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Research Journal of Agriculture and Biological Sciences, 4(5): 434-446, 2008 © 2008, INSInet Publication

Optimization, Immobilization of Extracellular Alkaline Protease and Characterization of its Enzymatic Properties 1

Samia A. Ahmed, 2Ramadan A. Al-domany, 3Nefisa M.A. El-Shayeb, 4 Hesham H. Radwan and 5Shireen A. Saleh. 1,3,5

Department of Chemistry of Natural and Microbial Products National Research Center, Cairo, Egypt. 2,4 Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University, Egypt. Abstract: Streptomyces avermectinus NRRL B-8165 was the most potent for production of extracellular alkaline protease among 6 strains of Streptomycetes using shaken culture technique (180 rpm) after 72h at 37°C. Highest yield of extracellular enzyme (3.62 U/ml) was achieved using 7% sucrose and 0.5435% alkaline extracted soybean as carbon and nitrogen sources, respectively. Addition of wheat bran extract in concentration of 20 g/L increased alkaline protease production by 24.29% than control. Adding 0.25% corn oil and MnCl2 (0.1mM) as trace element to the medium caused a slight increase in enzyme activity. The enzyme was partially purified with 50% acetone and showed 4.1-fold purification. Alkaline protease was immobilized on different carriers by different methods and its specific activity for casein hydrolysis was studied. Immobilization of alkaline protease on sponge (covalent binding) had the highest immobilization yield (86.19%) using glutaraldehyde (0.25% concentration). It was further observed that thermal stability of the immobilized enzyme was higher compared to free enzyme. The immobilized enzyme had higher K m (Michaelis constant) and lower Vm ax (Maximum rate of reaction) than the free enzyme. Activation energy (E A) of the free enzyme was 6.206 Kcal/mol which was higher than the immobilized enzyme. Half-life (t 1/2 ) of immobilized enzyme was 808.6 and 108.6 at 65 and 70°C, respectively, which was higher than that of free enzyme. Immobilized enzyme showed the highest operational stability for up to 15 reuses with 49.74% residual activity. On the other hand, some applications of enzyme were carried out (dehairing, decomposition of gelatin layers on X-ray films and detergent stability). Key words: Alkaline protease, immobilization, Streptomyces, application numerous secondary metabolites and particularly antibiotics. The ability of these bacteria to produce large amounts of enzymes, as proteases with varied substrate specificities, offers another potentially interesting use [40] . For industrial applications, the immobilization of proteases on a solid support can offer several advantages, included repeated usage of the enzyme, ease of product separation and improvements in enzyme stability. Enzyme stabilization will thus continue to be a key issue in biotechnology. In the present study, certain environmental and nutritional parameters controlling the biosynthesis of extracellular alkaline protease from Streptomyces avermectinus NRRL B-8165, partial purification and the immobilization of partially purified enzyme on different carriers using diff e r e n t m e t hods of immobilization including physical adsorption, ionic binding, covalent binding and entrapment were fully

INTRODUCTION P roteases are the most important kind of industrial enzymes[22] and account for about 65% of the worldwide sale of industrial enzymes in the world market [21] . Microbial alkaline proteases dominate the worldwide enzyme market, about two-third share of the detergent [18] . Applications of alkaline protease are concentrated in laundry detergents, leather processin g, b r e wing, food and pharmaceutical industries[26] . The enzyme should be stable and active in the presence of typical detergent ingredients for use in detergent [37]. Microorganisms served as an important source of proteases mainly due to their shorter generation time, the ease of bulk production and the ease of genetic and environmental manipulation [39] . Streptomyces species are the most industrially important actinomyces, due to their capacity to produce Corresponding Author:

N e fis a M . A . El-Shayeb, Department of Chemistry of Natural and Microbial Products National Research Center, Cairo, Dokki, Egypt. E-mail: [email protected]

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investigated. The immobilized enzyme by covalent binding on sponge had the highest immobilization yield (86.19% ). The refore, this immobilized enzyme preparation was used as a typical example for Streptomyces avermectinus NRRL B-8165 immobilized alkaline protease and its properties was investigated and compared with the free one. Also, in this paper, we present potential applications of extracellular alkaline protease for different industrial purposes.

tested. The precipitation was done in a sequential manner. The precipitated fractions were removed by centrifugation using a refrigerated centrifuge at 4000 rev/min for 15 min. The active fractions were lyophilized and assayed for caseinolytic activity. Immobilization Techniques: Physical Adsorption: It was carried out according to Woodward (1985), one gram of each carrier was incubated with the 50% acetone enzyme fraction (66.5 U/g carrier) dissolved in 5 ml glycine-NaOH buffer (0.1 M, pH 10) overnight at 4°C.

MATERIALS AND METHODS Organism: Streptomyces avermectinus NRRL B-8165 was obtained from Northern Regional Research Laboratory (NRRL), Peoria, Illinois, USA.

Ionic Binding: It was carried out according to W oodward[50] in which 0.1 g of the cation or anion exchanger was equilibrated with glycine-NaOH buffer (0.1 M, pH 10) and incubated with 1 ml of enzyme solution (6.65 U) overnight at 4°C.

Carriers for Enzyme Immobilization: Polystyrene, Dowex 50 Wx4 (50-100) mesh, Dowex 1x8 (20-50) mesh, cellulose triacetate, DEAE-Sephadex A-50, chitin, agarose, chitosan were from Fluka Company, Switzerland. Sephadex G-100 was obtained from Pharmacia fine chemicals, Uppsala, Sweden. DEAEcellulose 53 was obtained from Whatman Biosystems Ltd.

Covalent Binding: This method was carried out as follows: One gram of were treated with 5ml of 0.25% (v/v) glutaraldehyde for 2 h at 30°C. Then washed with distilled water to remove the excess glutaraldehyde. The carriers were mixed with 5 ml of enzyme solution (66.5 U) and incubated overnight at 4°C [3].

Medium Composition and Cultivation: The basal medium for liquid cultures consisted of (g/L): (NH4)2SO 4, 1; KH2PO 4, 0.5; CaCO 3 , 1; NaCl, 1; MgSO4 , 1; peptone, 1; dextrin (white pract), 30;[2] . The pH was adjusted to 8.0. The same medium was also used for inoculum preparation for all the fermentation experiments. Cultivation was in 250 Erlenmeyer flasks containing 50 ml of sterile medium. The flasks were inoculated with 2 ml of 24h old culture. The flasks were incubated at 37°C with shaking at 180 rpm. The cells were harvested by centrifugation at 4000 rev/min. The clear culture filtrates were assayed for enzyme activity.

Entrapment: In Agar and Agarose: 10ml of different concentrations of agar (2, 3 and 4%) or agarose (1, 2 and 3 %) solutions were mixed with enzyme solution (2.66U). The mixture was quickly cooled to 4°C and cut into small cubes[50]. In Ca-alginate Beads: 10ml of different concentrations of sodium alginate solution (1, 2 and 3%) were mixed with enzyme solution (2.66U). The entrapment was carried out by dropping the alginate solution into 0.15 M CaCl2 . The resulting beads (0.5-1.0mm diameter) were collected[8] .

Assay of Protease Activity: It was determined using casein as a substrate according to the method reported by Gessesse and Gashe [17 ] with some modifications and protease activity was determined as released tyrosine. One unit of enzyme activity was defined as the amount of enzyme that librates 1µmole of tyrosine per min under the above mentioned conditions.

Thermal Stability: The thermal stability of free and immobilized protease were tested by incubating the enzymes in 0.1 M glycine-NaOH buffer (pH 10) at different temperatures (30-75°C) for different time intervals (15-120 min) before activity assay.

Protein Determination: This was estimated according to the method of Lowry et al.[32] .

Effect of the Reaction Time: The enzyme assay was conducted at 55°C and pH 10 for different time intervals (5-60 min).

Partial Purification of the Enzyme: This was done by fractional precipitation using ethanol or acetone or by salting-out with ammonium sulphate using cell free culture filtrate of the optimized fermentation medium. Different acetone concentrations (30-60 %v/v) were

Effect of the Addition of Some Salts of Metal Ions: Free and immobilized enzymes were incubated, in absence of substrate, with metal ions in different concentrations (0.1, 1.0 and 10.0 mM) at 30°C for 1 h.

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compare d t o t h a t found by Kathiresan and Marivannan [24] who reported the production of alkaline protease at 5.0% sucrose by Streptomyces sp. Also, different nitrogen sources in the culture medium were investigated. This included organic (yeast extract, backer's yeast, peptone, casein, alkaline extracted soybean and urea) and inorganic (ammonium sulphate and ammonium chloride), nitrogen sources used in the production medium on equal nitrogen basis. It seemed that the alkaline extracted soybean was more suitable for the enzyme production than the other nitrogen sources. Ammonium chloride gave the least enzyme productivity, while ammonium sulphate inhibited the enzyme production completely. This result is similar to that reported by Kaur et al.[25] and Chauhan and Gupta [11]. They found that organic sources were better suited to Bacillus sp. for enzyme production. It has been reported that the effects of a specific nitrogen supplement on protease production differ from organism to organism although complex nitrogen sources are usually used for alkaline protease production [29] . However, addition of inorganic sources resulted in decreased production of enzyme. Similar results also have been obtained with other Bacillus sp. [ 4 1 ] . In contrast to our results, Sinha and Satayanarayana [ 4 4 ] and Nascimento and Martins[38] reported that higher protease production with inorganic nitrogen sources such as ammonium sulphate and ammonium nitrate. Therefore, alkaline extracted soybean was used as the most suitable nitrogen source for maximal enzyme production. This result is in agreement with that obtained by Mabrouk et al.[33]. The study also included the effect of different concentrations of alkaline extracted soybean as nitrogen source in the culture medium. The succeeding experiments were performed using alkaline extracted soybean at 5.435 g/L concentration in the culture medium. This concentration was shown to be the most effective for the enzyme production (3.62 U/ml). It is worthy to note that calcium carbonate in the basal medium was used as a buffering agent. Therefore, the effect of different levels of CaCO3 was also investigated. The maximal extracellular alkaline protease production was obtained when its level was 0.5% g/L. Increasing CaCO 3 level from 0.0 to 0.5 g/L resulted in 10.44-fold increase in enzyme production. However, the higher levels showed an adverse effect on the enzyme production Fig3. In this respect, Laan and Konings[30] reported that higher concentration of Ca 2+ ions negatively affected the release of proteinase from Lactococcus lactis. This indicated that CaCO3 at 0.1% was below the critical level at which the inhibition of protease occurred.

Operational Stability of the Immobilized Enzyme: 2.5 grams of immobilized enzyme (wet) containing about 66.5 U was incubated with substrate in glycineNaOH buffer (0.1M at pH 10) at 55°C for 20 min. At the end of the reaction period, the carrier was squeezed washed with buffer and the immobilized enzyme was resuspended in freshly prepared substrate to start a new run. The wash was assayed for alkaline protease activity. Some Applications: Detergent Stability of Crude Extracellular Alkaline Protease: This was carried out according to Hadj-Ali et al.[20] . Decomposition of Gelatin Layers on X-ray Films: This was done according to Masui et al.[34] method with some modifications. Dehairing Agent: This was done according to Zambare et al.[51] method. RESULTS AND DISCUSSION The ability of 6 Streptomycetes strains to produce active extracellular alkaline protease was investigated. They were cultured on the basal medium for different incubation periods under shaking conditions and in each case the culture filtrate was assayed for caseinase activity. The results indicated that Streptomyces avermectinus NRRL B-8165 was the most potent producer of extracellular alkaline protease (2.04 U/ml) after 72 h incubation at 37°C. These results are similar to those reported by Mohamedin [36] from Streptomyces ther monitrificans and Gupta et al.[19] from Virgibacillus pantothenticus for alkaline protease production. Substitution of the dextrin in the culture medium by other carbon sources (sucrose, glucose, soluble starch, lactose, maltose, molasses and casein) indicated that lactose and casein significantly inhibited the alkaline protease production. Sucrose proved to be the most favorable carbon source for the production of active alkaline protease by Streptomyces avermectinus NRRL B-8165 (2.61 U/ml). On the other hand, maltose, molasses, dextrin and glucose gave lower levels of enzyme productivity compared to sucrose. These results are similar to that reported by Chi and Zhao [ 1 4 ] who found that different carbon sources have different influence on extracellular enzyme production by different strains. The results indicated that gradual increase in sucrose concentration from 0.0 to 7.0% resulted in 12.5-fold increase in alkaline protease production. Higher levels however showed an adverse effect on enzyme production. The result is highly

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Some experiments were done in order to explore the effect of external additives on the enzyme production. The addition of wheat bran extract and corn oil was studied. The results showed that wheat bran extract in concentration of 20 g/L gave alkaline protease of (5.03 U/ml) with increase in the enzyme activity by 24.20% than the control. Also, adding 0.25% corn oil to the medium as a surfactant led to a little increase in productivity (1.38%). This result is in agreement with that obtained by Mabrouk et al.[33] by Bacillus licheniformis ATCC 21415. With regard to the effect of addition of some salts of metal ions indicated that Mn 2+ (1mM) enhanced the production of extracellular alkaline protease. On the other hand, Zn 2 + , Fe 3+ and Cu 2+ had adverse effects on the enzyme production. This result is similar to that reported by Shafee et al. [ 42] for a Bacillus sp. alkaline protease. Production of extracellular alkaline protease by Streptomyces avermectinus NRRL B-8165 was considerably affected by the initial pH values. It can be noticed that pH 7.5 was the most favorable for the alkaline protease production. Above and below initial pH values showed a gradual decrease in enzyme production. This result is similar to those reported by Banik and Prakash [7] who produced alkaline protease maximally at pH 7.5 after 72h incubation from Bacillus cereus. After optimization of the chemical composition of the production medium for the biosynthesis of extracellular alkaline protease by Streptomyces avermectinus NRRL B-8165, the results indicated that the culture filtrate possessed extracellular alkaline protease of 5.22 U/ml. This culture medium was therefore used in all succeeding work. Alkaline protease from Streptomyces avermectinus NRRL B-8165 was partially purified by ethanol, acetone and ammonium sulphate. The most of proteolytic activity was recovered at 50% acetone (21.02) and showed 4.10-fold purification. The results presented in Table1 points to the most active fractions produced by ethanol, acetone and ammonium sulphate. Immobilization of the partially purified extracellular alkaline protease was studied using the 50% acetone fraction. This was done using different supports and different methods of immobilization such as physical adsorption, ionic binding, covalent binding and entrapment. The efficiency of enzyme immobilization was evaluated different parameters including the retained enzyme activity, the specific activity of the immobilized enzyme and the loading efficiency (immobilized activity/g carrier). The immobilization yield is the key parameter since it represents the

general output of the efficiency of the immobilization process[9] . The data for immobilization of the extracellular partially purified alkaline protease by covalent binding (Fig1) indicated a good immobilization yield with sponge through a spacer groups (glutaraldehyde) which sh o w e d go o d lo a d in g e f fic ie n c y a n d go o d immobilization yield. The good loading efficiency for the immobilization by covalent binding may be due to the formation of stable crosslinking between carrier and enzyme through a spacer group (glutaraldehyde). In addition, covalent binding through a spacer group probably increased the surface area of the carrier and consequently reduced the steric hindrance in the immediate vicinity of the enzyme molecule. These results are similar to those reported by Siso et al.[45] . The results also showed that 0.25% glutaraldehyde was the best concentration to activate support (sponge) for giving the highest enzyme immobilization yield (77.47 %). Carrara and Rubiolo [10] reported that the best concentration of glutaraldehyde was 1% for support (chitosan) activation. While Tanksale et al.[48] and Ferreira et al.[16] found the maximum immobilization yield was obtained with 1.76 and 0.5% glutaraldehyde, respectively. The decrease in immobilization yield by increasing glutaraldehyde concentration may be due to the reticulation among enzyme molecules favored by the use of a bifunctional molecule and therefore, affecting the overall activity [16]. The data for the immobilization of alkaline protease by ionic binding (Fig2) showed low immobilization yield (11.03%) on DEAE- Sephadex. On the other hand, the immobilized enzyme on Amberlite IR-120 showed the highest immobilization yield (34.25%). Similar results were previously reported for Bacillus mycoides protease immobilized by ionic binding on Amberlite IR-120 [1] . Immobilization of Streptomyces avermectinus NRRL B-8165 alkaline protease by physical adsorption (Fig 3) was employed on different carriers including wool, stone, sponge, loofa, corn pulps, chitin and cellulose. The immobilization of the enzyme by physical adsorption on sponge had the highest immobilization yield (42.36%) and the highest loading efficiency (37.77 U/g carrier). The immobilization of the alkaline protease by entrapment in calcium alginate, agar or agarose was done using different concentrations (Fig 4). The results indicated that agarose (1%) gave the highest immobilization yield (49.79% ). At higher carrier concentration the immobilization yield decreased. This may be due to decreased gel porosity with the increase in gel concentration and consequently the diffusion limitation was developed. Similar observations

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were previously reported for other entrapped enzymes[31]. From all immobilization methods and carriers, the enzyme immobilized by covalent binding on sponge was used as a typical example for immobilized Streptomyces avermectinus NRRL B-8165 alkaline protease. The properties were investigated and compared with those of the free one (Table 2). The specific activity of immobilized was about 71.1% of the free enzyme which was higher than that obtained by Mittal et al.[35] with immobilized enzyme (56% ). while Suh et al.[47] reported that the specific activity of immobilized enterokinase was 25% . Investigation of the effect of the reaction time of immobilized enzyme and free enzyme were 20min and 10min (data not shown). In this respect, Mittal et al.[35] reported a c a lc iu m a lgin a t e im m o b ilize d dipeptidylpeptidase which had maximum activity of 90min compared to 60min for the free. They explained that this was possibly due to limited diffusion of substrate initially, but once a sufficient amount of substrate had entered the beads then the enzymatic activity increased because of the close proximity of substrate and enzyme. The optimum temperature of the reaction was not affected by immobilization process (55°C). This result is similar to that reported by Abdel-Naby et al.[1] who reported that both free and immobilized alkaline protease covalently linked to chitosan had the same optimum temperature (50°C). The calculated values of the activation energies for the free and immobilized enzymes were 6.206 and 5.688 Kcal/mol, respectively. The lower value of activation energy of the immobilized enzyme compared to the free enzyme may be attributed to the mass transfer limitations. Kitano et al.[27] and Allenza et al.[5] reported that the activation energies of the immobilized enzymes were lower because the internal diffusion is the rate limiting step. The decrease of the activation energy of immobilized protease was also reported by [16,49]. The rate of heat inactivation of the free and immobilized enzyme was investigated at 65 and 70°C. In general, the immobilization process protected the enzyme against heat inactivation. For example, the calculated half-live values at 65 and 70°C for the immobilized enzyme on sponge were 808.6 and 108.6 min, respectively, which were higher than that of the free enzyme. The result is higher than that reported by Ahmed et al.[4] on wool immobilized alkaline protease (t 1 /2 at 60 and 70°C were 100 and 25, respectively). Also, Silva et al.[43] reported lower half live of immobilized protease at 60°C (438 min) than our result.

The calculated deactivation rate constant showed that the thermal stability of the immobilized enzyme was superior to that of free one due to protection that provided by immobilization to enzyme [28]. The calculated deactivation rate constants at 65 and 70°C for the free enzyme were 4.20 x 10 -3 and 9.33 x 10-3 and for the immobilized enzyme 0.857 x 10-3 and 6.38 x 10-3, respectively. The investigation of the free and immobilized enzymes at different pH values showed that the optimum pH values of the free and immobilized enzymes were the same (pH 10.0) Table (2). This result is similar to that reported by Chellapandian and Sastry[12] who reported that alkaline protease covalently linked on nylon had optimum pH 8.5 which was similar to that of free 8.5-9.0. Thermal stability of the immobilized enzyme was also studied (Fig 5 & 6). In general, the thermal stability of the immobilized enzyme was significantly improved in comparison to the free enzyme. Thus after heating the enzyme, in the absence of the substrate, the adverse effect of temperature began with the free enzyme when incubated at 45°C for 60min and more. On the other hand, the immobilized enzyme was slightly affected when heated at 65°C for 90min leading to loss of only 10.37% of its activity. On heating free enzyme at 65°C and immobilized at 70°C and higher, the adverse effect of temperature were more pronounced in case of free than the immobilized enzyme. These results agreed with those reported for immobilization of Bacillus subtilis alkaline protease by Davidenko [15] . The immobilized enzyme showed 2-fold increase in thermal stability, compared to free one, after 2h at 50°C. This result is similar to Ferreira et al.[16]. Chellapandian and Sastry [12] stated that the increase in thermal stability of the immobilized protease is due to reduction in autolysis and this is possibly due to (i) enzyme-enzyme interaction and (ii) re st ricting conformational flexibility. The restriction of molecular flexibility might be due to multipoint covalent interaction with the matrix. The effect of the metal salts on the activity of immobilized and free alkaline protease was investigated in Tables (3). The activation by metal ions (Ca 2+ , Mg2+ , Mn 2+ and Na + ) became more pronounced with the i m m o b ilize d e n zy m e . O n t h e o t h e r h a n d , immobilization protected the enzyme from the inhibition by some metal ions (Co 2+ , Cu 2+ and Fe 3+ ). These results similar to Wehidy [49]. On the other hand, Amaral et al.[6] reported the inhibition of trypsin by Al3+ , Cu2+ , Zn 2+ and Mg2+ , but immobilization protected the enzyme to some extent from Ca 2+ and Mg2+ inhibition.

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Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008 Table 1: Partial purification of Streptomyces avermectinus NRRL B-8165 alkaline protease. Purification Total protein Total activity Recovered Specific activity Purification (mg/fraction) (U) activity (%) (U/mg protein) fold Culture filtrate 247.00 807.69 100 3.27 1 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ethanol 50% 19.82 77.79 9.63 3.92 1.20 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Acetone 50% 12.65 169.80 21.02 13.42 4.10 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ammonium sulphate 50% 7.59 55.50 6.87 7.31 2.24 Table 2: Properties of free and immobilized extracellular partially p urified alkaline protease from Streptomyces avermectinus NRRL B-8165 Property Free enzyme Immobilized enzyme Specific activity (U/mg protein) 13.42 9.54 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Optimum temperature (°C) 55 55 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Optimum pH 10 10 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Activation energy (Kcal/mole) 6.206 5.688 Half life t1 /2 (min) at 65 °C 165.0 808.6 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------70 °C 74.3 108.6 Deactivation rate constant(min-1 ) at 65 °C 4.20 x 10-3 0.857 x 10-3 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------70 °C 9.33 x 10-3 6.38 x 10-3 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Km (mg/ml) 0.59 4.55 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vm ax (U/mg protein) 16.95 12.50 Effect of addition of different concentrations of some metal salt on the ac tivity of free and immobilized alkaline protease of Streptomyces avermectinus NRRL B-8165. Metal salt conc. (mM) Relative activity (%) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Metal salts Free enzyme Immobilized enzyme --------------------------------------------------------------------------------------------------------------------------------0.1 mM 0.1 mM 0.1 mM 0.1mM 1 mM 10 mM Control 100.00 100.00 100.00 100.00 100.00 100.00 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CaCl2 100.41 106.48 90.97 111.10 119.66 90.25 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CoCl2 .6H2 O 94.80 90.03 73.42 100.00 93.68 89.07 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CuCl2 .2H2 O 93.44 100.81 99.74 100.25 100.89 100.50 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------FeCl3 86.72 86.13 84.59 100.55 98.07 90.78 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------LiSO 4 92.72 96.83 101.45 96.62 104.75 123.11 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------MgCl2 .6H2 O 98.02 98.99 98.08 104.50 112.4 159.57 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------MnCl2 .4H2 O 89.78 95.80 116.35 101.08 107.28 134.29 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------KCl 92.35 112.01 114.95 96.46 102.97 119.74 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------NaCl 98.98 100.70 99.64 100.35 139.09 115.66 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ZnCl2 97.93 100.97 102.50 99.64 98.86 100.86 *Control was carried out without any tested substances. Table 3:

Lineweaver-Bürk plots of the free enzyme gave Km (Michaelis constant) of 0.59mg/ml with casein, which was 7.7-fold lower than that of the immobilized enzyme (4.55mg/ml). The calculated value of Vm ax

(maximum reaction rate) of the free enzyme was 16.95U/mg protein which was 1.36-fold higher than that of the immobilized (12.5U/mg protein). So, the Vm ax value of immobilized enzyme is lower than that 439


Fig. 1: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus NRRL B-8165 using covalent binding.

Fig. 2: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus NRRL-B-8165 using ionic binding.

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Fig. 3: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus NRRL B-8165 using physical adsorption.

Fig. 4: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus NRRL B-8165 using entrapment in agar, agarose and calcium alginate.

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Fig. 5: Thermal stability of immobilized extracellular partially purified alkaline protease produced by Streptomyces avermectinus NRRL B-8165.

Fig. 6: Thermal stability of immobilized extracellular partially purified alkaline protease produced by Streptomyces avermectinus NRRL B-8165. of the free one [35,43 ] . In this respect, Wehidy [49] found Km of free was 1.2-fold lower than the immobilized protease. While Vm a x of free and immobilized was 7.67 and 7.27 U/mg protein, respectively. The operational stability of immobilized enzyme is one of the most important factors affecting the utilization of an important enzyme system. Therefore, the operational stability of the immobilized alkaline protease was evaluated in repeated batch process

(Fig. 7). The result indicated the durability of the catalytic activity in repeated use for more than 15 cycles. The retained catalytic activity after being used for 15 cycles was 50% of the initial activity. In this respect, it was reported by Chellapandian [13] that the vermiculite-bound alkaline protease retained 78% of its original activity after five repeated uses. Our result is higher than that obtained by, Mittal et al. [ 3 5] when reused immobilized dipeptidylpeptidase 6 cycles (retained activity 30%).

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Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008 Table 4: Detergent stability of crude extracellular alkaline protease produced by Streptomyces avermectinus NRRL B-8165. Time (min) --------------Relative activity % Detergent --------------------------------------------------------------------------------------------------------------------------------------------------0 15 30 45 60 Control 100.00 100.00 100.00 100.00 100.00 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ariel® 100.00 100.00 97.05 86.68 73.46 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Persil color® 100.00 100.00 100.00 98.66 82.03 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Tide® 100.00 85.25 65.42 60.71 53.89 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------X.TRA® 100.00 100.00 100.00 95.38 79.44

Fig. 7: Operational stability of immobilized extracellular partially purified alkaline protease on sponge using covalent binding produced by Streptomyces avermectinus NRRL B-8165.

Photo 1&2: Decomposition of X-ray films by partially purified extracellular alkaline protease produced by Streptomyces avermectinus NRRL B-8165. (1) control and (2) experiment. was most stable with Persil color ® up to 60 min where it lost only 17.97% of its activity. On the other hand, the lowest enzyme stability was observed with Tide ® . However, it lost 20.56% and 26.54% of its activity after 60 min incubation with X-TRA® and Ariel® , respectively at 40°C Table (4). In this respect, Hadj-Ali et al.[20] reported a detergent stable protease which

The overall performance of the immobilized alkaline protease is promising than the free enzyme. Thus it is suggested that Streptomyces avermectinus NRRL B-8165 alkaline protease immobilized on sponge by covalent binding is suitable for practical application. In our study, the enzyme compatibility with commercial detergents was investigated. The enzyme 443


Photo 3:

Dehairing of buffalo hide by partially purified extracellular alkaline protease produced by Streptomyces avermectinus NRRL B-8165. (1) control, (2) after 12h and (3) after 24h.

retained 65-95% activity after 1h incubation with commercial detergents at 40°C and Gupta et al.[19] reported a protease which retained 30-40% activity after 30min incubation with Ariel® and Surf Exel® but lost activity after that. On the other hand, it lost only 10-30% activity with other commercial detergents after 2h incubation at 40°C. In our studying the ability of the partially purified enzyme to decompose the gelatin layer of X-ray film in order to recover both silver and polyethylene tetraphthalate film base within only 15 min (Photo 1&2). This result is similar to Masui et al.[34] and Karadzic et al.[23]. They reported a protease that decomposed the gelatin layer on Xray film from Bacillus sp. and Pseudomonas aeruginosa, respectively. Also, studying the ability of the partially purified enzyme to dehair buffalo hide was investigated (Photo 3). The enzyme removed intact hairs completely from the hide after 24h incubation at 37°C. In this respect, Zambare et al.[51] reported an alkaline protease that was applied as a dehairing agent. The enzyme acted on the epidermis and hair was removed entirely; the intact hair can be used as a value-added byproduct. Using enzyme will reduce the time of the process and yield better quality leather. However, Sivasubramanian et al.[46] reported that the use of protease in deharing of goat skins and cow hide revealed complete absence of fine hair and epidermis and the pelts were whiter than that dehaired by chemical methods. At last, it is worthy to note that the results of the present work point out to the suitability of the strain Streptomyces avermectinus NRRL B-8165 as a new promising producer of highly active extracellular alkaline protea se u sin g inexpensive materials. Furthermore, enzyme immobilization proved to be a good technique for increasing enzyme thermostability and its several reuse.

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