Jun 1, 2005 - Purification of proteases. This was achieved by salting out with ammonium sulphate and gel filtration. In case of ammonium sulphate, different.
Purification and characterisation of extracellular neutral protease from Streptomyces microflavus (Received: 01.06.2005; Accepted: 15.06.2005) Hala M. Rifaat*, Saadia M. Hassanein**, Osama Hamed El-Said***, Soad A. Saleh***, Manal S. M. Selim*** *Microbial Chemistry Department, National Research Centre, Cairo, Egypt **Microbiology Department, Ain Shams University, Cairo, Egypt ***Microbial Biotechnology Department, National Research Centre, Cairo, Egypt
ABSTRACT A neutral protease was detected in the culture medium of Streptomyces microflavus isolated from some Egyptian soils. The enzyme was purified by precipitation with ammonium sulphate and gel filtration on Sephadex G-75. The optimal pH and temperature for catalytic activity of protease was pH 7 and 40 0C respectively. Calcium and manganese stimulated protease activity while Ag+ inhibited the enzyme. The proteolytic activity of protease was strongly inhibited by 0.8 mM of Para chloromercuribenzoic acid (P. CMBA) and phenyl methyl sulfonyl floride (PMSF). Key words: Streptomyces, protease, purification, characterisation.
INTRODUCTION
A
ctinomycetes, particularly strepto_ mycetes, are known to secrete multiple proteases into the culture medium (Kalisz, 1988). Some of these proteases, the serine proteases of Streptomyces griseus (Olafson et al., 1975) and Streptomyces fradiae (Kitadokora et al., 1994), have been characterised enzymatically. There also many descriptions of isolation and partial characterisation of protease activities from other members of the genus Streptomyces (Bockle et al., 1995 and Wang et al., 1999). In these prokaryotic microorganisms, extracellular proteases are involved in hydrolysis of large polypeptide substrates into smaller molecular entities which can subsequently be recognised by the cells (Cohen, 1990). They usually show low Arab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
substrate specificities and can degrade most of the non-structural proteins such as fibrin, fibrinogen but also elastin, collagen or laminin (Monod et al., 1991). In bacteria, production of extracellular proteinases also coincides with sporulation and germination (Li and Yousten, 1975). In the present study, neutral protease was isolated, purified and characterised from Streptomyces microflavus. MATERIALS AND METHODS Microorganism, culture conditions and extraction of protease enzyme Streptomyces microflavus was isolated and identified from some Egyptian soil samples. It was grown in a medium containing (per litre of distilled water): 20g starch, 3.8g yeast, 1.0g K2HPO4, 3.0g CaCO3 and 0.01g
52
Hala M. Rifaat et al.
FeSO4. 7H2O at pH 7 (Rifaat et al., 2005). The cells (107/ml) were grown in flasks containing 50 ml medium and maintained at 28 0C under constant shaking 150 rpm (rotation/minute). At the end of the incubation period (3 days), the crude enzyme was recovered in the supernatant by centrifugation (27500 Xg, 15 min, 4 0C). Purification of proteases This was achieved by salting out with ammonium sulphate and gel filtration. In case of ammonium sulphate, different concentrations of 20, 40, 60 and 80% (w/v) were used according to Dixon (1953). For each concentration of ammonium sulphate, the protein content and proteolytic activity were measured (Bradford, 1976 and Yang and Huang, 1994). Besides, gel filtration was applied. The dialysate ammonium sulphate fractions were applied to a column (1.6 X 60 cm) of Sephadex G-75 equilibrated with 0.05 M phosphate buffer (pH 7.2) and eluted with one liter of the same buffer at flow rate of 35 ml/hr. Elute (5ml fractions) was collected for measurement of absorbance at 280 nm. The enzyme activity was assyed as well adopting the procedure of Kang et al. (1995). The active fractions were pooled and dialysed against distilled water. The protein content was measured according to the method given by Bradford (1976). Determination of protein content To determine the protein concentration, a standard curve of protein concentration was established using Coomassie brilliant blue (CBB) and Bovine serum albumin (BSA), according to Bradford (1976). Briefly, protein solutions with 1 to 20 µg were made up to a total volume of 0.5 by distilled water. 0.5ml of CBB dye solution was added to these solutions, mixed well and the absorbance was measured after 10 minutes at 595 nm. The rab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
absorbance was plotted against the protein content. The protein content of the unknown samples was assayed as above and calculated from the standard curve. Protease assay Protease activity of culture broth was determined by the modified Anson's method (Yang and Huang, 1994). One unit of protease activity was defined as the amount of enzyme that produced an absorbance at 280 nm equivalent to 1 µ mole of tyrosine in one minute under the assay conditions. Preparation of standard tyrosine solution The tyrosine standard was prepared by weighting out 289.7 mg of pure, dry tyrosine, dissolved in one litre of 0.2 M HCl with 0.5% formaldehyde followed by dilution ten fold in 0.2 M HCl. The optical density was measured at 280 nm. Results were expressed in terms of micrograms of tyrosine released per ml (µg/ml) of reaction mixture. Properties of proteases Thermal stability The purified enzyme was incubated at different temperatures ranging from 30 to 60 5 C for 10 to 60 min. pH Three different buffers with different pH estimates were used; citrate phosphate at pH 4 (0.05M) (Gupta et al., 1999), phosphate at pH 7.2 (0.05M) (Yang and Huang, 1994) and carbonate bicarbonate at pH 9 (0.05M) (Mahmoud et al., 1977). Inhibitors Various inhibitors were used, among this Tween 80, urea, EDTA (ethylenediaminetetra -acetic acid), para chloromercur-ibenzoic acid (P.CMBA) and phenylmethylsulfonyl floride (PMSF) were applied with concentration of 0.08 mM. Metal ions A number of metal ions; Mn2+ (Mncl2), Ca2+ (Cacl2), Zn2+ (Zncl2), Ag+ (AgNO3) with
Extracellular neutral protease from S. microflavus
different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1 mM) was used. RESULTS Purification of neutral protease produced by Streptomyces microflavus Ammonium sulphate Different concentrations of ammonium sulphate were added to the crude protease produced by Streptomyces microflavus (Table 1). Fractions
53
from 40 and 60% ammonium sulphate saturation showed high proteolytic activity and specific activities in comparison with crude protease and other concentrations. On the other hand, concentration of 20% showed extremely high protein content rather than other concentrations. However, such concentration revealed no protease and specific activities. There was no precipitation obtained with 80% ammonium sulphate.
Table (1): Characteristics of protease from Streptomyces microflavus by ammonium sulphate. Ammonium sulphate
Protease activity
Protein content
Specific activity
concentration (%)
(U ml-1)
(mg ml-1)
(U ml-1)
Crude
0.726
0.300
2.42
20
0
5.930
0
40
4.200
0.343
12.20
60
5.500
0.233
23.60
80
0
0
0
Gel filtration The obtained neutral proteases produced by Streptomyces microflavus after partial purification with 40 and 60% ammonium sulphate were subjected on column of Sephadex G-75. In both fractions (40 and 60%), a single peak co-eluting with the protease activity was observed (Figs. 1 and 2). Results summarised in Table (2) indicate an over all recovery of 5.28 and 5.71 as well as 9.05 and 30.63 of both fractions respectively. The neutral protease preparation had a specific activity of 21.92 and 74.14 Umg-1 of both fractions respectively. Properties of purified neutral proteases Thermal stability Proteases were stable at 40 and 30 0C in both protease fractions (40 and 60%) at
Arab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
different time limits as shown in Table (3). The residual activity exhibited a drastic reduction in fraction 40% ammonium sulphate from 85% after 10 minutes to 8.2% after 60 minutes at 50 0C. Protease of fraction 60% ammonium sulphate seemed more tolerant to high temperature than fraction 40%. At 60 0C, only 4% activity was detected after 20 min. in case of protease fraction 40%, while 5% activity was observed after 40 min. in protease fraction 60%.PH Protease activity is generally higher in fraction 60% compared to 40% ammonium sulphate at different pH values (Table 4). The maximum protease activity was detected at pH 7 for protease fractions 40 and 60% reaching 1.096 and 2.076 Uml-1 respectively. Very low activity was obtained at pH values of 4 and 9 for both fractions.
Hala M. Rifaat et al.
O.Dat 280 nm
3.0
3. 0
Activity (U ml-1) O.D at 280 nm
2.5
2. 5
2.0
2. 0
1.5
1. 5
1.0
1. 0
0.5
0. 5
Protease activity (U/ml)
54
0. 0
0.0 0
10
20
30
40
50
60
No. of fractions
No..of fractions Fig. ( 1): Elution profile of neutral protease ( 40% sat urat ion) on Sephadex G -75
Fig.(1): Elution profil of neutral protease (40% saturation) on Sephadex G-75.
3.0
Activity (U/ml) O.D at 280 nm
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
Protease activity (U/ml)
O.Dat 280 nm
3.0
0.0 0
20
40 No. of fractions
60
80
No.of fractions
Fig. ( 2): Elution profile of neutral protease ( 60% sat urat ion) on Sephadex G -75
Fig.(2): Elution profil of neutral protease (60% saturation) on Sephadex G-75.
rab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
Extracellular neutral protease from S. microflavus
55
Table (2): Purification of neutral protease from Streptomyces microflavus. Volume
Total protease activity
Total protein
-1
Specific activity
-1
Purification
Yeild
-1
(ml) 1000
(U ml ) 726
(U ml ) 300
(U mg ) 2.42
(fold) 1.0
(%) 100
Fraction 40%
20
84
6.86
12.24
5.05
11.57
Fraction 60% Sephadex G-75
20
110
4.66
23.6
9.75
15.15
Fraction 40%
35
38.36
1.75
21.92
9.05
5.28
Fraction 60%
20
41.52
0.56
74.14
30.63
5.71
Crude Ammonium sulphate
Table (3): Thermal stability of the purified neutral protease from Streptomyces microflavus. Residual protease activity (%) of fraction 40% Time (minutes) 10 20 30 40 50 60
Time (minutes) 10 20 30 40 50 60
Temperature 0C 40
30
95.00 98.28 93.22 97.24 93.00 97.00 92.02 96.77 92.00 96.50 91.87 96.00 Residual protease activity (%) of fraction 60% Temperature 0C 30 40 96.00 98.79 95.23 98.00 94.78 97.41 93.60 97.00 92.00 96.22 91.62 95.98
50
60
85.00 72.00 51.30 39.05 15.00 8.20
17.03 4.29 0 0 0 0
50 87.00 80.00 75.61 70.62 67.09 63.00
60 40.20 25.30 13.33 5.00 0 0
Table (4): Effect of pH on the purified two fractions of neutral protease from Streptomyces microflavus.
Buffers
pH value
Fraction (40%) Concentration of Protease tyrosine activity (µg ml-1)
(U ml-1)
Fraction (60%) Concentration of tyrosine Protease activity (µg ml-1)
(U ml-1)
Citrate-phosphate
4
4.01
0.088
4.22
0.093
Phosphate
7
49.62
1.096
93.97
2.076
Carbonate-bicarbonate
9
3.59
0.079
8.34
0.180
Arab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
Hala M. Rifaat et al.
56
noticed with Mn2+ ions. Generally speaking, the residual activity increased as the metal ions increased from 0.1 to 0.8 mM. On the other hand, Zn2+ ions variably decreased the residual activity, while Ag+ did inhibit protease production.
Metal ions The highest estimate of residual activity was recorded in presence of Ca2+ ions reaching 298.70 and 319.11% at concentration 0.8 mM in both fractions of ammonium sulphate respectively (Table 5). Considerable increases in the residual activity were also
Table (5): Effect of various inhibitors on the purified two fractions of neutral protease from Streptomyces microflavus. Inhibitors
Residual protease activity (%)
0.08 mM
fraction 40%
fraction 60%
Tween 80
45.0
47.0
Urea
39.0
41.5
EDTA
20.0
22.0
P.CMBA
6.5
8.0
PMSF
2.0
3.0
DISCUSSION The purification of proteases is important from the perspective of developing a better understanding of the functioning of the enzyme (Tsai et al., 1988). Precipitation is the most commonly used method for the isolation and recovery of proteins from crude biological mixtures (Bell et al., 1983). It also performs both purification and concentration steps. Precipitation by ammonium sulphate is used in acidic and neutral pH solutions (Aunstrup, 1980). The obtained data showed that the fractions from 40 to 60% ammonium sulphate saturation correlated with high proteolytic and specific activities compared with the crude protease and other concentrations. Kang et al. (1995) found that proteases from Streptomyces albidoflavus was precipitated by 45% saturation of ammonium sulphate. Moreover, proteases from Streptomyces alboniger
rab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
precipitated by 40% saturation of ammonium sulphate (Lopes et al., 1999). For further purification, gel filtration technique was applied for both fractions 40 and 60% ammonium sulphate. Reports on the purification of proteases by different affinity chromatographic methods showed that Sephadex was the best to separate proteases. Sephadex G-75 showed single peak co-eluting with protease activity of both fractions. Kang et al (1995) and Hatanaka et al., (2005) reported as well the use of Sephadex G7-5 for the purification of proteases from Streptomyces albidoflavus. The properties of partially purified neutral protease were examined. Streptomyces microflavus protease was stable (ca. 96% activity) at 30 and 40 0C after 1 hour incubation for both fractions. Similar results were obtained for Streptomyces spp. by Azeredo et al. (2004). The optimum pH of proteolytic activity was recorded at 7.0, but reduction in activity
Extracellular neutral protease from S. microflavus
was detected at pH values of 4 and 9 for both fractions. Sampth et al. (1997) reported that the optimum pH range of Streptomyces proteases was 6.0 - 12.0. Inhibition of enzymatic activity was noticed only for 0.08 mM P.CMFS and PMSF (6.5, 2.0 and 8.0, 3.0% of residual activity respectively) of both fractions. Similar results were previously obtained by Demina and Lysenko (1995). Tween 80, urea and EDTA at 0.08 mM reduced the residual activity of protease to different extents. Thus, it is concluded that the sensitivity of the enzyme to inhibitors is identical to those of other serine proteases. Microbial proteases are best classified by pH optimum and inhibitor sensitivity rather than by most readily hydrolysed substrate (Ong and Gaucher, 1973). The obtained results indicate the presence of neutral serine protease (s). Protease activity enhanced by Ca2+ and 2+ Mn and completely inhibited by Ag+. Best results were obtained with Ca2+ with an increase in activity around three-fold. These results are in accordance with Tsujibo et al. (1990) and El-Raheem et al. (1995) who stated that proteases from Streptomyces spp. are stimulated by Mn2+ and Ca2+. James et al. (1991) suggested that presumably Ca2+ stabilises the protein through specific or nonspecific binding site, and may also allow for additional bonding within the enzyme molecule, preventing unfolding at higher temperatures. Streptomyces microflavus synthesizes multiple proteases, serine and metal requiring proteases which offer an interesting potential for enzymatic and/or micobiological hydrolysis at industrial level. REFERENCES Aunstrup, K. (1980). Proteinases. In: Rose, A. H., (ed). Economic Microbiology: Arab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
57
Microbial Enzymes and Bioconversions, New York: Academic Press., 5: 50-114. Azeredo, L. A., Freire, D. M., Soares, R. M., Leite, S. G., and Coelho, R. R. (2004). Production and partial characterisation of thermophilic proteases from Streptomyces sp. isolated from Brazilian cerrado soil. Enz. Microbiol. Technol., 34: 354-358. Bell, D. J., Hoare, M. and Dunnill, P. (1983). The formation of protein precipitates and their centrifugal recovery. Adv. Biochem. Eng. Biotechnol., 26: 1-72. Bockle, B.; Galunsky, B. and Muller, R. (1995). Characterisation of a keratinolytic serine proteinase from Streptomyces pactum. Appl. Environ. Microbiol., 61: 3705-3710. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Cohen, B. L. (1990). Transport and utilisation of proteins by fungi, p. 411. In: J. W. (Ed.), Microorganisms and Nitrogen Sources. J. Wiley & Sons, London, UK. Demina, N. S. and Lysenko, S. V. (1995). Exoproteases of Streptomyces lavendulae. Microbiology, 64: 385-387. Dixon, M. (1953). Nomogram for saturation of ammomium sulphate at room temperature. J. Biochem., 54: 457-459. El-Raheem, A., El- Shanshoury, A. R., ElSayed, M. A., Sammour, R. H. and ElShonny, W. A. (1995). Purification and partial characterisation of two extracellular alkaline proteases from Streptomyces corchorusii ST36. Can. J. Microbiol., 41: 99104. Gupta, R., Gupta, K., Saxena, R. K., and Khan, S. (1999). Bleach stable alkaline protease from Bacillus spp. Biotechnol. Lett., 21: 135-138. Hatanaka, T., Yoshiko Uesugi, J. A and Iwabuchi M. (2005). Purification,
58
Hala M. Rifaat et al.
characterisation cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2. Arch. Biochem. Biophys., 434: 289-298. James, P. D. A., Iqbal, M., Edwards, G. and Miller, P. G. G. (1991). Extracellular protease activity in antibiotic producing Streptomyces thermoviolaceus. Curr. Microbiol., 22: 377-382. Kalisz, H. M. (1988). Microbial proteases. In: Advances in Biochemical Engineering Biotechnology (Fisher, A. Ed.), SpringerVerlaqg, Frankfurt, 37: 126-134. Kang, S., Kim, I., Rho, Y. and Lee, K. (1995). Production dynamics of extracellular proteases accompanying morphological differentiation of Streptomyces albidoflavus SMF301. Microbiology, 141: 3095-3103. Kitadokora, K., Tsuzuki, H., Nakamura, E., Sato, T. and Teraoka, H. (1994). Purification, characterisation, primary structure, crystallisation and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae. Eur. J. Biochem., 220: 55-61. Li, F. and Yousten, A. (1975). Metalloprotease from Bacillus thuringiensis. Appl. Microbiol., 30: 354. Lopes, A., Coelho, R., Meirelles, M., Branquinha, M. and Vermelho, A. (1999). Extracellular serine-proteinases isolated from Streptomyces alboniger: Partial characterisation and effect of aprotinin on cellular structure. Mem. Inst. Oswaldo Cruz., 94: 763-770. Mahmoud, S. A. Z., Abdel Hafez, A. M., El Sawy, M. and Fatma, R. A. (1977). Production of fungal proteinase. II. Effect of carbon source, phosphate and pH. Egypt. J. Microbiol., 1: 129-137. Monod, M., Togni, G., Rahalison, L. and Freuk, E. (1991). Isolation and
rab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
characterisation of an extracellular alkaline protease of Aspergillus fumigatus. J. Med. Microbiol., 35: 23-35. Olafson, R. W., Jurasek, L., Carpenter, M. R. and Smillie, M. (1975). Amino acid sequence of Streptomyces griseus tyrpsin cyanogens bromide fragments and complete sequence. Biochemistry, 14: 1168-1177. Ong, P. S. and Gaucher, G. M. (1973). Protease production by thermophilic fungi. Can. J. Microbiol., 19: 129-133. Rifaat, H. M., Hassanein, S. M., El-Said, O. H., Saleh, S. A. and Selim, M. S. M. (2005). Factors affecting the production of extracellular neutral protease by Streptomyces microflavus. Acta. Micrpbiol. Imm.(in press). Sampth, P., Subramanicin, C. and Chandrakasan, G. (1997). Extracellular proteases from Streptomyces sp. Purification and characterisation. Biotechnol. Appl. Biochem., 26: 85-90. Tsai, Y. C., Juang, R. Y., Lin, S. F., Chen, S.W., Yamasaki, M. and Tamura, G. (1988). Production and characterisation of an alkaline elastase produced by alkalophilic Bacillus strain Ya-B. Appl. Environ. Microbiol., 54: 3156-3161. Tsujibo, H., Miyamoto, K., Hasegawa, T. and Inamori, Y. (1990). Purification and characterisation of two types of alkaline serine proteases produced by alkalophite actinomycete. J. Appl. Bacteriol., 69: 520529. Wang, J. Wang, M. and Wang, Y. (1999). Purification and characterization of a novel fibrinolytic enzyme from Streptomyces spp. Chin. J. Biotechnol., 15: 83-89. Yang, S. and Huang, C. I. (1994). Protease production by amylolytic fungi in solid state fermentation. J. Chin. Agric. Chem. Soc., 32: 589-601.
59
Extracellular neutral protease from S. microflavus
א מא
א
א א
ﻫﺎﻟﻪ ﻤﺤﻤﺩ ﺭﻓﻌﺕ *ﺴﻌﺩﻴﺔ ﻤﺤﻤﺩ ﺤﺴﻨﻴﻥ** ﺃﺴﺎﻤﻪ ﺤﺎﻤﺩ ﺍﻟﺴﻴﺩ*** ﺴﻌﺎﺩ ﺃﺤﻤﺩ ﺼﺎﻟﺢ*** ﻭﻤﻨﺎل ﻤﺤﻤﺩ ﺴﻠﻴﻡ*** *ﻗﺴﻡ ﻜﻴﻤﻴﺎﺀ ﺍﻟﻜﺎﺌﻨﺎﺕ ﺍﻟﺩﻗﻴﻘﺔ-ﺍﻟﻤﺭﻜﺯ ﺍﻟﻘﻭﻤﻰ ﻟﻠﺒﺤﻭﺙ **ﻗﺴﻡ ﺍﻟﻤﻴﻜﺭﻭﺒﻴﻭﻟﻭﺠﻰ-ﻜﻠﻴﺔ ﺍﻟﻌﻠﻭﻡ ﺠﺎﻤﻌﺔ ﻋﻴﻥ ﺸﻤﺱ ***ﻗﺴﻡ ﺍﻟﺘﻜﻨﻭﻟﻭﺠﻴﺎ ﺍﻟﺤﻴﻭﻴﺔ ﺍﻟﻤﻴﻜﺭﻭﺒﻴﺔ-ﺍﻟﻤﺭﻜﺯ ﺍﻟﻘﻭﻤﻰ ﻟﻠﺒﺤﻭﺙ
ﺘﻡ ﺍﺴﺘﺨﻼﺹ ﺇﻨﺯﻴﻡ ﺍﻟﺒﺭﻭﺘﻴﺯ ﺍﻟﻤﺘﻌﺎﺩل ﻤﻥ ﻨﻭﻉ ﺇﺴﺘﺭﺒﺘﻭﻤﻴﺴﺱ ﻤﻴﻜﺭﻭﻓﻼﻓﻭﺱ ﺍﻟﻤﻌﺯﻭل ﻤﻥ ﺍﻟﺘﺭﺒﺔ ﺍﻟﻤﺼﺭﻴﺔ ،ﻭﺘﻡ ﺘﻨﻘﻴﺔ ﺍﻹﻨﺯﻴﻡ ﺒﻁﺭﻴﻘﺘﻰ ﺍﻟﺘﺭﺴﻴﺏ ﺒﻭﺍﺴﻁﺔ ﻜﺒﺭﻴﺘﺎﺕ ﺍﻷﻤﻭﻨﻴﻭﻡ ﻭﺍﺴﺘﺨﺩﺍﻡ ﻋﻤﻭﺩ ﺍﻟﻔﺼل ﺍﻟﻠﻭﻨﻰ ﺍﻟﻤﻌﺒﺄ ﺒﻤﺎﺩﺓ ﺴﻴﻔﺎﺩﻜﺱ ﺠﻰ .75ﻭﺒﺩﺭﺍﺴـﺔ ﺒﻌﺽ ﺨﻭﺍﺹ ﺍﻹﻨﺯﻴﻡ ﺘﺒﻴﻥ ﺃﻥ ﻨﺸﺎﻁ ﺍﻹﻨﺯﻴﻡ ﻴﻜﻭﻥ ﻋﻨﺩ ﺍﻷﺱ ﺍﻟﻬﻴﺩﻭﺠﻴﻨﻰ 7ﻭﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ 40ﺩﺭﺠﺔ ﻤﺌﻭﻴﺔ ،ﻜﻤﺎ ﻴﺯﺩﺍﺩ ﻨـﺸﺎﻁ ﺍﻹﻨﺯﻴﻡ ﻓﻰ ﻭﺠﻭﺩ ﺃﻴﻭﻨﺎﺕ ﺍﻟﻜﺎﻟﺴﻴﻭﻡ ﻭﺍﻟﻤﻨﺠﻨﻴﺯ ﻓﻰ ﺤﻴﻥ ﺃﻥ ﺃﻴﻭﻥ ﺍﻟﻔﻀﺔ ﻴﻭﻗﻑ ﻨﺸﺎﻁ ﺍﻹﻨﺯﻴﻡ ،ﻜﻤﺎ ﺘﺄﺜﺭ ﻨﺸﺎﻁ ﺍﻹﻨﺯﻴﻡ ﺒـﺸﺩﺓ ﻓـﻰ ﻭﺠﻭﺩ ﻤﺎﺩﺘﻰ ﺒﺎﺭﺍ ﻜﻠﻭﺭﻭﻤﻴﺭﻜﺭﻴﺒﻨﺯﻭﻙ ﻭ ﻓﻴﻨﻴل ﻤﻴﺜﻴل ﺴﻠﻔﻨﻴل ﻓﻠﻭﺭﻴﺩ ﺒﺘﺭﻜﻴﺯ 0.8ﻤﻠﻠﻰ ﻤﻭل.
Arab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
60
rab J. Biotech., Vol. 9, No. (1) Jan. (2006): 51-60.
Hala M. Rifaat et al.
