Purification of phytase from aspergillus fumigatus

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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY PURIFICATION OF PHYTASE FROM Aspergillus fumigatus SUPERVISOR STUDENT Dr. DUONG THI HUONG GIANG HUYNH THAO TIEN Student code: 3083766 Session: 34 (2008-2013) Can Tho, 2013 APPROVAL SUPERVISOR STUDENT Dr. DUONG THI HUONG GIANG HUYNH THAO TIEN Can Tho, May 10, 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract Aspergillus fumigatus, a potential phytase producing isolate, which has recently been found in the Laboratory of Enzymology, Biotechnology R&D Institure, Can Tho University. Preliminary studies showed that the phytase enzymes from this fungi strain adopted high thermostability, which is favorable for animal feed production. Since there was little information about the phytase form this species this thesis aimed at purification of phytase from the A. fumigatus isolate. The results showed that ammonium sulfate fractionation gave two phytases of different molecular mass precipitated at 20%  50% and 60%  90% AS saturation. Furthermore, Ammonium sulfate precipitation in combination with cation exchange chromatography on SP-Streamline allowed purifying a high molecular mass phytase from the 60  90% AS precipitate to homogenous protein, which adopted a molecular mass of 87.7 kDa. This phytase of Aspergillus fumigatus was purified to about 2.68-fold and exposed a high specific activity of 4.398 U/mg protein. Key words: Aspergillus fumigatus, ammonium sulfate fractionation, cation exchange chromatography, phytase, SP-Streamline. i CONTENTS Abstract i CONTENTS ii 1. INTRODUCTION 1 2. MATERIALS AND METHODS 3 2.1. Materials 3 2.2. Methods 3 2.2.1. Preparation of fungi strain and culture medium 3 2.2.2. Extraction of the crude enzyme phytase 4 2.2.3. Phytase purification 4 Ammonium sulfate fractionation 4 Phytase purification by hydrophobic interaction chromatography on Phenyl Sepharose 5 Phytase purification by cation-exchange chromatography on SP-Streamline column. 5 3. RESULTS AND DISCUSSION 7 3.1. Extraction of crude enzyme 7 3.2 Phytase purification 7 3.2.1. Ammonium sulfate fractionation 7 3.2.2 Purification of phytase by hydrophobic interaction chromatography on Phenyl Sepharose. 9 3.2.2. Purification of phytase by cation exchange chromatography on SP-Streamline. 10 4. CONCLUSIONS AND SUGGESTIONS 14 4.1. Conclusions 14 4.2. Suggestions 14 REFERENCES 15 ii 1. INTRODUCTION Phosphorus (P) plays major structural and metabolic roles in living cells. It is a structural component of important molecules, such as nucleic acid (DNA, RNA), phospholipid membranes, high-energetic molecules (ATP, NADPH,...) (Jahnke, 2000). Phytases are myo-inositol-1,2,3,4,5,6hexakisphosphate phosphohydrolases that catalyze the degradation of phytic acid to myo-inositol-1,2,3,4,5,6-pentakisphosphate and orthophosphate, which is easily to be absorbed in animal digestive tract. However, monogastric animals, such as pigs and poultry, are not able to utilize phytate, so phytate in their digestive tracts cannot be absorbed. The unutilized phytate is excreted to the environment lead to pollution in areas of intensive husbandry. Besides, phytic acid can act as an anti-nutrient factor by chelating with minerals, such as zinc, iron, calcium and magnesium (Cheryan, 1980). Phytate-degrading enzymes have been studied intensively in recent years because of the interest in such enzymes for reducing the levels of inorganic as well as organic phosphate pollution in livestock areas, and also improving nutritional values of organic phosphate (phytate) (Lei and Porres, 2003). Phytases are found naturally in plants and microorganisms, particularly fungi. Most of the studies focused on phytases produced from Aspergillus sp., they are high phytase producers (Rao et al., 2009). Among them A. fumigatus is a potential fungi strain producing phytase of valuable properties such as heat-labile, broad pH activity, broad substrate specificity v.v... Up until present, very limited information about the purification of phytase from A. fumigatus is available. In order to obtain pure enzyme phytase from A. fumigatus to apply in feed/food industries, the research on “Purification of phytase from Aspergillus fumigatus” has been carried out. 1 Aim of the thesis: Purification of the phytase form A. fumigatus by combination of the methods including ammonium sulfate precipitation, Ionexchange chromatography and hydrophobic interaction chromatography. 2 2. MATERIALS AND METHODS 2.1. Materials - Aspergillus fumigatus was provided by Enzyme Technology Laboratory. - Phytate substrates such as wheat powder, rice husk were purchased in Xuan Khanh market, Ninh Kieu District, Can Tho City. - Equipment: pH Meter Lab 850 (Schott, Germany), Eppendorf – Germany, spectrometer (Hitachi – Japan), centrifuge (Rotor – Germany) and other lab facilities. - Chemicals: Sodium phytate (C6H6Na12O24P6H2O), L(+)- Ascorbic acid (C6H8O2) (Sigma), Sodium acetate (Merck), Trichloroacetic acid (TCA) (Merck), Acetone (China),… - Medium:  PGA – Potato Glucose Agar: 2% D-glucose, 1.8% (w/v) agar, 20% (w/v) potato.  Semi-solid substrate medium (Arpana et al., 2012): 30g phytate substrate, 15g rice husk, 25 mL mineral solution (Spieck and Lipski, 2011) pH 5.0.  Mineral solution: MgSO4.7H2O (0.1g/L), KCl (0.5g/L), FeSO4 (0.01g/L), MnSO4 (0.01g/L), NaCl (0.1g/L), CaCl2 (5g/L), KH2PO4 0.1%, glucose : sucrose (1:1) 1% 2.2. Methods 2.2.1. Preparation of fungi strain and culture medium Aspergillus fumigatus was maintained in PGA medium and incubated in 45°C for 2 days. Determine the centration of A. fumigatus spore by hemocytometer and adjust to the concentration of 108 spores/mL. For phytase production, semi-solid substrate medium was used. 1 mL A. fumigatus spore (108 spores/mL) was inoculated to semi-solid substrate 3 medium and incubated in 35°C. After 2 days incubation, fungi fresh biomass was collected and the crude enzyme was extracted. 2.2.2. Extraction of the crude enzyme phytase Enzyme extraction was performed accordingly to the method of Nguyễn Văn Tính (2012). Semi-solid substrate medium Sterilized in 20 minutes, 121°C Inoculation of 1mL spore (108/mL) of A. fumigatus Incubated for 2 days, 35°C Homogenization of fungi biomass in 50mL sodium acetate buffer 0.02M, pH 5.5 Centrifugation for 20 minutes, 13000 rpm, 4°C Take off the pellet Crude phytase extract Figure 1. Phytase extraction procedure Protein content of crude phytase extract were measured by Bradford method (1976) and the enzyme activity was determined by the method of Heinonen and Lahti, (1981). 2.2.3. Phytase purification Ammonium sulfate fractionation Ammonium sulfate (AS) was added into the crude enzyme extract with the concentration from 0, 20, 30, 40, 50, 60, 70, 80 and 90% saturation (Appendix 1). The solution was stirred and kept at 4°C for 1 – 2 hours, then centrifuged at 13000 rpm for 20 minutes. The pellet of each ammonium 4 sulfate fraction was collected and determined the protein content by Bradford method, enzyme activity was determined by the method of Heinonen and Lahti, (1981). Phytase purification by hydrophobic interaction chromatography on Phenyl Sepharose The crude enzyme extract was precipitated with ammonium sulfate saturation chosen from, and centrifuged at 7000 rpm for 20 minutes. Dissolving the pellet in the buffer Tris-HCl 0.02M pH 7.6 + AS 30% saturation, and dialyzed against the same buffer in the fridge for 24 hours. After dialysis, the enzyme solution was loaded onto the Phenyl Sepharose column. Washing the column with buffer Tris-HCl 0,02M pH 7.6 + AS 30% to remove unbound proteins. Bound proteins were eluted by decreasing AS concentration gradient from 30% – 0%. Protein content and specific activity of precipitated fractions were determined by Bradford (1976) and Heinonen and Lahti, (1981) methods respectively. SDS-PAGE was used to check for phytase purity. Phytase purification by cation-exchange chromatography on SP-Streamline column. The crude enzyme extract was precipitated with the ammonium sulfate saturation concentration chosen from, centrifuged at 7000 rpm for 20 minutes at 4°C. The obtained pellet was dissolved in Tris-HCl 0,02M pH 7.6 and dialyzed against the same buffer in the fridge for 24 hours. After dialysis, the enzyme solution was passed through SPStreamline column with the rate of 0.8 mL/minute. The column was washed with buffer Tris-HCl 0.02M, pH 7.6 to remove the unbound proteins. The bound proteins were released by increasing NaCl concentration gradient from 0 – 0.5M with the rate of 1 mL/minute. 5 Eluted protein fractions were determined protein content by Bradford (1976) and phytase activity by Heinonen and Lahti, (1981). The enzyme purity was checked by SDS-PAGE. 6 3. RESULTS AND DISCUSSION 3.1. Extraction of crude enzyme The crude enzyme extracts 620 mL were collected from 900 g fresh fungi biomass, the total protein content was 235.743 mg, and the enzyme specific activity was 1.643 U/mg protein. Similar result was reported by Đỗ Thị Thu Trang (2011) studying phytase from A. niger PE1, the specific activity of this fungi was 1.65 U/mg protein. This result was lower than phytase from A. niger 11T53A9 (2.6 U/mg protein) (Greiner et al., 2009). Wyss et al., (1999) studied several A. fumigatus strains and concluded that A. fumigatus phytase specific activity was lower than A. niger strains. 3.2 Phytase purification 3.2.1. Ammonium sulfate fractionation The phytases from A. fumigatus were preliminary purified from the crude enzyme extract with ammonium sulfate concentration varied from 20%  90% saturation. Interestingly, there were two phytases were obtained, one phytase was precipitated at AS concentration from 20  50% and the other phytase precipitated at AS 60  90% saturation (2.337 U/mg protein) (Figure 2). SDS-PAGE of protein fractions precipitated by AS of different saturation levels showed that these two protein fractions were different mainly by the protein band of about 87.7 kDa (Figure 3, lane 7,8,9,10). In the 20  50% AS precipitate the other, lower molecular mass phytase of about 66.2 kDa and others. (Figure 3, lane 4,5,6,7). The existence of different phytases was reported in the work of Vats and Banerjee (2004), and it could be due to the culture conditions. 7 a b b b c c c c Figure 2. Preliminary purification of phytases from A. fumigatus by ammonium sulfate fractionation phytase 1 2 3 4 5 6 7 8 9 10 Figure 3. SDS-PAGE of ammonium sulfate precipitated fractions. 1. Protein standard 2. Crude enzyme extract 3. AS 20%. 4. AS 30% 5. AS 40% 6. AS 50%. 7. AS 60% 8. AS 70% 9. AS 80% 10. AS 90% 8 3.2.2 Purification of phytase by hydrophobic interaction chromatography on Phenyl Sepharose. The 60  90% AS saturation protein fraction was loaded onto Phenyl Sepharose column. It was separated into four peaks– unbound, FI, FII and FIII (Figure 4). The three protein fractions unbound, FII and FIII had no enzyme activity. The only FI fraction showed low enzyme activity, about 0.380 U/mg protein. Unbound FI FII FIII Figure 4. Chromatogram of 60  90% AS phytase fraction on Phenyl Selpharose column SDS-PAGE showed very high impurity of the phytase fraction (Figure 7, lane 5) with many protein bands. It appeared that hydrophobic interaction chromatography was not an appropriate method for phytase purification from the phytase fraction precipitated at AS 60  90% saturation. 9 3.2.2. Purification of phytase by cation exchange chromatography on SP-Streamline. Unbound FI FII Figure 5. Chromatogram of phytase fraction precipitated with 60-80% ammonium sufate saturation on cation exchange column SP-Streamline The phytase fraction precipitated by AS 60  90% saturation was applied on cation exchange SP-Streamline column. The chromatogram revealed that there were three protein fractions (unbound, FI and FII) (Figure 4). Surprisingly, the unbound fraction was the phytase that exposed high specific phytase activity about 4.398 U/mg protein. While the bound proteins (fraction I&II) did not have enzyme activity. It seemed that this phytase adopted the pI > 7.6, under the chromatography pH condition (pH 7.6), it was generally charged negative, due to this, it could not bind to the 10 SP-streamline column and eluted as the unbound fraction. SDS-PAGE analysis showed that the purified phytase was homogenous with molecular mass of about 87.7 kDa (Figure 6, lane 6). Similarly, Wang et al., (2007) studied on phytases from A. fumigatus WY-2 also showed that the molecular mass of the phytase is about 88 kDa. Other research on phytase from A. fumigatus isolate found another phytase of 60 kDa (Pasamontes et al., 1997). Wyss et al., (1999) studied on phytases from six fungi strains revealed that molecular mass of phytases from the two different A. fumigatus batches were 72.3 kDa and 60.7 kDa. Figure 6 also revealed that cation exchange chromatography by SPStreamline was an appropriate method for purification of a high molecular phytase of the fraction 60  90% AS precipitation from A. fumigatus. 11 Phytase 87.7 kDa 1 2 3 4 5 6 7 Figure 6. SDS-PAGE analysis of phyase fractions from Phenyl sepharose and SP-Streamline column. 1. Protein standard. 2. Crude phytase extract. 3. 60-80% AS preciptated phytase. 4. Unbound fraction from Phenyl Sepharose column. 5. Bound fraction from phenyl sepharose column. 6. Unbound fraction from SP-streamline column. 7. Bound fraction from SP-Streamline column. Based on the purification scheme of the high molecular phytase (87,7kDa) from A. fumigatus (Table 1), it can be concluded that this phytase can be successfully purified by combination of the two methods ammonium sulfate fractionation and cation exchange chromatography. The phytase was purified 2.68 fold in comparison with the crude extract. 12 Table 1. Purification scheme of a high molecular mass phytase from A. fumigatus by hydrophobic interaction chromatography Step Crude enzyme extract 60  90% AS precipitation Total Total Specific protein activity activity (mg) (U) (U/mg protein) 285.173 143.211 1.643 1 61.840 15.754 2.338 1.42 15.381 12.400 0.806 0.5 Purification (folds) Hydrophobic interaction chromatography Table 2. Purification scheme of a high molecular mass phytase from A. fumigatus by ion exchange chromatography Step Crude enzyme extract 60  90% AS precipitation Ion exchange chromatography Total Total Specific protein activity activity (mg) (U) (U/mg protein) 235.743 387.321 1.643 1 31.515 145.147 4.624 2.81 3.810 16.758 4.4 2.68 13 Purification (folds) 4. CONCLUSIONS AND SUGGESTIONS 4.1. Conclusions - Ammonium sulfate fractionation allowed separating the two phytases from the crude enzyme extract of fresh A. fumigatus biomass. - A high molecular mass phytase (87.7 kDa) was completely purified by AS fractionation following cation exchange chromatography. The purification factor of the enzyme phytase was about 2.68-fold with high specific activity (4.398 U/mg protein). 4.2. Suggestions - Purifying the low molecular mass phytase in 20  50% AS precipitate - Optimizing the purification procedure to get higher phytase yield. - Characterizing phytases from A. fumigatus such as optimum pH and temperature, and the effect of metal ions on the enzyme activity. - Application of phytase in animal feed. 14 REFERENCES Vietnamese Đỗ Thị Thu Trang, 2011. Tinh sạch và khảo sát một số đặc điểm của enzyme phytase từ nấm Aspergillus niger. Luận văn Thạc sĩ. Đại học Cần Thơ. 27-36. Dương Thị Hương Giang. 2010. Bài giảng hóa protein. Viện nghiên cứu và phát triển công nghệ sinh học, Đại học Cần Thơ. English Arpana, M., S. Gulab, V.G., A.Y., N.K and N.K. Aggarwal. 2012. Production of phytase by acido-thermophilic strain of Klebsiella sp. DB-3FJ711774.1 using orange peel flour under submerged fermentation. Inovative Romanian Food Biotechnology 10:18-27. Cheryan, M. 1980. Phytic acid interactions in food systems. Crit Rev Food Sci Nutr 13(4):297-335. Greiner, Ralf, Lucineia Gomes da Silva and Sonia Couri. 2009. Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9. Brazilian Jf Microbiol 40:795-807. Heinonen, J. K. and R. J. Lahti. 1981. A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 113(2):313-317. Jahnke, R.A. (2000). The phosphorus cycle, Earth System Science, pp.360376. Lei, X. G. and J. M. Porres. 2003. Phytase enzymology, applications, and biotechnology. Biotechnol Lett 25(21):1787-1794. Pasamontes, L., M. Haiker, M. Wyss, M. Tessier and A. P. van Loon. 1997. Gene cloning, purification, and characterization of a heat-stable 15 phytase from the fungus Aspergillus fumigatus. Appl Environ Microbiol 63(5):1696-1700. Rao, D. E., K. V. Rao, T. P. Reddy and V. D. Reddy. 2009. Molecular characterization, physicochemical properties, known and potential applications of phytases: An overview. Crit Rev Biotechnol 29(2):182-198. Spieck, E. and A. Lipski. 2011. Cultivation, growth physiology, and chemotaxonomy of nitrite-oxidizing bacteria. Methods Enzymol 486:109-130. Vats, P., U. C. Banerjee. (2004). Production studies and catalytic properties of phytases (myo-inositolhexakisphosphate phosphohydrolases): an overview. Enzyme Microbial Technol 35: 3–14 Wang, Y., X. Gao, Q. Su, W. Wu and L. An. 2007. Cloning, expression, and enzyme characterization of an acid heat-stable phytase from Aspergillus fumigatus WY-2. Curr Microbiol 55(1):65-70. Wyss, M., L. Pasamontes, A. Friedlein, R. Remy, M. Tessier, A. Kronenberger, A. Middendorf, M. Lehmann, L. Schnoebelen, U. Rothlisberger, E. Kusznir, G. Wahl, F. Muller, H. W. Lahm, K. Vogel and A. P. van Loon. 1999. Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance. Appl Environ Microbiol 65(2):359-366. 16
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