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Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters

Journal of Industrial Microbiology & Biotechnology volume 37, pages 129–138 (2010)Cite this article

Abstract

Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30°C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 × 105 U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31°C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 × 105 U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 × 105 U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48–56, 1977].

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References

  1. Anbu P, Annadurai G, Lee J, Hur B (2008) Optimization of alkaline protease production from Shewanella oneidensis MR-1 by response surface methodology. J Chem Tech Biotechnol 84:54–62. doi:10.1002/jctb.2004

    Google Scholar 

  2. Chakraborty R, Srinivasan M, Sarkar SK, Raghavan KV (1995) Production of acid protease by a new Aspergillus niger by solid state fermentation. J Microbiol Biotechnol 10:17–30

    CAS  Google Scholar 

  3. Chandel AK, Rudravaram R, Rao LV, Ravindra P, Narasu ML (2007) Industrial enzymes in bioindustrial sector development, An Indian perspective. J Commer Biotechnol 13(4):283–291

    Article  Google Scholar 

  4. Chauhan B, Gupta R (2004) Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14. Process Biochem 39:2115–2122. doi:10.1016/j.procbio.2003.11.002

    Article  CAS  Google Scholar 

  5. Dahiya N, Tewari R, Tiwari RP, Hoondal GS (2005) Chitinase production in solid state fermentation by Enterobacter sp. NRG4 using statistical experimental design. Curr Microbiol 51:222–228. doi:10.1007/s00284-005-4520-y

    Article  CAS  PubMed  Google Scholar 

  6. Datta A (1992) Purification and characterization of a novel protease from solid substrate cultures of Phanerochaete chrysosporium. J Biol Chem 267:728–736

    CAS  PubMed  Google Scholar 

  7. Davidson R, Gertler A, Hofmann T (1975) Aspergillus oryzae acid proteinases: purification and properties and formation of π-chymotrypsin. Biochem J 147:45–53

    CAS  PubMed  Google Scholar 

  8. Dutta JR, Dutta PK, Banerjee R (2004) Optimisation of culture parameters for extracellular protease production from a newly isolated Pseudomonas sp. using response surface and artificial neural network models. Process Biochem 39:2193–2198. doi:10.1016/j.procbio.2003.11.009

    Article  CAS  Google Scholar 

  9. Ferreira SLC, Bruns RE, Ferreira HS, Matos GD, David JM, Brandao GC, da Silva EGP, Portugal LA, dos Reis PS, Souza AS, dos Santos WNL (2007) Box-Behnken design, an alternative for the optimization of analytical methods. Anal Chim Acta 597:179–186. doi:10.1016/j.aca.2007.07.011

    Article  CAS  PubMed  Google Scholar 

  10. Germano S, Pandey A, Osaku CA, Rocha SN, Soccol CS (2003) Characterization and stability of proteases from Penicillium sp. produced by solid-state fermentation. Enzyme Microb Technol 32:246–251. doi:10.1016/S0141-0229(02)00283-1

    Article  CAS  Google Scholar 

  11. Ikasari L, Mitchell DA (1996) Leaching and characterization of Rhizopus oligosporus acid protease from solid-state fermentation. Enzyme Microb Technol 19:171–175. doi:10.1016/0141-0229(95)00227-8

    Article  CAS  Google Scholar 

  12. Kitano H, Kataoka K, Furukawa K, Hara S (2002) Specific expression and temperature-dependent expression of the acid protease-encoding gene (pepA) in Aspergillus oryzae in solid-state culture (Rice-Koji). J Biosci Bioeng 93:563–567. doi:10.1016/S1389-1723(02)80238-9

    CAS  PubMed  Google Scholar 

  13. Klapper BF, Jameson DM, Mayer RM (1973) The purification and properties of an extracellular protease from Aspergillus oryzae NRRL 2160. Biochim Biophys Act 304:505–512

    CAS  Google Scholar 

  14. Krishna C (2005) Solid-state fermentation systems—an overview. Critical Rev Biotech 25:1–30

    Article  CAS  Google Scholar 

  15. Krishnan S, Vijayalakshmi MA (1985) Purification of an acid protease and a serine carboxypeptidase from Aspergillus niger using metal chelate affinity chromatography. J Chromatogr 329:165–170. doi:10.1016/S0021-9673(01)81911-7

    Article  CAS  PubMed  Google Scholar 

  16. Kundu AK, Manna S (1975) Purification and characterization of extracellular proteinases of Aspergillus oryzae. Appl Environ Microbiol 30(4):507–513

    CAS  Google Scholar 

  17. Lowry OH, Rosenbergh NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  18. Mabrouk SS, Hashem AM, El-Shayeb NMA, Ismail M, Abdel-Fattah AF (1999) Optimisation of alkaline protease productivity by Bacillus licheniformis ATCC 21415. Bioresource Tech 69:155–159. doi:10.1016/S0960-8524(98)00165-5

    Article  CAS  Google Scholar 

  19. Mohammed H, Ahmed R, Neji G, Moncef N (2008) Optimisation of alkaline protease production by Aspergillus clavatus ES1 in Mirabilis jalapa powder using statistical experimental design. Appl Microbiol Biotech 79:915–923. doi:10.1007/s00253-008-1508-0

    Article  Google Scholar 

  20. Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid-state fermentation for the production of industrial enzymes. Curr Sci 77:149–162

    CAS  Google Scholar 

  21. Rao MB, Tanksale AM, Ghatge MS, Deshpande V (1998) Molecular and biotechnology aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635

    CAS  PubMed  Google Scholar 

  22. Senthilkumar SR, Ashokkumar B, Raj CK, Gunasekaran P (2005) Optimization of medium composition for alkali-stable xylanase production by Aspergillus fisheri Fxn 1 in solid-state fermentation using central composite rotary design. Bioresource Tech 96:1380–1386. doi:10.1016/j.biortech.2004.11.005

    Article  CAS  Google Scholar 

  23. Tari C, Genckal H, Tokatli F (2006) Optimisation of growth medium using a statistical approach for the production of an alkaline protease from a newly isolated Bacillus sp. L21. Process Biochem 41:659–665. doi:10.1016/j.procbio.2005.08.012

    Article  CAS  Google Scholar 

  24. Tello-Solis S, Rodriguez-Romero A, Hernandez Arana A (1994) Circular dichroism studies of acid proteases from Aspergillus niger and Aspergillus awamori. Biochem Mol Biol Int 33:759–768

    CAS  PubMed  Google Scholar 

  25. Thys RCS, Guzzon SO, Olivera FC, Brandelli A (2006) Optimization of protease production by Microbacterium sp. in feather meal using response surface methodology. Process Biochem 41:67–73. doi:10.1016/j.procbio.2005.03.070

    Article  CAS  Google Scholar 

  26. Tremacoldi CR, Watanabe NK, Carmona EC (2004) Production of extracellular proteases from Aspergillus clavatus. World J Microbiol Biotech 20:639–642. doi:10.1023/B:WIBI.0000043194.21080.c1

    Article  CAS  Google Scholar 

  27. Tsujita Y, Endo A (1977) Extracellular acid protease of Aspergillus oryzae grown on liquid media: multiple forms due to association with heterogeneous polysaccharides. J Bacteriol 130(1):48–56

    CAS  PubMed  Google Scholar 

  28. Tsujita Y, Endo A (1978) Purification and characterization of two molecular forms of membrane acid protease from Aspergills oryzae. Eur J Biochem 84:347–353

    Article  CAS  PubMed  Google Scholar 

  29. Tsujita Y, Endo A (1978) Presence and partial characterization of internal acid protease of Aspergillus oryzae. Appl Environ Microbiol 36(2):237–242

    CAS  PubMed  Google Scholar 

  30. Tunga R, Banerjee R, Bhattacharyya BC (2001) Optimization of some additives to improve protease production under SSF. Ind J Exp Biol 39:1144–1148

    CAS  Google Scholar 

  31. Villegas E, Aubegue S, Alcantara L, Auria R, Revah S (1993) Solid state fermentation, acid protease production in controlled CO2 and O2 environments. Biotech Adv 11:387–397. doi:10.1016/0734-9750(93)90008-B

    Article  CAS  Google Scholar 

  32. Vishwanatha KS, Appu Rao AG, Singh SA (2009) Characterization of acid protease expressed from Aspergillus oryzae MTCC 5341. Food Chem 114:402–407. doi:10.1016/j.foodchem.2008.09.070

    Article  CAS  Google Scholar 

  33. Wu LC, Hang MD (2000) Acid protease production from Neosartorya fischeri. Lebensmittel-Wissenschaft und-Technologie 33:44–47. doi:10.1006/fstl.1999.0608

    Article  CAS  Google Scholar 

  34. Yagi F, Fan J, Tadera K, Kobayashi A (1986) Purification and characterization of carboxyl proteinase from Aspergillus kawachii. Agric Biol Chem 50(4):1029–1033

    CAS  Google Scholar 

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Acknowledgments

The authors wish to thank Dr. V. Prakash, Director, CFTRI, for his valuable support and suggestions. The authors thank Dr. M. C. Varadaraj, Head, Human Resource Development, CFTRI, for help in isolation and identification of Aspergillus oryzae, MTCC 5341. The authors thank Dr. N. Bhaskar, Department of Meat, Fish & Poultry Technology, CFTRI, for helpful discussion.The Department of Biotechnology, Govt. of India provided the funds for this research. K.S.V. thanks CSIR for a research fellowship.

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Authors and Affiliations

  1. Department of Protein Chemistry and Technology, Central Food Technological Research Institute (A Constituent Laboratory of the Council of Scientific and Industrial Research), Mysore, 570 020, India

    K. S. Vishwanatha, A. G. Appu Rao & Sridevi Annapurna Singh

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  1. K. S. Vishwanatha
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  2. A. G. Appu Rao
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  3. Sridevi Annapurna Singh
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Correspondence to Sridevi Annapurna Singh.

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Vishwanatha, K.S., Rao, A.G.A. & Singh, S.A. Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters. J Ind Microbiol Biotechnol 37, 129–138 (2010). https://doi.org/10.1007/s10295-009-0654-4

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Keywords

  • Acid protease
  • Solid-state fermentation
  • Aspergillus oryzae
  • Media optimization