Statistical Optimization of Cold Adapted á-amylase Production by Free and... Nocardiopsis aegyptia
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Statistical Optimization of Cold Adapted á-amylase Production by Free and... Nocardiopsis aegyptia
Journal of Applied Scienes Research, 5(3): 286-292, 2009 © 2009, INSInet Publication Statistical Optimization of Cold Adapted á-amylase Production by Free and Immobilized Cells of Nocardiopsis aegyptia Abou-Elela G.M., Nermeen A. El-Sersy, and Wefky S.H. National Institute of Oceanography and Fisheries, Microbiology lab. Environmental Division, Alexandria, Egypt Abstract: á-Amylase production has a wide range of applications in many industries such as baking, brewing, ethanolic production, wine and textile industries. The cold adapted á-amylase production from Nocardiopsis aegyptia was dependent on the fermentation conditions. Plackett-Burman statistical design was used to optimize culture conditions and evaluate the most significant variables affecting enzyme production. Potassium nitrate concentration (1.5 g/l) and inoculum size (1.5 ml /50 ml medium) were positively affecting the production which was increased up to 1.12 fold. The experimental results showed that optimum temperature and pH values were 25°C and 5.0 respectively. Immobilized Nocardiopsis aegyptia cells on luffa pulp achieved it´s highest productivity (2255 unit) after 2 days of incubation this yield increased up to 1.16 fold. Key words: á-amylase-Nocardiopsis aegyptia - Statistical Optimization- immobilization as saline soils or marine sediments [9 ,2 ] . The nutritional and environmental conditions have a great influence on production of enzymes. In order to develop an efficient production of enzymes, knowledge regarding the environmental factors affecting this process needs to be well identified. Experimental designs are excellent techniques for optimization of culture conditions to achieve optimal production [7 ,2 4 ,3 ] . and was recently used for the production of amylase by actinomycetes [1 3] . Modification of biotechnology and processes, using immobilized biocatalysts, has recently gained the attention of many biotechnologists. Application of immobilized enzymes or whole cells is advantageous, because such biocatalysts display better operational stability[1 0 ] . and higher efficiency of catalysis [2 2] . Immobilization of whole cells for extracellular enzyme production offers several advantages i.e. the ease to separate cell mass from the liquid culture for possible reuse, facilities continuous operation over a prolonged period, enhances reactor productivity [2 6 ] . In the present study screening of different actinomycetes isolates has been carried out to explore the possibility of amylase production. In addition fermentation parameters were optimized by applying Plackett-Burman experimental design. Moreover, cell immobilization technique was carried out aiming for maximum amylase production. INTRODUCTION Amylase constitute one of the important groups of enzymes that are used in a wide range of starch industries i.e. baking, brewing, starch liquefaction and distillery. Amylase has numerous biotechnological applications in the production of syrups containing oligosaccharides, maltose and glucose. Another product from starch hydrolysis by amylases is dextrin, which is important in food processing as viscosity improver, filter or ingredient. In the textile industry, amylase is used in resizing process to degrade starch from clothing materials [1 1 ,3 1 ]. Several amylolytic enzymes, with different specificities can contribute to starch degradation. á amylases are widely distributed in microorganisms. Industrial á amylases are produced by bacteria and fungi, e.g. Bacillus subtilis, B. licheniform is, Aspergillus oryzae, A. niger, Micrococcus halobius, etc. Production of amylases was also reported by some strains of actinomycetes [1 2 ,1 3 ] . in addition to plants, and animals. Actinomycetes are one of the most investigated groups because they constitute a potencial source of b io techno lo gically interesting sub sta n c e s[1 7 ]. Nocardiopsis strains are distributed ubiquitously in the environment[1 5 ] . They are frequently isolated from habitats with moderate to high salt concentrations such Corresponding Author: Nermeen A. El-Sersy, Associate prof. National Institute of Oceanography and Fisheries, Microbiology lab. Environmental Division, Alanfushy Qayt bay, Alexandria, Egypt. Tel: 0106620217 E-mail: [email protected] 286 J. Appl. Sci. Res., 5(3): 286-292, 2009 W here Exi is the variable main effect, and Mi+, Mi- are the enzyme activity (units) in the trials, where the independent variable was present in high and low concentrations, respectively, and N is the number of trials divided by 2. Statistical t-values for equal unpaired samples were calculated using Microsoft Excel to determine the variable significance. M ATERIALS AND M ETHODS M icro-organisms: All tested organisms were isolated from aquatic sources. Nocardiopsis aegyptia was isolated from marine sediment of Abu Qir Bay on the western seashore, Alexandria, Egypt [ 2 5 ] . while Streptoverticillum morookaense, Streptomyces globosus, Streptom yces ruber, Streptom yces alanosinicus, Streptomyces gancidicus, and Nocardia brasiliansis, were isolated from Burullus lake sediments [1 ]. Effect of PH on Amylase Production by N.aegyptia: The optimized culture medium was adjusted at different pHs (4.0-8.0). The pH measurements were carried out with Beckman digital pH meter using a glass electrode. The pHs of 4.0-6.0 was maintained with acetate buffer (0.2M) while pH 6.0-8.0 were achieved with phosphate buffer (0.1M) [1 3 ]. Screening for á-amylase Production by Different Actinomycetes Strains: The organisms were inoculated on starch nitrate culture medium plates [1 9 ]. contained (gl-1 ): soluble starch, 20.0 ; KNO 3,1.0; K 2HPO 4,0.5; MgSO 4 .7H 2 O, 0.5 ; FeSO 4 .7H 2 O, 0.05; and agar, 20 ; sea water or lake water 1L. Incubation at 30°C was carried out for 7 days after which the plates were flooded with Gram's iodine solution (0.1% I 2 and 1% KI) and the colonies with the largest halo-forming zone were chosen for further investigations [1 8]. Effect of Immobilization Technique on á-amylase Production by N.aegyptia: Immobilization was carried out by adsorption of N. aegyptia cells on different solid porous supports, 1.5 ml of bacterial suspension were added to 100 ml sterile flasks containing 50 ml of optimized culture medium and five grams of porous support materials (Luffa pulp, sponge and pumice). Luffa pulp and sponge were cut to small pieces, washed several times with water before use. The size of pumice particles was around 0.5 cm in diameter. The flasks were then shacked slowly at 120 rpm. After different time intervals of incubation (2-6) days, am ylase production was estim ated. R e p e a te d fermentation of immobilized cells in shacked flasks up to 6 th run carried out in the modified starch nitrate medium. Effect of Incubation Period on á-amylase Activity of Nocardiopsis aegyptia: Nocardiopsis eagyptia was investigated for amylase production at different incubation periods. Amylase production was estimated daily for 8 days using the method described by [3 0 ]. Inoculum size was 1ml (10 5 CFU/ml) Enzyme Assay: Amylase assay was based on the reduction in blue colour intensity resulting from enzymatic hydrolysis of starch and formation of starchiodine complex [3 0 ] . The reaction mixture consisted of 0.2 ml enzyme (cell free supernatant), 0.25 ml of starch solution and 0.5 ml of phosphate buffer (0.1 M, pH 6). Incubated at 50 º C for 10 min. The reaction was stopped by adding 0.25 ml of 0.1 N HCl and the colour was developed by adding 0.25 ml of I/KI solution (2% KI in 0.2% I). The optical density (O.D.) of the colour solution was determined using a UV-Vis Spectrometer at 690 nm. One unit of the enzyme activity is defined as the quantity of enzyme that causes 0.01% reduction of blue colour intensity of starch iodine solution at 50 º C in one min. per ml [3 0 ]. RESULTS AND DISCUSSION Screening for Extra-cellular Amylase Production: Among the tested isolates for their ability to degrade starch, Nocardiopsis aegyptia showed the largest degradation zone as shown in Table 1. It was found that degradation zone of Nocardiopsis aegyptia were 30 mm, in spite of, the diameter of that zone equal that produced by Streptomyces rubber, Streptomyces alanosinicus and Nocardia brasiliansis, but it was noticed that, the diameter of Nocardiopsis aegyptia colony was the lowest (12 mm).. Optimization of Growth Culture Conditions Using Plackett Burman Experimental Design: The PlackettBurman design [2 1 ,3 2 ] . was applied to reflect the relative importance of various environmental factors involved in the production of á-amylase by Nocardiopsis aegyptia. For each variable a high (+) and low (-) levels were tested. The examined variables in this experiment and their levels are shown in Table 2. Eight different trials were performed in duplicates. Rows in Table 3. represent the different trials (Row no. 9 represents the basal control). The main effect of each variable was determined with the following equation: Exi = (Mi+ – Mi-) / N Determination of the Optimum Incubation Period: The optimum incubation period for á-amylase production by Nocardiopsis aegyptia was after 6 days representing (1949 units) of productivity as shown in (Figure 1), no significant difference in the productivity between 6 th and 7 th day (1940 units).The productivity decreased significantly at the 8 th day (1800 units). 287 J. Appl. Sci. Res., 5(3): 286-292, 2009 Table 1: Screening of different actinom ycetes isolates for starch degradation after 7 days of incubation. Strain Colony diam eter Zonediam eter (m m ) (m m ) Streptoverticillum m orookaense 13 22 Streptom yces globosus 22 26 Streptom yces ruber 24 30 Streptom yces alanosinicus 20 30 Streptom yces gancidicus 18 25 Nocardia brasiliansis 16 30 Nocardiopsis aegyptia 12 30 Fig. 1: Effect of incubation periods production by N. aegyptia. significant variables. Their interaction was illustrated in Figure 3, which showed amazing increase of enzyme production, indicating direct relationship between these two factors. Table 2: Independent variables affecting á-am ylase production and their levels in the Plackett-Burm an design Factor Sym bol Level ---------------------------------------------------------1 0 1 KN O 3 (g/l) KN 0.5 1 1.5 M g SO 4 (g/l) Mg 0.25 0.5 0.75 K 2 H PO 4 ((g/l) K2 0.25 0.5 0.75 Sea water conc. SW 50% 100% >100% * FeSO 4 ((g/l) Fe 0.025 0.05 0.075 Inoculum size (m l) Is 0.5 1 1.5 Tem perature (o C) T 25 30 35 *150 m l of sea water was concentrated by evaporation to 100 m l Table 3: The applied Plackett- Burm an experim ental design for seven cultural variables. Trials Factor Level ----------------------------------------------------------------------------KN M g K2 SW Fe Is T Enzym e production (U nits) 1 -1 -1 -1 1 1 1 -1 2189 2 1 -1 -1 -1 -1 1 1 2254 3 -1 1 -1 -1 1 -1 1 1869 4 1 1 -1 1 -1 -1 -1 2202 5 -1 -1 1 1 -1 -1 1 2179 6 1 -1 1 -1 1 -1 -1 2224 7 -1 1 1 -1 -1 1 -1 2215 8 1 1 1 1 1 1 1 2208 9 0 0 0 0 0 0 0 2178 on amylase Optimization of Amylase Production Using Plakett -Burman Design: This design was applied with nine different fermentation conditions as shown in Table 3. All experiments were performed in duplicates and the averages of results (enzyme activity expressed in production units) are presented as the response in Table 3. The main effect of each variable on enzyme production as well as t-values were estimated for each independent variable as shown in Table 4 and graphically presented in Figure 2. Results in Figure 2 indicated that the presence of high levels of KNO 3, Mg SO 4, sea water concentration and inoculum size in the growth medium affects enzyme production positively. On the other hand, the presence of K 2 HPO 4 , FeSO 4 and temperature at their lowest levels would result in high enzyme production. According to the obtained results, it can be predicted that the optimum medium composition for áamylase production by Nocardiopsis aegyptiae is as follows gl-1 : KNO 3 , 1.5; Mg SO 4, 0.75; K 2HPO 4, 0.25; FeSO 4 , 0.025; concentrated sea water ( >100%); adjusted to pH 7 and inoculum size 1.5 ml for each 50 ml medium, incubated for 7 days at 25 o C. In order to evaluate the accuracy of the applied Plackett-Burman statistical design, a verification experiment was applied to compare between the predicted optimum levels of independent variables and the basal condition settings. It was found that the production of the enzyme (expressed in units) increased to (2187) unit with (1.12) fold increase when compared to it's production under the basal conditions. On the basis of the calculated ttest (Table 4) KNO 3 and inoculum size were positive Table 4: Statistical analysis of the Plackett- B urm an experim ental design Variables M ain Effect t-value* KN O 3 109 1.3 M g SO 4 78 0.88 K 2 H PO 4 -88 -1.02 Sea water 54 0.6 FeSO 4 -90 -1.04 Inoculum size 98 1.156 Tem perature -80 -0.91 *t-value significant at the 1% level = 3.70 t-value significant at the 5% level = 2.45 t-value significant at the 10% level = 1.94 t-value significant at the 20% level = 1.37 Standard t-values are obtained from statistical m ethods [4 ] . Fig. 2: Elucidation of fermentation conditions affecting á-amylase production by N. aegyptia. 288 J. Appl. Sci. Res., 5(3): 286-292, 2009 Repeated fermentation of immobilized cells up to 6 th run carried out in modified starch nitrate medium showed that N. aegyptia were physiologically active. Table 5: á-am ylase production by cells of N.aegyptia Im m obilizing substrate Luffa Pum ice Enzym e production(unit) 2255 2248 Discussion: Amylases have most widely been reported to occur in microorganisms, although they are also found in plants and animals. Two major classes of amylases have been identified in microorganisms, namely á -amylase and Glucoamylase [2 3 ]. á -Amylase may be derived from several bacteria, yeasts and fungi. Bacterial amylase, however, is generally preferred over fungal amylase due to several characteristic advantages that it offers. Strain of Aspergillus sp and Bacillus sp mainly Bacillus amyloliquefaciens and B.licheniformis, are e m p lo ye d fo r com m ercial applications [ 6 ] . Thermostable á- amylases are generally preferred as their application minimizes contamination risk and reduces reaction time, thus providing considerable energy saving. Hydrolysis carried out at higher temperature also minimizes polymerization of Dglucose to Isomaltose. Commercial production of amylases is carried out in various steps, essentially because the environmental factors required for the optimum growth for microorganism being employed factors required for the production of enzymes. These parameters include nutrient supplementation, pH of the medium, osmotic relationship, and degree of aeration, temperature and the control of contamination during fermentation. Maintaining the purity of the medium is also a very important factor, especially when the fermentation is carried out under aerobic conditions. Actinomycetes are Gram positive bacteria with a high G+C content, have the capacity to produce a vast array of secondary metabolites and extracellular proteins. The latter comprise many hydrolytic enzymes such as amylases, cellulases, chitinases, and xylanases allowing actinomycetes to grow on polymeric substrates. Here we first report the production of amylase secreted by Nocardiopsis aegyptia that was isolated as novel species from marine sediments [2 5] . In the present study, Plackett-Burman design was applied which was successfully employed in enzyme production and other optimization experiments [3 2 ,7 ,8 ]. Results revealed that concentration of potassium nitrate and inoculum size are the most highly positive significant factors which affect enzyme production by Nocardiopsis aegyptia More over, using the high settings of potassium nitrate, inoculum size, magnesium Fig. 3: Inoculum size and K NO 3 concentrations response as function of á-amylase production. Effect of different pHs on á -amylase production: The data presented in fig.4, showed that, production of the enzyme required acidic conditions, and maximum yield (2255 unit) was achieved at pH 5, i.e., at pH 5, the production of á -amylase was 16% high, above and lower this value, the yield decreased. The lowest production was estimated at pH 8 (1400 unit). Fig. 4: Effect of production different pHs on Sponge 2250 á -amylase Effect of different immobilizing substrates on á amylase production: Living cells of N.aegyptia were sub jected to im mo b ilization using adsorption techniques. Adsorption was carried out using luffa pulp, pumice and sponge as supporting materials. The results in (table 5) showed that, no increase in enzyme production by using immobilized cells and no significant differences between the estimated values resulted from using different substrates. On the other hand,estimation á-amylase production by using of luffa pulp immobilized cells at different time intervals (2-6 days) showed that, the highest productivity (2255 unit) was achieved after only 2days. 289 J. Appl. Sci. Res., 5(3): 286-292, 2009 sulphate and concentrated sea water,would positively affect production by Nocardiopsis aegyptia while the using of low settings of dipotassium hydrogen phosphate, ferrous sulphate and temperature, would also promote enzyme production.. Therefore, results concluded that to achieve the highest productivity by No card iop sis aeg yp tia , the o p tim um m edium composition should be as follows: (g l-1 ) Starch, 20; potassium nitrate, 1.5; magnesium sulphate,0.75;dipotassium hydrogen phosphate, 0.25; ferrous sulphate, 0.025; concentrated sea water, with inoculum size 1.5 ml (for 50 ml medium) at 25 o C . Under such conditions, the enzyme production by Nocardiopsis aegyptia was 2187 unit i.e (1.12 fold increase) than that obtained using the basal growth medium. Narayana and Vijayalakshmi.[2 0] reported that, among the studied carbon sources used for amylase production starch was found to be the best substrate, showing maximum enzyme activity K uo and Hartman[1 6 ] . showed that thermoactinomyces vulgaris produces best yields of á – amylase when starch or maltose is used as a carbon source. Simpson and McCoy,[2 8 ] . studied the influence of incubation period on á – amylase produced by Streptomyces albidoflavus. The production of á – amylase began after 24 h of cultivation and reached to peak levels after 48 h and declined there after. The highest yield has been found after 52 to 56h while in this study the maximum production achieved after 6 days of incubation and after applying the immobilization techniques, the maximum productivity (2255 units) was achieved after 2 days. K uo and H artm an [ 1 6 ] . also found that thermoactinomyces vulgaris synthesized amylase most rapidly at pH values ranging from 6.5 and 7.5 and that amylase inactivation occurred rapidly if pH rose above 7 .5 . A m yla se p ro d uctio n b y S trep to m yces aureofaciens77 has been increased gradually as the initial pH values ascend from 5 to 7 . Shatta, et al., [2 7 ]. found that á – amylase are generally stable at pH ranged from 5.5 to 8.0 and optimal activity of á – amylase occurs between pH 4.8 and 6.5 these results agreed with our results where maximum production achieved at pH 5 and the yield increased to (2255 unit) i.e (1.16) fold increase. Only two reports on production of á – amylase from Nocardiopsis species up to now Stamford, et al. [29] . showed the highest activity at 70 º C and pH 5.0 while the second study by[3 3 ] . on production of cold adapted á – amylase by Nocardiopsis species 7326 gave its highest activity at low temperature(35 ºC) and stability at alkaline pH(8), which also permitted its biotechnological applications in various studies. For example, it could be applied as a detergent additive, as a resizing agent in textile processing, and in food industry. Also Narayana and Vijayalakshmi[2 0 ] . reported that the optimum temperature for maximum enzyme activity was found to be at 30 º C and enzyme stable up to 35º C and at higher temperature, the enzyme activity decreased sharply and most purified enzymes lose activity rapidly above 50 º C. It is amazing that our strain N.aegyptia achieved the highest productivity at lower temperature (25 º C) in acidic conditions (pH 5.0). 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