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

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Journal of Industrial Microbiology & Biotechnology

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.

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References

  1. Andrews P (1964) Estimation of molecular weight of proteins by Sephadex gel filtration. Biochem J 92:222–223

    Google Scholar 

  2. Bogar B, Szakacs G, Tengerdy RP, Linden JC, Pandey A (2003) Optimization of phytase production by solid substrate fermentation. J Ind Microbiol Biotech 30:183–189

    CAS  Google Scholar 

  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 19:312–319

    Article  PubMed  CAS  Google Scholar 

  4. Box GEP, Hunter JS (1957) Multifactor experimental design for exploring response surfaces. Ann Math Stat 28:95–241

    Article  Google Scholar 

  5. Chadha BS, Gulati H, Mandeep M, Saini SH, Singh N (2004) Phytase production by the thermophilic fungus Rhizomucor pusillus. World J Microbiol Biotechnol 20:105–109

    Article  CAS  Google Scholar 

  6. Davis BJ (1964) Disc electrophoresis II. Method and application to human serum proteins. Ann N Y Acad Sci 121:404–427

    Article  PubMed  CAS  Google Scholar 

  7. Deutscher MP (1990) Guide to protein purification, methods enzymology, vol 182. Academic, Toronto, p 430

    Google Scholar 

  8. Durand D, Broise D, Blachere H (1988) Laboratory scale bioreactor for solid state process. J Biotechnol 8:59–66

    Article  CAS  Google Scholar 

  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 70:3041–3046

    Article  PubMed  CAS  Google Scholar 

  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 52:491–497

    Article  Google Scholar 

  11. Gokhale DV, Puntambekar US, Deobagkar DN, Peberdy JF (1988) Production of cellulolytic enzymes by mutants of Aspergillus niger NCIM 1207. Enzy Microbiol Technol 10:442–445

    Article  CAS  Google Scholar 

  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 35:1587–1596

    Article  PubMed  CAS  Google Scholar 

  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 80:417–426

    Article  Google Scholar 

  14. Konietzny U, Greiner R (2002) Molecular and catalytic properties of phytate-degrading enzymes (phytases). Int J Food Sci Technol 37:791–812

    Article  CAS  Google Scholar 

  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 26:161–170

    Article  PubMed  CAS  Google Scholar 

  16. Lineweaver H, Burk D (1934) Determination of enzyme dissociation constants. J Am Chem Soc 56:658–666

    Article  CAS  Google Scholar 

  17. Lei X, Stahl CH (2001) Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 57:474–481

    Article  PubMed  CAS  Google Scholar 

  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RL (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  19. Mandviwala TN, Khire JM (2000) Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid-state fermentation. J Ind Micrbiol Biotechnol 24:237–243

    Article  CAS  Google Scholar 

  20. Miller GL, Lorenz G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  21. McCleary BV, Sheehan H (1987) Measurement of cereal alpha amylase: a new assay procedure. J Cereal Sci 6:237–251

    Article  CAS  Google Scholar 

  22. Ole K, Torben VB, Claus CF (2002) Industrial enzyme applications. Curr Opinion Biotechnol 13:345–351

    Article  Google Scholar 

  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 82:1053–1061

    PubMed  CAS  Google Scholar 

  24. Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation. Proc Biochem 35:397–402

    Article  Google Scholar 

  25. Pandey A, Szakacs G, Soccol CR, Rodriguez-Leon JA, Soccol VT (2001) Production, purification and properties of microbial phytases. Bioresour Technol 77:203–214

    Article  PubMed  CAS  Google Scholar 

  26. Plackett RL, Burman JP (1946) The design of optimum multifactor experiments. Biometrika 33:305–325

    Article  Google Scholar 

  27. Prabhakar A, Krishnaiah K, Janaun J, Bono A (2005) An overview of engineering aspects of solid state fermentation. Malaysian J Microbiol 1:10–16

    Google Scholar 

  28. Raboy V (2001) Seeds for a better future: “low phytate” grains help to overcome malnutrition and pollution. Trend Plant Sci 6:458–462

    Article  CAS  Google Scholar 

  29. Reissig JL, Stromiger JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217:959–966

    PubMed  CAS  Google Scholar 

  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 101:344–352

    Article  PubMed  CAS  Google Scholar 

  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 23:1585–1593

    Article  CAS  Google Scholar 

  32. Terebiznik MR, Pilosof AMR (1999) Biomass estimation in solid state fermentation by modeling dry matter weight loss. Biotech Tech 13:215–221

    Article  CAS  Google Scholar 

  33. Vats P, Banerjee UC (2004) Production studies and catalytic properties of phytases (myoinositolhexakisphosphate phosphohydrolases): an overview. Enzy. Microbiol Technol 35:3–14

    Article  CAS  Google Scholar 

  34. Vohra A, Satyanarayana T (2003) Phytases: microbial sources, production, purification and potential biotechnological applications. Crit Rev in Biotech 23:29–60

    Article  CAS  Google Scholar 

  35. Wodzinski RJ, Ullah AHJ (1996) Phytase. Adv Appl Microbiol 42:263–302

    Article  PubMed  CAS  Google Scholar 

  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 45:76–84

    Article  CAS  Google Scholar 

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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.

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Authors and Affiliations

  1. NCIM Resource Center, National Chemical Laboratory, Pune, 411 008, India

    K. Bhavsar & J. M. Khire

  2. Chemical Engineering and Process Development Division, National Chemical Laboratory, Pune, 411 008, India

    V. Ravi Kumar

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  1. K. Bhavsar
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  2. V. Ravi Kumar
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  3. J. M. Khire
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Correspondence to J. M. Khire.

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Bhavsar, K., Ravi Kumar, V. & Khire, J.M. High level phytase production by Aspergillus niger NCIM 563 in solid state culture: response surface optimization, up-scaling, and its partial characterization. J Ind Microbiol Biotechnol 38, 1407–1417 (2011). https://doi.org/10.1007/s10295-010-0926-z

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