Abstract
Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30 °C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH2PO4 (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60 °C while Phy II was 4.0 and 60 °C, respectively. Phy I was stable in the pH range 1.5–3.5 while Phy II was stable in the wider pH range, 2.0–7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg2+, Mn2+, Ca2+ and Fe3+ ions and inhibited by Zn2+ and Cd2+ ions while Phy II activity was moderately stimulated by Fe3+ ions and was inhibited by Hg2+, Mn2+ and Zn2+ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 μmols/min/mg protein, respectively.
Similar content being viewed by others
References
Andrews P (1964) Estimation of molecular weight of proteins by Sephadex gel filtration. Biochem J 92:222–223
Bitar K, Reinhold JG (1972) Phytase and alkaline phosphate activities in intestinal mucosa of rat, chicken, calf and man. Biochim Biophys Acta 268:442–452
Common FH (1989) Biological availability of phosphorus for pigs. Nature 143:370–380
Davis BJ (1964) Disc electrophoresis II. Method and application to human serum proteins. Ann N Y Acad. Sci 121:404–427
Deutscher MP (1990) Guide to protein purification, Methods enzymol, vol 182. Academic press, Toronto, p 430
Dvorakova J (1998) Phytase: source, preparation and exploitation. Folia Microbiol 43:323–338
Erdman JW, Poneros-Schneier A (1989) Phytic acid interactions with divalent cations in food and in the gastrointestinal tract. Adv Exp Med Biol 249:167–171
Fox MRS, Tao SH (1989) Antinutritive effects of phytate and other phosphorylated Derivatives. Nutr Toxicol 3:59–96
Fujita J, Yamane Y, Fukuda H, Kizaki Y, Wakabayashi S, Shigeta S, Suzuki O, Ono K (2003) Production and properties of phytase and acid phosphatase from a sake koji mold, Aspergillus oryzae. J Biosci Bioeng 95:348–353
Gargova S, Sariyska M (2003) Effect of culture conditions on the biosynthesis of Aspergillus niger phytase and acid phosphatase. Enzyme Microb Technol 32:231–235
Heinohen JK, Lathi RJ (1981) A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay to inorganic pyrophosphatase. Anal Biochem 113:313–317
Kim YO, Kim HK, Bae KS, Yu JH, Oh TK (1998) Purification and properties of a thermostable phytase from Bacillus spp DS11. Enzyme Microb Technol 22:2–7
Lineweaver H, Burk D (1934) Determination of enzyme dissociation constants. J Amer Chem Soc 56:658–666
Lowry OH, Rosebrough NJ, Farr AL, Randall RL (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Mahadik ND, Bastawde KB, Puntambekar US, Khire JM, Gokhale DV (2004) Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation. Process Biochem 39:2031–2034
Mandviwala TN, Khire JM (2000) Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation. J Ind Microbiol Biotechnol 24:237–243
Mitchell DB, Vogel K, Weimann BJ, Pasamontes L, van Loon A (1997) The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from fungi Aspergillus terreus and Myceliophthora thermophila. Microbiology 143:245–252
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:427–431
Mullaney EJ, Daly C, Ullah AB (2000) Advances in phytase research. Adv Appl Microbiol 47:157–199
Oh BC, Choi S, Park S, Kim YO, Oh TK (2004) Biochemical properties and substrate specificities of alkaline and histidine acid phytases. Appl Microbiol Technol 63:362–372
Radcliffe JS, Zhang Z, Kornegay ET (1998) The effect of microbial phytase, citric acid and their interaction in a corn soybean meal based diet for weaning pigs. J Anim Sci 76:1880–1886
Reddy NR, Sathe SK, Salunkhe DK (1982) Phytases in legumes and cereals. Adv Food Res 82:1–92
Shieh TR, Ware JH (1968) Survey of microorganisms for the production of extracellular phytase. Appl Microbiol 16:1348–1351
Ullah AHJ, Gibson DM (1987) Extracellular Phytase (EC 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization. Prep Biochem 17:63–91
Ullah AHJ (1988) Production, rapid purification and catalytical characterization of extracellular Phytase from Aspergillus ficuum. Prep Biochem 18:443–458
Vats P, Banerjee UC (2002) Studies on the production of phytase by a newly isolated strain of Aspergillus niger van Teigham obtained from rotten wood-logs. Process Biochem 38:211–217
Vats P, Banerjee UC (2004) Production studies and catalytic properties of phytases (myoinositolhexakisphosphate phosphohydrolases): an overview. Enzyme Microb Technol 35:3–14
Vats P, Sahoo DK, Banerjee UC (2004) Production of phytase (myo-Inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter. Biotechnol Prog 20:737–743
Vohra A, Satyanarayana T (2003) Phytases: microbial sources, production, purification and potential biotechnological applications. Crit Rev Biotechnol 23:29–60
Volfova O, Dvorakova J, Hanzlikova A, Jandera A (1994) Phytase from Aspergillus niger. Folia Microbiol 39:479–482
Walsh GA, Power RF, Headon DR (1994) Enzymes in the animal feed industry. Trends Food Sci Technol 5:81–87
Wodzinski R, Ullah AHL (1996) Phytase. Adv Appl Microbiol 42:263–283
Zyta K (1992) Mold phytases and their application in the food industry. World J Microbiol Biotechnol 8:467–472
Acknowledgements
The authors gratefully acknowledge financial support provided by the Department of Biotechnology, New Delhi, India. One of the authors, Mr. Sarvesh Kumar Soni thanks Council of Scientific and Industrial Research, Government of India for the financial assistance.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Soni, S.K., Khire, J.M. Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions. World J Microbiol Biotechnol 23, 1585–1593 (2007). https://doi.org/10.1007/s11274-007-9404-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-007-9404-9