1, 5 and 10 mmol of each metal. The α-amylase activity was determined as previously.

K_m and V_max: The K_m and V_max of α-amylase were determined at pH 7 and 40°C by varying the substrate concentration from 1.0 to 15 mg of starch per mL. Data plotted by the method of Lineweaver-Burk plot.

Partial purification of the amylase enzyme: The crude enzyme was precipitated with ammonium sulphate. The precipitation was carried out at 4°C under constant stirring and the precipitated proteins were centrifuged at 10,000 rpm for 10 min. The fraction was dialyzed against 0.1 M phosphate buffer (pH 7) to remove the remaining salt. Specific activity of the enzyme was estimated in all precipitates. This partially purified enzyme was used for the preparation of the immobilized enzyme.

Protein estimation: The quantitative estimation of protein was carried out according to Bradford (1976). Protein concentrations were determined from a standard curve with Bovine Serum Albumin (BSA).

Immobilization methods

Immobilization on sodium-alginate (Entrapment): An equal volume of tested enzyme solution and sodium alginate solution was mixed to give a 4% (w/v) final concentration of sodium alginate solution. The mixture obtained was extruded drop wise through a Pasteur pipette (1 mm diameter) into a gently stirred 2% (w/v) CaCl₂.2H₂O solution 2 h to give bead size of 3 mm. The calcium alginate beads containing the enzyme were thoroughly washed with distilled water and used for further studies (Roy et al., 2000).

Immobilization on chitin (physical adsorption): Chitin beads were prepared as follows; 0.5 g of chitin was shacked in 10 mL phosphate buffer containing 2.5% glutaraldehyde for 2 h at room temperature. The solid material was filtered and washed to remove excess of glutaraldehyde. The treated chitin was mixed with 30 mL of the enzyme solution for 24 h at 4°C. The mixture was centrifuge at 10,000 rpm for 5 min and the supernatant was used for further study. The precipitated was washed by phosphate buffer containing small amount of 0.1 M sodium chloride then subjected to the immobilized enzyme activity (Ohtakara et al., 1988).

Immobilization on sephadex: One gram of Sephadex was added to 30 mL of the partial purified enzyme solution in phosphate buffer (pH 7) and kept at 4°C for 24 h. The unbound enzymes were removed by washing three times with the same buffer (Kusunoki et al., 1982).

Immobilization on dowex (ionic binding): One gram Dowex was incubated with 30 mL of the partial purified enzyme solution in phosphate buffer (pH 7) and kept at 4°C for 24 h. The unbound enzymes were removed by washing three times with the same buffer to remove unbounded protein.

Physical properties of the immobilized α-amylase: Effect of pH, temperature, thermal stability and kinetic constant K_m and V_max and some metal on physical properties of the immobilized α-amylase were studied as mentioned before.

Statistical analysis and data presentation: The experiments were carried out in three replicates with standard deviation and standard error presented the statistical procedure used where appropriate was the t-test.

RESULTS

The time course for the production of α-amylase by Bacillus subtilis in the basal liquid medium containing 1% soluble starch as a substrate is shown in the Fig. 1. The α-amylase activity of the Bacillus subtilis increased during the growth phase of the culture and the optimum period for the production was reached after 48 h. After 60 h, α-amylase production declined significantly (p<0.05, t-test). α-amylase production reached the minimum level after 72 h. The maximum growth measured after 24 h and the growth declined significantly to reach the minimum level after 48 to 60 h.

As shown in the Table 1 Bacillus subtilis was able to grow in basal liquid medium supplied with the different carbon sources which were glucose, xylose, galactose, mannitol, rice, flower, potato, sucrose, fructose, lactose and starch. The best carbon source for production of α-amylase for Bacillus subtilis was starch with activity (3.19 μ mL^-1) and the maximum growth measured with galactose. Glucose, xylose, galactose, sucrose, mannitol, fructose and lactose were found to be good sources for the production of α-amylase. The growth was recorded when using flower powder xylose, galactose, rice powder, potato powder, starch and lactose. Little growth was observed with glucose, mannitol, sucrose and fructose.

The optimum concentration of starch for α-amylase production was 0.25 mg mL^-1 with activity of 3.6 μ mL^-1 (Fig. 2). At the concentration of 0.375 mg mL^-1 the activity could be decreased or still constant, no change in the α-amylase production. Also at 0.125 mg mL^-1 the activity well be increased until reached to 0.25 mg mL^-1.

The optimum pH for α-amylase production was at pH 7 with activity 1.95 μ mL^-1 (Fig. 3.). The activity of α-amylase was decreased significantly at pH 9.0 (p<0.05, t-test). The range of maximum growth was found from pH 5 to 8, respectively.

An optimum temperature for α-amylase production from Bacillus subtilis was 45°C (Fig. 4.). At 50°C the activity was declined significantly (p<0.05, t-test). The range of maximum growth from 40 to 45°C, respectively.

Table 1:	Effect of different carbon sources on α-amylase production and growth of Bacillus subtilis
Each results is the means of three replicates±standard error (SE)

Table 2:	Effect of different carbon sources on α-amylase production and growth of Bacillus subtilis
Each results is the means of three replicates±standard error (SE)

Fig. 1:	Effect of different incubation periods on α-amylase production and growth of Bacillus subtilis. Values are shown as means of triplicates±standard error

Fig. 2:	Effect of different starch concentration on α-amylase production and growth of Bacillus subtilis. Values are shown as means of triplicates±standard error

The best nitrogen source for production of α-amylase from Bacillus subtilis was peptone with activity (2.71 μ mL^-1) and the maximum growth measured with beef extract as shown in Table 2.

Physicochemical properties of free and immobilized α-amylase: Free α-amylase activity increased with increasing pH within the range pH (3-7) till reached to the optimum point of pH 7 (Fig. 5). Generally, the optimum pH of the immobilized α-amylase was shifted to acidic range in comparison to the free enzyme (Fig. 5). After the optima pH of the free and immobilized α-amylase, the relative activity decreased and reached to minimum level at pH 8 and 9.5 as shown in Fig. 5.

Fig. 3:	Effect of different pH values on α-amylase production and growth of Bacillus subtilis. Values are shown as means of triplicates±standard error

Fig. 4:	Effect of different temperatures on α-amylase production and growth of Bacillus subtilis. Values are shown as means of triplicates±standard error

The activity of free α-amylase increased with increasing temperature starting from 40°C till reached to the optimum activity at 50°C (Fig. 6). The optimal reaction temperature of the free α-amylase shifted from 50°C to 70°C for the enzyme immobilized on Dowex and Ca alginate and to 80°C for immobilization on sephadex and chitin (Fig. 6). The minimum level of the free and immobilized α-amylase activity occurred at 90°C as shown in Fig. 6.

Generally, some metals increased the activity of free and immobilized α-amylase such as CaCl₂ and NaCl with the three used concentrations (1, 5 and 10 mM). The other metals, notably CoCl₂, NiCl₂ and EDTA inhibited the activity of free and immobilized α-amylase. The minimum level of the activity was reported when using EDTA in all concentrations (Table 3).

Table 3:	Effect of metal ions on relative activity (%) of free and immobilized α-amylase of Bacillus subtilis. Values are shown as means of triplicates

Fig. 5:	Effect of pH on activity of α-amylase of Bacillus subtilis before and after immobilization. Values are shown as means of triplicates±standard error (SE)

Fig. 6:	Effect of temperature on activity of α-amylase of Bacillus subtilis before and after immobilization. Values are shown as means of triplicates±standard error (SE)

Fig. 7:	Thermal stability of α-amylase of Bacillus subtilis before and after immobilization. Values are shown as means of triplicates±standard error (SE)

In general, the calculated K_m and V_max values of the immobilized enzymes are higher than that of the free enzyme (Table 4).

Thermal stability of free and immobilized α-amylase was completely stable at 60°C for 10 min (Fig. 7). The free enzyme lost about 50% of the relative activity after 15 min while the immobilized ones retain 60% in case of utilization of sephadex and dowex; to 80% of the activity after 30 min when chitin and Ca-alginate were used for immobilization, respectively (Fig. 7). After 60 min, the free and immobilized enzyme lost more than 50% of the activity.

Table 4:	Km and Vmax values of free and immobilized α-amylase of Bacillus subtilis. Values are shown as means of triplicates

DISCUSSION

Extracellular α-amylase production and growth of Bacillus subtilis was maximal at pH 7 and this result is in accordance with the work of Bajpai and Bajpai (1989) and Lin et al. (1998). The composition of cell wall and plasma membrane of microorganisms is known to be affected by the culture pH (Ellwood and Tempest, 1972a, b). The change of the initial pH of the medium may be leads to change of the nature of the cell membrane and/or cell wall and hence affecting the α-amylase production and the growth of Bacillus subtilis. On the other hand, Mamo and Gessesse (1999) reported that high level of α-amylase at pHs 5.0 and 6.0 and when the pH was increased low level of α-amylase was obtained.

The optimal temperature for production of α-amylase and growth of B. subtilis occurred at 45°C. Similar observations were recorded by Bajpai and Bajpai (1989), Lin et al. (1998) and Anyangwa et al. (1993). Bacterial cells have various mechanisms that allow them strictly to control excretion (Mamo amd Gessesse, 1999). Change in the nature of cell envelope can affect the release of extracellular enzymes to the culture medium (Antranikian, 1990). Temperature is one of the factors that induces such changes on cell membranes and cell walls (de Vrij et al., 1990; Nordstrom, 1993). It is also reported that in Bacillaceae, the surface protein layer (S-layer) in involved in the control of exoenzyme release (Egelseer et al., 1996a, b).

The best carbon source for α-amylase production from B. subtilis was found to be starch. Starch is known to induce amylase production in different bacterial strains (Saito and Yamamoto, 1975; Fogarty and Bourke, 1983; Wind et al., 1994; Aiyer, 2004; Ryan et al., 2006). Low levels of α-amylase were detected when the organism was grown with mannitol. Similar observations were recorded by Wind et al. (1994), Chandra et al. (1980) and Meers (1972). Glucose and fructose showed repression effects on enzyme production. It has been reported that the synthesis of carbohydrate degrading enzymes in most species is subjected to catabolite repression by readily metabolize substance such as glucose and fructose. Among the undefined carbon source that led to high levels of amylase were flower, potato and rice. Undefined carbon sources are known to induce a high level of amylase production in many bacterial strains (Fogarty et al., 1974; Mamo and Gessesse, 1989; Tonkova et al., 1993; Ryan et al., 2006).

α-amylase production and growth of Bacillus subtilis increased after 24 to 48 h. After this period, the production of the enzyme decreased. The observed peaking and throughing of the production of extracellular enzymes can be attributed to (1) The products of action of one component inducing the synthesis of another, (2) Differential inhibition by products of substrate hydrolysis, (3) A decrease in growth was observed after 48 h of growth of B. subtilis. This probably resulted from cellular lysis, an observation previously reported. These results indicate that the production of extracellular α-amylase by B. subtilis was growing associated and this is agreement with other investigators (Bajpai and Bajpai, 1989; Stephenson et al., 1998; Riaz et al., 2003).

The maximum starch concentration for the optimal α-amylase production from Bacillus subtilis was 1.0%. Similar results were reported by Bajpai and Bajpai (1989). Bacillus subtilis produced different levels of α-amylase with varying C: N ratio and this may be explained as different types of induction of enzymes. This may suggest different mechanisms of production of extracellular enzymes of Bacillus subtilis when grown with different C: N ratio.

The optimal production of α-amylase and growth of B. subtilis were obtained when peptone was used as nitrogen sources and this is in accordance similar with Aiyer (2004), Chandra et al. (1980) and Babu and Satyanarayana (1995). Peptone containing medium is complex and contains high nutritional amino acids and this may be lead to high α-amylase production and good growth of Bacillus subtilis. Proteose peptone was recorded as good nitrogen source for amylase production and growth of thermophilic Bacillus sp. WN11 (Bajpai and Bajpai, 1989).

The pH optimal for the free α-amylase of B. subtilis was found to occur at pH 7. These results are in accordance with those reported by Vihinen and Mäntsälä (1989), Hamitton et al. (1999), Saito (1973) and Khoo et al. (1994). The immobilized-amylase showed optimum pH in the acidic range in comparison to the free enzyme. These effects may be dependent on the ionic environment around the active site of the enzyme bound to the carrier (Abdel-Naby, 1993). Similar shifts of pH optima were reported by Sadhukhan et al. (1993) and Yoshida et al. (1989).

The free α-amylase of B. subtilis showed temperature optima at 50°C and this result is in accordance with Mamo and Gessesse (1999) and Vihinen and Mäntsälä (1989). The immobilized α-amylase showed higher relative activity at the temperatures 70 and 80°C than that of free enzyme. Increase in temperature optima had been found in immobilized enzymes (Sadhukhan et al., 1993; Yoshida et al., 1989). The increase in temperature optima for the activity of immobilized enzyme could be due to the fact that actual temperature in the micro-environment of the gel matrix incases of Ca-alginate as a carrier was lower than in the bulk solution (Kennedy, 1987).

Free α-amylase of B. subtilis, the relative activity was declined to 44% after 15 min, to 38% after 25 min at 60°C, these results are also in accordance with that to Gerhartz, (1990), Shaw and Chuang (1982) and Shaw and Ou-Lee (1989). The thermoinactivation at 60°C of free and immobilized enzyme showed remarkable achievement of thermostability by the immobilization with Bacillus subtilis with different carriers. The high thermostability of immobilized-amylase is consistent with the results obtained for other enzymes (Sheffield et al., 1995). The thermal stability of the enzyme increased the tolerance to thermal denaturation, therefore, had been imparted by the gel entrapment covalent cross-linking of the enzyme protein.

The free α-amylase from Bacillus subtilis showed high affinity towards starch with a K_m and V_max values of 8.3 mg mL^-1 and 10.0 μ mole min^-1 mL^-1, respectively. The K_m and K_max of the immobilized α-amylase increased with the four carriers. This increase is most likely a consequence of either structural changes in the enzyme introduced by the applied immobilization procedure and/or lower accessibility of the substrate to the active site of the immobilized enzyme (Abdel-Naby, 1993). The latter, as explained above, may result either from diffusional resistance of the matrix or steric hinderance in the immediate vicinity of the enzyme molecules (Abdel-Naby, 1993).

The results obtained in the our study were similar to those that have been reported for immobilized glucoamylase (Koji et al., 1999). For practical application, an immobilized system with lower K_m value and faster rate of reaction is preferred.

Among the tested metals, CaCl₂ was the most activator metals. The relative activity of free and immobilized α-amylase from B. subtilis increased in the presence of all concentration of calcium. These results are in accordance with the work of other investigators (Lin et al., 1998). Ca²⁺ ions has a positive effect on the activity of the α-amylase enzyme. The binding of Ca²⁺ ions has been shown to increase the-helical structure of the α-amylase enzyme leading to increased stability. Na⁺ ions similar to Ca²⁺ increased the activity of the α-amylase. The ions likes Co²⁺, Ni²⁺ slightly inhibited amylase activity. Some amylases are metalloenzymes, containing a metal ion with a role in catalytic activity by Ni⁺⁺ and Co⁺⁺ ions could be due to competition between the exogenous cations and the protein-associated cation-resulting in decreased metalloonzyme activity. The inhibitory effect of the chelating agent EDTA demonstrated the ion requirement of this amylase similarly. EDTA has been found to be a potent inhibitor of amylases from other bacteria (Sadhukhan et al., 1993; Yoshida et al., 1989; Shaw et al., 1995).