Advertisement

Journal of Industrial Microbiology & Biotechnology

, Volume 38, Issue 9, pp 1407–1417 | Cite as

High level phytase production by Aspergillus niger NCIM 563 in solid state culture: response surface optimization, up-scaling, and its partial characterization

  • K. Bhavsar
  • V. Ravi Kumar
  • J. M. KhireEmail author
Original Paper

Abstract

Phytase production by Aspergillus niger NCIM 563 was optimized by using wheat bran in solid state fermentation (SSF). An integrated statistical optimization approach involving the combination of Placket–Burman design (PBD) and Box–Behnken design (BBD) was employed. PBD was used to evaluate the effect of 11 variables related to phytase production, and five statistically significant variables, namely, glucose, dextrin, NaNO3, distilled water, and MgSO4·7H2O, were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a BBD. Phytase production improved from 50 IU/g dry moldy bran (DMB) to 154 IU/g DMB indicating 3.08-fold increase after optimization. A simultaneous reduction in fermentation time from 7 to 4 days shows a high productivity of 38,500 IU/kg/day. Scaling up the process in trays gave reproducible phytase production overcoming industrial constraints of practicability and economics. The culture extract also had 133.2, 41.58, and 310.34 IU/g DMB of xylanase, cellulase, and amylase activities, respectively. The partially purified phytase was optimally active at 55°C and pH 6.0. The enzyme retained ca. 75% activity over a wide pH range 2.0–9.5. It also released more inorganic phosphorus from soybean meal in a broad pH range from 2.5 to 6.5 under emulated gastric conditions. Molecular weight of phytase on Sephacryl S-200 was approximately 87 kDa. The K m and V max observed were 0.156 mM and 220 μm/min/mg. The SSF phytase from A. niger NCIM 563 offers an economical production capability and its wide pH stability shows its suitability for use in poultry feed.

Keywords

Phytase Aspergillus niger Solid state fermentation Statistical methods Response surface optimization 

Notes

Acknowledgments

One of the authors, Ms Kavita Bhavsar, thanks Council of Scientific and Industrial Research, Government of India for the financial assistance. We also gratefully acknowledge support and facilities provided by the Center of Excellence in Scientific Computing, National Chemical Laboratory, India.

References

  1. 1.
    Andrews P (1964) Estimation of molecular weight of proteins by Sephadex gel filtration. Biochem J 92:222–223Google Scholar
  2. 2.
    Bogar B, Szakacs G, Tengerdy RP, Linden JC, Pandey A (2003) Optimization of phytase production by solid substrate fermentation. J Ind Microbiol Biotech 30:183–189Google Scholar
  3. 3.
    Bogar B, Szakacs G, Pandey A, Abdulhameed S, Linden J, Tengerdy R (2003) Production of phytase by Mucor racemosus in solid state fermentation. Biotechnol Prog 19:312–319PubMedCrossRefGoogle Scholar
  4. 4.
    Box GEP, Hunter JS (1957) Multifactor experimental design for exploring response surfaces. Ann Math Stat 28:95–241CrossRefGoogle Scholar
  5. 5.
    Chadha BS, Gulati H, Mandeep M, Saini SH, Singh N (2004) Phytase production by the thermophilic fungus Rhizomucor pusillus. World J Microbiol Biotechnol 20:105–109CrossRefGoogle Scholar
  6. 6.
    Davis BJ (1964) Disc electrophoresis II. Method and application to human serum proteins. Ann N Y Acad Sci 121:404–427PubMedCrossRefGoogle Scholar
  7. 7.
    Deutscher MP (1990) Guide to protein purification, methods enzymology, vol 182. Academic, Toronto, p 430Google Scholar
  8. 8.
    Durand D, Broise D, Blachere H (1988) Laboratory scale bioreactor for solid state process. J Biotechnol 8:59–66CrossRefGoogle Scholar
  9. 9.
    Garrett JB, Kretz KA, O’Donoghue E, Kerovuo J, Kim W, Barton NR, Hazlewood GP, Short JM, Robertson DE, Gray KA (2004) Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement. Appl Environ Microbiol 70:3041–3046PubMedCrossRefGoogle Scholar
  10. 10.
    Gilati HK, Chadha BS, Saini HS (2007) Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by Mucor indicus MTCC 6333: purification and characterization of phytase. Folia Microbiol 52:491–497CrossRefGoogle Scholar
  11. 11.
    Gokhale DV, Puntambekar US, Deobagkar DN, Peberdy JF (1988) Production of cellulolytic enzymes by mutants of Aspergillus niger NCIM 1207. Enzy Microbiol Technol 10:442–445CrossRefGoogle Scholar
  12. 12.
    Gunashree BS, Venkateswaran G (2008) Effect of different cultural conditions for phytase production by Aspergillus niger CFR 335 in submerged and solid-state fermentations. J Ind Microbiol Biotechnol 35:1587–1596PubMedCrossRefGoogle Scholar
  13. 13.
    Huoqing H, Huiying L, Wang Y, Fu D, Shao N, Wang G, Yang P, Yao B (2008) A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. Appl Microbiol Biotechnol 80:417–426CrossRefGoogle Scholar
  14. 14.
    Konietzny U, Greiner R (2002) Molecular and catalytic properties of phytate-degrading enzymes (phytases). Int J Food Sci Technol 37:791–812CrossRefGoogle Scholar
  15. 15.
    Krishna C, Nokes SE (2001) Predicting vegetative inoculum performance to maximize phytase production in solid-state fermentation using response surface methodology. J Ind Microbiol Biotechnol 26:161–170PubMedCrossRefGoogle Scholar
  16. 16.
    Lineweaver H, Burk D (1934) Determination of enzyme dissociation constants. J Am Chem Soc 56:658–666CrossRefGoogle Scholar
  17. 17.
    Lei X, Stahl CH (2001) Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 57:474–481PubMedCrossRefGoogle Scholar
  18. 18.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RL (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  19. 19.
    Mandviwala TN, Khire JM (2000) Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid-state fermentation. J Ind Micrbiol Biotechnol 24:237–243CrossRefGoogle Scholar
  20. 20.
    Miller GL, Lorenz G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  21. 21.
    McCleary BV, Sheehan H (1987) Measurement of cereal alpha amylase: a new assay procedure. J Cereal Sci 6:237–251CrossRefGoogle Scholar
  22. 22.
    Ole K, Torben VB, Claus CF (2002) Industrial enzyme applications. Curr Opinion Biotechnol 13:345–351CrossRefGoogle Scholar
  23. 23.
    Omogbenigun FO, Nyachoti CM, Slominski BA (2004) Dietary supplementation with multienzyme preparations improves nutrient utilization and growth performance in weaned pigs. J Anim Sci 82:1053–1061PubMedGoogle Scholar
  24. 24.
    Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation. Proc Biochem 35:397–402CrossRefGoogle Scholar
  25. 25.
    Pandey A, Szakacs G, Soccol CR, Rodriguez-Leon JA, Soccol VT (2001) Production, purification and properties of microbial phytases. Bioresour Technol 77:203–214PubMedCrossRefGoogle Scholar
  26. 26.
    Plackett RL, Burman JP (1946) The design of optimum multifactor experiments. Biometrika 33:305–325CrossRefGoogle Scholar
  27. 27.
    Prabhakar A, Krishnaiah K, Janaun J, Bono A (2005) An overview of engineering aspects of solid state fermentation. Malaysian J Microbiol 1:10–16Google Scholar
  28. 28.
    Raboy V (2001) Seeds for a better future: “low phytate” grains help to overcome malnutrition and pollution. Trend Plant Sci 6:458–462CrossRefGoogle Scholar
  29. 29.
    Reissig JL, Stromiger JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217:959–966PubMedGoogle Scholar
  30. 30.
    Singh B, Satyanarayan T (2006) A marked enhancement in phytase production by a thermophilic mould Sporotricum thermophile using statistical designs in a cost effective cane molasses medium. J Appl Microbiol 101:344–352PubMedCrossRefGoogle Scholar
  31. 31.
    Soni SK, Khire JM (2007) Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under SF conditions. World J Microbiol Biotechnol 23:1585–1593CrossRefGoogle Scholar
  32. 32.
    Terebiznik MR, Pilosof AMR (1999) Biomass estimation in solid state fermentation by modeling dry matter weight loss. Biotech Tech 13:215–221CrossRefGoogle Scholar
  33. 33.
    Vats P, Banerjee UC (2004) Production studies and catalytic properties of phytases (myoinositolhexakisphosphate phosphohydrolases): an overview. Enzy. Microbiol Technol 35:3–14CrossRefGoogle Scholar
  34. 34.
    Vohra A, Satyanarayana T (2003) Phytases: microbial sources, production, purification and potential biotechnological applications. Crit Rev in Biotech 23:29–60CrossRefGoogle Scholar
  35. 35.
    Wodzinski RJ, Ullah AHJ (1996) Phytase. Adv Appl Microbiol 42:263–302PubMedCrossRefGoogle Scholar
  36. 36.
    Wu YB, Ravindran V, Thomas DG, Birtles MJ, Hendricks WH (2004) Influence of phytase and xylanase, individually or in combination, on performance, apparent metabolizable energy, digestive tract measurements and gut morphology in broilers fed wheat-based diets containing adequate level of phosphorus. Brit Poultry Sci 45:76–84CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2010

Authors and Affiliations

  1. 1.NCIM Resource Center, National Chemical LaboratoryPuneIndia
  2. 2.Chemical Engineering and Process Development DivisionNational Chemical LaboratoryPuneIndia

Personalised recommendations