Annals of Microbiology

, Volume 64, Issue 2, pp 859–867 | Cite as

Thermostable alkaline protease from Thermomyces lanuginosus: optimization, purification and characterization

  • Mohamed Ghareib
  • Eman M. Fawzi
  • Nouf A. Aldossary
Original Article
First Online:
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Accepted:

Abstract

A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-100 and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K m), maximal reaction velocity (V max) and turnover number (K cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s−1, respectively. The best enzyme activators were K+, Ca2+ and Mn2, respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry.

Keywords

Thermostable Alkaline protease Thermomyces lanuginosus Optimization Purification Characterization 
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References

  1. Aftab S, Ahmed S, Saeed S, Rasool SA (2006) Screening, isolation and characterization of alkaline protease producing bacteria from soil. Pak J Biol Sci 9(11):2122–2126CrossRefGoogle Scholar
  2. Agarwal D, Patidar P, Banerjee T, Patil S (2004) Production of alkaline protease by Penicillium sp. under SSF conditions and its application to soy protein hydrolysis. Process Biochem 39:977–981CrossRefGoogle Scholar
  3. Anandan D, Marmer W, Dudely RL (2007) Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii. Ind J Microbiol Biotechnol 34:339–347CrossRefGoogle Scholar
  4. Arlumani M, Aparanjini K, Vasanthi K, Arumugam P, Arivuchelvi P, Kalaichelvan PT (2007) Purification and partial characteriuzation of serine protease from thermostable alkalophilic Bacillus laterosporus-Ak1. World J Microbiol Biotechnol 23:475–481CrossRefGoogle Scholar
  5. Barata RA, Andrade MHG, Rodrigues RD, Castro IM (2002) Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini. J Biosci Bioeng 94(4):304–308PubMedCrossRefGoogle Scholar
  6. Cerovsky V (1992) Protease-catalysed peptide synthesis in solvent-free system. Biotechnol Tech 6:155–160CrossRefGoogle Scholar
  7. Charles P, Devanathan V, Anbu P, Ponnuswamy MN, Kalaichelvan PT, Hur BK (2008) Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA-10. J Basic Microbiol 48:347–352PubMedCrossRefGoogle Scholar
  8. Damare S, Raghukumar C, Muraleedhran UD, Raghukumar S (2006) Deep-sea fungi as sources of alkaline and cold-tolerant proteases. Enzyme Microb Technol 39:172–181CrossRefGoogle Scholar
  9. Deore GB, Limaye AS, Dushing YA, Dhobale SB, Kale S, Laware SL (2013) Screening of protease producing fungi for microbial digestion of seed proteins and synthesis of amino acids-metalnutrient chelates. Pak J Biol Sci 16:86–91PubMedCrossRefGoogle Scholar
  10. Dunaevsky YAE, Matveeva AR, Beliakova GA, Domash VI, Domash VI, Belozersky MA (2007) Extracellular alkaline proteinase of Colletotrichum gloeosporodes. Biochemistry (Moscow) 72:345–350CrossRefGoogle Scholar
  11. Ghareib M, Youssef K, Nour El-Dein M (1988) Isolation and characterization of intracellular phytase from Macrophomina phaseolina. Zentralbl Mikrobiol 143:397–403Google Scholar
  12. Ghareib M, Khalil AA, Saad El-Dein HS (1999) Dextranase production by Aspergillus flavipes in presence of some important additives. Az J Microbiol 46:144–152Google Scholar
  13. Ghareib M, Khalil A, Hamdy HS, Abu Tahoon M (2011) Potentiality of some alkaliphilic fungi isolated from Egyptian soil as producers of alkaline detergent enzymes. New Egypt J Biotechnol 28:267–279Google Scholar
  14. Ghareib M, Fawzi EM, Aldossary NA (2012) Thermostable alkaline protease producing mycoflora from the soils of the Eastern Province of Saudi Arabia. Wulfenia 19(12):95–111Google Scholar
  15. Glantz AS (1992) Primer of biostatistics. McGraw Hill, New York, pp 2–18Google Scholar
  16. Godfrey T, West S (1996) Industrial enzymology, 2nd edn. Macmillan, New York, pp 3–10Google Scholar
  17. Griffin HL, Greene RV, Cotta MA (1992) Isolation and characterization of an alkaline protease from the marine shipworm bacterium. Curr Microbiol 24:111–117CrossRefGoogle Scholar
  18. Gupta A, Joseph B, Malni A, Thomas G (2008) Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World J Microbiol Biotechnol 24:237–243CrossRefGoogle Scholar
  19. Ha M, Bekhit A, Carne A, Hopkins D (2013) Comparison of the proteolytic activities of new commercially available bacterial and fungal proteases toward meat proteins. J Food Science 78(2):170–177CrossRefGoogle Scholar
  20. Hamilton LM, Kelly CT, Fogarty WM (1998) Raw starch degradation by the non-raw starch-adsorbing bacterial alpha amylase of Bacillus sp. IMD. Carbohydr Res 314:251–257CrossRefGoogle Scholar
  21. Kalpana Devi MA, Rasheedha Banu A, Gnanaprabhal GR, Pradeep BV, Palaniswamy M (2008) Purification, characterization of alkaline protease enzyme from native isolate Aspergillus niger and its compatibility with commercial detergents. Ind J Sci Technol 1(7):1–7Google Scholar
  22. Keay L, Moser PW, Wildi BS (1970) Proteases of the genus Bacillus II. Alkaline proteases. Biotechnol Bioeng 12:213–249PubMedCrossRefGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during assembly of head bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  24. Lee JK, Kim YO, Kim HK, Park YS, Oh TK (1996) Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. Biosci Biotechnol Biochem 60:840–846PubMedCrossRefGoogle Scholar
  25. Li DC, Yang YJ, Shen CY (1997) Protease production by the thermophilic fungus Thermomyces lanuginosus. Mycol Res 101:18–22CrossRefGoogle Scholar
  26. Mane M, Mahadik K, Kokare C (2013) Purification, characterization and application of thermostable alkaline protease from marine Streptomyces sp. Int J Pharm Bio Sci 4(1):572–582Google Scholar
  27. Manivannan S, Kathiresan K (2007) Alkaline protease production by Penicillium fellutanum isolated from mangrove sediment. Int J Biol Chem 1(2):98–103CrossRefGoogle Scholar
  28. Monod M, Togni G, Rahalison L, Frenk E (1991) Isolation and characterization of an extracellular alkaline protease of Aspergillus fumigatus. J Med Microbiol 35:23–28PubMedCrossRefGoogle Scholar
  29. Nehra KS, Dhillon S, Chaudhary K, Singh R (2002) Production of alkaline protease by Aspergillus species under submerged and solid state fermentation. Ind J Microbiol 42:43–47Google Scholar
  30. Nehra KS, Singh A, Sharma J, Kumar R, Dhillon I (2004) Production and characterization of alkaline protease from Aspergillus species and its compatibility with commercial detergents. Asian J Microbiol Biotechnol Environ Sci 6:67–72Google Scholar
  31. Phadatare SU, Deshpande VV, Srinivasan MC (1993) High activity alkaline protease from Conidobus coronatus (NCL 86.8.20): enzyme production and compatibility with commercial detergents. Enzyme Microb Technol 15:72–76CrossRefGoogle Scholar
  32. Saba I, Qazi PH, Rather SA, Dar RA, Qadri QA, Ahmad N, Johri S, Taneja SC, Shawl S (2012) Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India. World J Microbiol Biotechnol 28:1071–1079PubMedCrossRefGoogle Scholar
  33. Sharma J, Singh A, Kumar R, Mittal A (2006) Partial purification of an alkaline protease from a new strain Of Aspergillus oryzae AWT 20 and its enhanced stabilization in entrapped ca-alginatebeads. Int J Microbiol 2(2):165–176Google Scholar
  34. Sindhu R, Suprabha GN, Shashidhar S (2009) Optimization of process parameters for the production of alkaline protease from Penicillium godlewskii SBSS 25 and its application in detergent industry. Afr J Microbiol Res 3(9):515–522Google Scholar
  35. Tanksale A, Chandra PM, Rao M, Deshpande V (2001) Immobilization of alkaline protease from Conidiobolus macrospous for reuse and improved thermal stability. Biogeosciences 23:51–54Google Scholar
  36. Tremacoldi CR, Monti R, Selistre-De-Araujo HS, Carmona EC (2007) Purification and properties of an alkaline protease of Aspergillus clavatus. World J Microbiol Biotechnol 23:295–299CrossRefGoogle Scholar
  37. Wang Y, Lee M (1996) Influence of culture and nutritional condition on the production of protease from thermophilic strain Aspergillus species NTIJ-FC-671. J Chinese Agric Chemical Society 34:732–742Google Scholar
  38. Wilson SA, Young OA, Coolbear T, Daniel RM (1992) The use of proteases from extreme thermophiles for meat tenderization. Meat Sci 32:93–103PubMedCrossRefGoogle Scholar
  39. Yadav SK, Bisht D, Shikha, Darmwal NS (2011) Oxidant and solvent stable alkaline protease from Aspergillus flavus and its characterization. Afr J Biotechnol 10(43):8630–8640Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Authors and Affiliations

  • Mohamed Ghareib
    • 1
  • Eman M. Fawzi
    • 1
  • Nouf A. Aldossary
    • 2
  1. 1.Biological & Geological Sciences Department, Faculty of EducationAin Shams UniversityRoxyEgypt
  2. 2.Ministry of Higher Education, Kingdom of Saudi ArabiaRiyadhSaudi Arabia

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