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Three-phase partitioning of α-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques

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Journal of Industrial Microbiology & Biotechnology

Abstract

Simple, attractive and versatile technique, three-phase partitioning (TPP) was used to purify α-galactosidase from fermented media of Aspergillus oryzae. The various conditions required for attaining efficient purification of the α-galactosidase fractions were optimized. The addition of n-butanol, t-butanol, and isopropanol in the presence of ammonium sulfate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The single step of three-phase partitioning, by saturating final concentration of ammonium sulfate (60%) with 1:1 t-butanol, gave activity recovery of 92% with 12-fold purification at second phase of TPP. The final purified enzyme after TPP showed considerable purification on SDS-PAGE with a molecular weight of 64 kDa. The enzyme after TPP showed improved activity in organic solvents. Results are compared with conventional established processes for the purification of α-galactosidase produced by Aspergillus oryzae and overall the proposed TPP technique resulted in 70% reduction of purification cost compared to conventional chromatographic protocols.

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References

  1. Sadana A, Beelaram A (1994) Efficiency and economics of bioseparation: some case studies. Bioseparation 4:221–235

    PubMed  CAS  Google Scholar 

  2. Roy I, Gupta MN (2000) Current trends in affinity base separation of proteins/enzymes. Curr Sci 78:587–591

    CAS  Google Scholar 

  3. Dennison C, Lovrein R (1997) Three phase-partitioning: concentration and purification of proteins. Protein Expr Purif 11:149–161. doi:10.1006/prep.1997.0779

    Article  PubMed  CAS  Google Scholar 

  4. Saxena L, Iyer BK, Ananthanarayan L (2007) Three phase-partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor. Process Biochem 42:491–595. doi:10.1016/j.procbio.2006.09.016

    Article  CAS  Google Scholar 

  5. Sharma A, Gupta MN (2001) Purification of pectinases by three-phase partitioning. Biotechnol Lett 23:625–1627. doi:10.1023/A:1011984517432

    Article  Google Scholar 

  6. Dey PM, Pridham JB (1972) Biochemistry of α-galactosidase. Adv Enzymol 36:91–130

    PubMed  CAS  Google Scholar 

  7. Ulezlo IV, Zaprometova OM (1982) Microbial α-Galactosidase (a review). Prikl Biokhim Mikrobiol 18:3–15

    PubMed  CAS  Google Scholar 

  8. Mulimani VH, Ramalingam (1995) Enzymic hydrolysis of raffinose and stachyose present in soymilk by crude α-galactosidase from Gibberella fujikuroi. Biochem Molecul Biol Intern 36:897–905

  9. Girigouda K, Prashanth SJ, Mulimani VH (2005) Oligosaccharides of black gram (Vigna Munga) as effected by processing methods. Plant Foods Hum Nutr 60:173–180. doi:10.1007/s11130-005-9552-3

    Article  Google Scholar 

  10. Morton RK (1950) Separation and purification of enzymes associated with insoluble particles. Nature 166:1092–1095. doi:10.1038/1661092a0

    Article  PubMed  CAS  Google Scholar 

  11. Pike RN, Dennison C (1989) Protein fractionation by three phase-partitioning (TPP) in aqueous t-butanol mixtures. Biotechnol Bioeng 33:221–228. doi:10.1002/bit.260330213

    Article  PubMed  CAS  Google Scholar 

  12. Prashanth SJ, Mulimani VH (2005) Soymilk oligosaccharides hydrolysis by Aspergillus oryzae α-galactosidase immobilized in calcium alginate. Process Biochem 40:1199–1205. doi:10.1016/j.procbio.2004.04.011

    Article  CAS  Google Scholar 

  13. Kulkarni DS, Kapanoor SS, Girigouda K, Kote NV, Mulimani VH (2006) Reduction of flatus inducing factors in soy milk. Biotechnol Appl Biochem 45:51–57. doi:10.1042/BA20060027

    Article  PubMed  CAS  Google Scholar 

  14. Sharma A, Sharma S, Gupta MN (2000) Purification of alkaline phosphatase from chicken intestine by three-phase partitioning and use of phenyl-Sepharose 6B in the batch mode. Bioseparation 9:155–161. doi:10.1023/A:1008195729472

    Article  PubMed  CAS  Google Scholar 

  15. Lowry AH, Rosebrough NJ, Far AL, Randall J (1950) Protein measurement with the phenol reagent. J Biol Chem 193:265

    Google Scholar 

  16. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0

    Article  PubMed  CAS  Google Scholar 

  17. Ramalingam G, Mulimani VH (2004) Purification and characterization of guar galactomannann degrading α-galactosidase form Aspergillus oryzae DR-5. J Microbiol Biotechnol 11:863–867

    Google Scholar 

  18. Kotwal SK, Gote MM, Khan IM, Khire JM (1999) Production purification and characterization of a constitutive intracellular α-galactosidase form the thermophilic fungus. Hemicola sp. J Ind Microbiol Biotechnol 23:661–667. doi:10.1038/sj.jim.2900680

    Article  PubMed  CAS  Google Scholar 

  19. Mi S, Meng K, Wang Y, Yuan T, Luo H, Yao B (2007) Molecular cloning and characterization of a novel α-galactosidase gene from Penicillium sp F63 CGMCC 1669 and expression in Pichia pastoris. Enzyme Microb Technol 1380:1373–1380. doi:10.1016/j.enzmictec.2006.10.017

    Article  Google Scholar 

  20. Lovrein RE, Goldensoph C, Andreson P, Odegard B (1987) Three phase partitioning (TPP) via t-butanol: enzyme separation from crudes. In: Burgess R (ed) Protein Purification Micro to Macro. Marcel Dekker, New York, pp 521–533

    Google Scholar 

  21. Dennison C, Moolman L, Pillay CS, Meinesz RE (2000) t-Butanol: Nature gift for protein isolation. S Afr J Sci 96:159–160

    CAS  Google Scholar 

  22. Szamos J, Kiss E (1995) Interfacial behavior of proteins in three phase partitioning using salt containing water/tert-butanol systems. J Colloid Interface Sci 170:290–292. doi:10.1006/jcis.1995.1101

    Article  CAS  Google Scholar 

  23. Sharma S, Gupta MN (2000) Three phase partitioning as a large scale separation method for purification of a wheat germ bifunctional protease/amylase inhibitors. Process Biochem 37:193–196. doi:10.1016/S0032-9592(01)00199-6

    Article  Google Scholar 

  24. Gupta MN (1992) Enzyme function in organic solvents. Eur J Biochem 203:25–32. doi:10.1111/j.1432-1033.1992.tb19823.x

    Article  PubMed  CAS  Google Scholar 

  25. Singh RK, Gourinath S, Sharma S, Roy I, Gupta MN, Betzel CH et al (2001) Enhancement of enzyme activity through three phase-partitioning: crystal structure of a modified serine protease at 1.5 A° resolution. Protein Eng 14:307–313. doi:10.1093/protein/14.5.307

    Article  PubMed  CAS  Google Scholar 

  26. Roy I, Sharma A, Gupta MN (2004) Obtaining higher transesterification rates with subtilisin Carlsberg in nonaqueous media. Bioorg Med Chem Lett 14:887–889. doi:10.1016/j.bmcl.2003.12.021

    Article  PubMed  CAS  Google Scholar 

  27. Tanuja S, Srinivas ND, Gowthman MK, Raghavarao KSMS (2000) Aqueous two phase extraction coupled with ultrafiltration for purification of amyloglucosidase. Bioprocess Eng 23:63–68. doi:10.1007/s004499900123

    Article  CAS  Google Scholar 

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Acknowledgments

One the author Dhananjay SK is thankful to Council of Scientific and Industrial Research (CSIR) government of India, for providing financial support in the form of Senior Research Fellowship (SRF).

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Authors and Affiliations

  1. Department of Biochemistry, Gulbarga University, Gulbarga, Karnataka, 585106, India

    S. K. Dhananjay & V. H. Mulimani

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  1. S. K. Dhananjay
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  2. V. H. Mulimani
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Correspondence to V. H. Mulimani.

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Dhananjay, S.K., Mulimani, V.H. Three-phase partitioning of α-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques. J Ind Microbiol Biotechnol 36, 123–128 (2009). https://doi.org/10.1007/s10295-008-0479-6

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  • DOI: https://doi.org/10.1007/s10295-008-0479-6

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