Advertisement

Purification and Characterization of a Novel Thermostable β-Amylase from Aspergillus foetidus MTCC-508. β-Amylase from Aspergillus foetidus MTCC-508

  • Sarad Kumar MishraEmail author
  • Kumar Shivam
  • Sanjeev Kumar Diwakar
  • Swati Shukla
Chapter

Abstract

An extracellular β-amylase was produced from Aspergillus foetidus MTCC-508, and was purified 254.8-fold with 14.6 yields by precipitation with acetone and by column chromatographies with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The band of enzyme was visible around 20 kDa on SDS-PAGE while around 80 kDa on Native-PAGE, showing its homotetrameric nature. The enzyme was optimally active at pH-6.0 and 50 °C temperature. It was fully stable at 50 °C for 2 h. The activity was strongly inhibited by Hg2+, Zn2+ and Co2+, while Mg2+ marginally enhanced the enzyme activity. The enzyme was able to hydrolyze the raw starches of potato, wheat, rice, maize, and Trapa natans, with the highest degree of saccharification of maize starch. The K m and V max values for this enzyme against boiled soluble starch were found 2.7 mg/mL and 2,100 U/mg of protein, respectively.

Keywords

β-amylase Aspergillus foetidus Enzyme activity Enzyme purification Native-PAGE 

Notes

Acknowledgments

The support from the Department of Biotechnology, DDU Gorakhpur University, Gorakhpur is duly acknowledged.

References

  1. Abouzeid AM (1997) Production purification and characterization of an extracellular α-amylase enzyme isolated from Aspergillus flavus. Microbios 89:55–66Google Scholar
  2. Alli AI, Ogbonna CIC, Rahman ATMF (1998) Hydrolysis of certain Nigerian cereal starch using crude fungal amylase. Nig J Biotechnol 9:24–36Google Scholar
  3. Bahrim GE, Scantees M, Negoitan T (2007) Biotechnological conditions of amylase and complex production and utilization involving filamentous bacteria. The Annals of the University Dunarea de jos of Gulati-, Fascicle iv- Food Technol 76–81Google Scholar
  4. Bernfeld P (1955) Amylase α/β. Methods Enzymol 1:149CrossRefGoogle Scholar
  5. Chang CT, Liou HY, Tang HL, Sung HY (1996) Activation, purification and properties of β-amylase from sweet potatoes (Ipomea batatas). Biotechnol App Bichem 24:113–118Google Scholar
  6. Colman P, Matthews B (1971) Symmetry, molecular weight and crystallographic Data for sweet potato beta-amylase. J Mol Biol 60:163PubMedCrossRefGoogle Scholar
  7. Dey PM, Pridham JB (1977) Biochemistry of alpha galactosidase. Adv Enzymol 15:91–130Google Scholar
  8. Hyun HH, Zeikus JG (1985) General biochemical characterization of thermostable extracellular β-amylase from Clostridium thermosulfurogenes. Appl Environ Microbiol 49:1162–1167PubMedCentralPubMedGoogle Scholar
  9. Kwan HS, So KH, Chan KY, Cheng SC (1994) Purification and properties of β-amylase from Bacillus circulans S31. World J Microbiol Biotechnol 10:597–598PubMedCrossRefGoogle Scholar
  10. Laemmli UK (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature 227:680–685Google Scholar
  11. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Sci 56:658–666CrossRefGoogle Scholar
  12. Lowry OH, Rosenbrough NJ, Farr Al, Randall RJ (1951) Protein estimation with the Folin-phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  13. Obi SKC, Odibo FJC (1984) Partial purification and characterization of a thermostable actinomycete β-amylase. Appl Environ Microbiol 47:571–575PubMedCentralPubMedGoogle Scholar
  14. Oboh G (2005) Isolation and characterization of amylase from fermented cassava (Manihot esculenta Crantz) waste water. Afr J Biotechnol 4:1117–1123Google Scholar
  15. Oboh G, Ajele JO (1997) Effect of some metallic chlorides on the activity of β-amylase from sweet potatoes. Nig J Biochem Mol Biol 12:73–75Google Scholar
  16. Okada y, Yoshigi N, Sahara H, Koshino S (1995) Increase in thermostability of recombinant barley beta-amylase by random mutagenesis. Biosci Biotechnol Biochem 59:1152–1153PubMedCrossRefGoogle Scholar
  17. Omemu AM, Akpan I, Bankole MO, Teniola OD (2005) Hydrolysis of raw tuber starches by amylase of Aspergillus niger AM07 isolated from the soil. Afr J Biotechnol 4:19–25Google Scholar
  18. Pandey A, Nigam P, Sccol CR, Sccol VT, Singh D, Mohan R (2000) Advances in microbial amylases (Review). Biotechnol Appl Biochem 31:135–152PubMedCrossRefGoogle Scholar
  19. Rama M, Swamy MV, Seenayya G (1998) Purification and characterization of thermostable β-amylase and pullulanase from high yielding Clostridium thermosulfurogenes SV2. World J Microbiol Biotechnol 14:89–94Google Scholar
  20. Schokker EP, Van Boekel AJS (1999) Kinetic of thermal inactivation of extracellular proteinase from Pseudomonas fluorescens 22F, Influence of pH, calcium and protein. J Agric Food Chem 47:1681–1686PubMedCrossRefGoogle Scholar
  21. Schuster E, Dunn-Coleman N, Frisvad JC, Van Dijck PWM (2002) On the safety of Aspergillus niger-a review. Appl Microbiol Biotechnol 59:426–435PubMedCrossRefGoogle Scholar
  22. Shen GJ, Saha BC, Lee YE, Bhatnagar L, Zeikus JG (1988) Purification and characterization of a novel thermostable β-amylase from Clostridium thermosulphurogenes, BiChemi. J 254:835–840Google Scholar
  23. Swamy MV, Sairam N, Seenayya G (1994) β-amylase from Clostridium thermocelluum SS8 a thermophillic, anaerobic, cellulolytic bacterium. Lett Appl Microbiol 18:301–304CrossRefGoogle Scholar
  24. Thoma JA, Spradlin JE, Dygert S (1997) Plant and animal amylases. In: Boyer PD (ed) The enzymes, 3rd edn. Academic Press, New York, pp 115–189Google Scholar
  25. Thoma JA, Spradlin JE, Dygert S (1971) Plant and animal amylases. In: Boyer PD (ed) The enzymes, 5th edn. Academic, New York, pp 115–189 Google Scholar
  26. Walker JM (2002) The protein protcols handbook. Humana Press Inc, New Jersey, pp 343–345CrossRefGoogle Scholar
  27. White JS, White DC (1997) Source book of enzymes. CRC Press, New York, p 572Google Scholar

Copyright information

© Springer India 2014

Authors and Affiliations

  • Sarad Kumar Mishra
    • 1
    Email author
  • Kumar Shivam
    • 1
  • Sanjeev Kumar Diwakar
    • 1
  • Swati Shukla
    • 1
  1. 1.Department of BiotechnologyD. D. U. Gorakhpur UniversityGorakhpurIndia

Personalised recommendations