Fundagül EREM, Mehmet İNAN, Muharrem CERTEL

OPTIMISATION OF Bacillus amyloliquefaciens FE-K1 EXTRACELLULAR PEPTIDASE PRODUCTION BY RESPONSE SURFACE METHODOLOGY

Bu çalışmada, daha önce sünmüş ekmekten izole edilmiş olan Bacillus amyloliquefaciens FE-K1’in ekstraselüler peptidaz üretiminin, merkezi kompozit tasarıma (MKT) dayalı yanıt yüzey yöntemi (YYY) ile optimize edilmesi amaçlanmıştır. Yanıt yüzey yönteminde sıcaklık (20-45°C), enzim üretim ortamının başlangıç pH değeri (pH 5-9) ve inokülasyon seviyesi (%1-5, v/v) faktör olarak kullanılmış, fermentasyon süresi her deneme için ayrı ayrı belirlenmiştir. Sonuçlar optimum peptidaz üretiminin 33,4°C, pH 6,62 ve %2,3 inokülasyon seviyesinde elde edildiğini göstermiştir. Optimum koşullar altında fermentasyon süresinin sadece 7 saat, ham enzimin aktivitesinin 49,17U/mL, spesifik aktivitesinin ise 504,77U/mg olduğu tespit edilmiştir.

Anahtar Kelimeler:

Bacillus, central composite design, peptidase, response surface methodology

OPTIMISATION OF Bacillus amyloliquefaciens FE-K1 EXTRACELLULAR PEPTIDASE PRODUCTION BY RESPONSE SURFACE METHODOLOGY

In this study, it was aimed to optimise the extracellular peptidase production of Bacillus amyloliquefaciens FE-K1, previously isolated from ropy wholemeal bread, by using response surface methodology (RSM) based on central composite design (CCD). The temperature (20-45°C), initial pH of the enzyme production medium (pH 5-9) and inoculation level (1-5%, v/v) were used as the factors for RSM, and the fermentation time was determined for each trial separately. Results showed that the optimum peptidase production occurred at 33.4°C, pH 6.62 and 2.3% inoculation. It was determined that the fermentation time was only 7h, the crude enzyme had a peptidase activity of 49.17U/mL and a specific activity of 504.77U/mg under the optimised conditions.

Keywords:

Bacillus, central composite design, peptidase, response surface methodology,

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1. Ahsan, T., Chen, J., Wu, Y. & Irfan, M. 2017. Application of response surface methodology for optimization of medium components for the production of secondary metabolites by Streptomyces diastatochromogenes KX852460. AMB Express, 7(96): 1-10.
2. Akbalik, G., Gunes, H., Yavuz, E., Yaşa, I., Harsa, S., Elmaci, Z.S. & Yenidünya, A.F. 2004. Identification of extracellular enzyme producing alkalophilic bacilli from Izmir province by 16S-ITS rDNA RFLP. Journal of Applied Microbiology, 97: 766-773.
3. Ali, N., Ullah, N., Qasim, M., Rahman, H., Khan, S.N., Sadiq, A. & Adnan, M. 2016. Molecular characterization and growth optimization of halo-tolerant protease producing Bacillus subtilis strain BLK-1.5 isolated from salt mines of Karah, Pakistan. Extremophiles, 20: 395-402.
4. Anandharaj, M., Sivasankari, B., Siddharthan, N., Rani, R.P. & Sivakumar, S. 2016. Production, purification, and biochemical characterization of thermostable metallo-protease from novel Bacillus alkalitelluris TWI3 isolated from tannery waste. Applied Biochemistry and Biotechnology, 178: 1666-1686.
5. Bailey, C.P. & von Holy, A. 1993. Bacillus spore contamination associated with commercial bread manufacture. Food Microbiology, 10: 287-294.
6. Beg, Q.K. & Gupta, R. 2003. Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme and Microbial Technology, 32: 294-304.
7. Beg, Q.K., Sahai, V. & Gupta, R. 2003. Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor. Process Biochemistry, 39: 203-209.
8. Beheshti Maal, K., Emtiazi, G. & Nahvi, I. 2011. Increasing the alkaline protease activity of Bacillus cereus and Bacillus polymyxa simultaneously with the start of sporulation phase as a defense mechanism. African Journal of Biotechnology, 10(19): 3894-3901.
9. Bhunia, B., Basak. B. & Dey, A. 2012. A review on production of serine alkali protease by Bacillus spp. Journal of Biochemical Technology, 3(4): 448-457.
10. Bradford, M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.
11. Chauhan, B. & Gupta, R. 2004. Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14. Process Biochemistry, 39: 2115-2122.
12. Chu, W.H. 2007. Optimization of extracellular alkaline protease production from species of Bacillus. Journal of Industrial Microbiology and Biotechnology, 34: 241-245.
13. Collins, N.E., Kirschner, L.A.M. & von Holy, A. 1991. Characterization of Bacillus isolates from ropey bread, bakery equipment and raw materials. South African Journal of Science, 87: 62-66.
14. Cupp-Enyard, C. 2008. Sigma’s non-specific protease activity assay-casein as a substrate. Journal of Visualized Experiments, 19: 899.
15. Denizci, A.A., Kazan, D., Abeln, E.C.A. & Erarslan, A. 2004. Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under highly alkaline conditions. Journal of Applied Microbiology, 96: 320-327.
16. Ellis, W.O., Obubuafo, A.K., Ofosu-Okyere, A., Marfo, E.K., Osei-Agyemang, K. & Odame-Darkwah, J.K. 1997. A survey of bread defects in Ghana. Food Control, 8: 77-82.
17. Erem, F., Certel, M. & Karakaş, B. 2009. Identification of Bacillus species isolated from ropey breads both with classical methods and API identification kits. Mediterranean Agricultural Sciences, 22(2): 201-210.
18. Erem, F. & Certel, M. 2018. Sünmüş ekmekten izole edilen Bacillus suşlarının peptidaz üretme potansiyellerinin belirlenmesi ve peptidaz üretimi için bazı kültür şartlarının optimizasyonu. Anadolu Üniversitesi Bilim ve Teknoloji Dergisi-C Yaşam Bilimleri ve Biyoteknoloji, 7(2): 160-179.
19. Genckal, H. & Tari, C. 2006. Alkaline protease production from alkalophilic Bacillus sp. isolated from natural habitats. Enzyme and Microbial Technology, 39: 703-710.
20. Gerze, A., Omay, D. & Guvenilir, Y. 2005. Partial purification and characterization of protease enzyme from Bacillus subtilis megatherium. Applied Biochemistry and Biotechnology, 121-124: 335-346.
21. Gorlach-Lira, K., Pedroza, M.L.V., Burdziej-Pokojska, A., Rozycki, H. & Dahm, H. 2010. Response surface analysis on the effect of temperature and pH on growth and proteolytic activity of thermophilic Bacillus sp. Brazilian Archives of Biology and Technology, 53(5): 1067-1072.
22. Guangrong, H., Dehui, D., Weilian, H. & Jiakin, J. 2008. Optimization of medium composition for thermostable protease production by Bacillus sp. HS08 with a statistical method. African Journal of Biotechnology, 7(8): 1115-1122.
23. Gupta, R., Gupta, K., Saxena, R.K. & Khan, S. 1999. Bleach-stable, alkaline protease from Bacillus sp. Biotechnology Letters, 21: 135-138.
24. Gupta, R., Beg, Q.K., Khan, S. & Chauhan, B. 2002. An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Applied Microbiology and Biotechnology, 60: 381-395.
25. Hammami, A., Bayoudh, A., Abdelhedi, O. & Nasri, M. 2018. Low-cost culture medium for the production of proteases by Bacillus mojavensis SA and their potential use for the preparation of antioxidant protein hydrolysate from meat sausage by-products. Annals of Microbiology, 68: 473-484.
26. Hamoen, L.W., Venema, G. & Kuipers, O.P. 2003. Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology, 149: 9-17.
27. Hanlon, G.W. & Hodges, N.A. 1981. Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources. Journal of Bacteriology, 147(2): 427-431.
28. Harwood, C.R. & Cranenburgh, R. 2008. Bacillus protein secretion: an unfolding story. Trends in Microbiology, 16(2): 73-79.
29. Hussain, F., Kamal, S., Rehman, S., Azeem, M., Bibi, I., Ahmed, T. & Iqbal, H.M.N. 2017. Alkaline protease production using response surface methodology, characterization and industrial exploitation of alkaline protease of Bacillus subtilis sp. Catalysis Letters, 147(5): 1204-1213.
30. Jaswal, R.K., Kocher, G.S. & Virk, M.S. 2008. Production of alkaline protease by Bacillus circulans using agricultural residues: A statistical approach. Indian Journal of Biotechnology, 7: 356-360.
31. Johnvesly, B. & Naik, G.R. 2001. Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochemistry, 37: 139-144.
32. Kim, M., Si, J.B., Reddy, L.V. & Wee, Y.J. 2016. Enhanced production of extracellular proteolytic enzyme excreted by a newly isolated Bacillus subtilis FBL-1 through combined utilization of statistical designs and response surface methodology. RSC Advances, 6: 51270-51278.
33. Kirschner, L.A.M. & von Holy, A. 1989. Rope spoilage of bread. South African Journal of Science, 85: 425-427.
34. Kumar, C.G., Tiwari, M.P. & Jany, K.D. 1999. Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties. Process Biochemistry, 34: 441-449.
35. Kolkman, M., Mejldal, R., Goedegebuur, F., Babe, L.M., Kellett-Smith, A.H., Mulder, H., Bott, R.R. & Scotcher, M.C. 2016. Serine proteases of the Bacillus gibsonii-clade. US Patent No. US20160319266A1.
36. Lakshmi, B.K.M. & Hemalatha, K.P.J. 2016. Production of alkaline protease from Bacillus licheniformis through statistical optimization of growth media by response surface methodology. Fermentation Technology, 5(2): 130-137.
37. Leong, L.B. 2006. Recombinant Bacillus proteases and uses thereof. US Patent No. US 7081359 B2.
38. Mabrouk, S.S., Hashem, A.M., El-Shayeb, N.M.A., Ismail, A.M.S. & Abdel-Fattah, A.F. 1999. Optimization of alkaline protease productivity by Bacillus licheniformis ATCC 21415. Bioresource Technology, 69: 155-159.
39. Manni, L., Jellouli, K., Agrebi, R., Bayoudh, A. & Nasri, M. 2008. Biochemical and molecular characterization of a novelcalcium-dependent metalloprotease from Bacillus cereus SV1. Process Biochemistry, 43: 522-530.
40. Matta, H. & Punj, V. 1998. Isolation and partial characterization of a thermostable extracellular protease of Bacillus polymyxa B-17. International Journal of Food Microbiology, 42: 139-145.
41. Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Biochemistry, 31(3): 426-428.
42. Molva, C., Sudagidan, M. & Okuklu, B. 2009. Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey. Food Control, 20: 839-834.
43. Nadeem, M., Qazi, J.I., Syed, Q.A. & Baig, S. 2008. Optimization of process parameters for alkaline protease production by Bacillus licheniformis N-2 and kinetics studies in batch fermentation. Turkish Journal of Biology, 32: 243-251.
44. O’hara, M.B. & Hageman, J.H. 1990. Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells. Journal of Bacteriology, 172(8): 4161-4170.
45. Orhan, E., Omay, D. & Guvenilir, Y. 2005. Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus. Applied Biochemistry and Biotechnology, 121-124: 183-194.
46. Outtrup, H., Dambmann, C. & Aaslyng, D.A. 1990. Alkaline protease from Bacillus J 20. US Patent No. US5358865A.
47. Pantamas, P., Chaiprasert, P. & Tanticharoen, M. 2003. Anaerobic digestion of glucose by Bacillus licheniformis and Bacillus coagulans at low and high alkalinity. Asian Journal on Energy and Environment, 4(1-2): 1-17.
48. Patel, R.K., Dodia, M.S., Joshi, R.H. & Singh, S.P. 2006. Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. Process Biochemistry, 41: 2002-2009.
49. Pepe, O., Blaiotta, G., Moschetti, G., Greco, T. & Villani, F. 2003. Rope-producing strains of Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria. Applied and Environmental Microbiology, 69(49): 2321-2329.
50. Prakasham, R.S., Rao, C.S. & Sarma, P.N. 2006. Green gram husk-an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation. Bioresource Technology, 97: 1449-1454.
51. Puri, S., Beg, Q.K. & Gupta, R. 2002. Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Current Microbiology, 44: 286-290.
52. Qadar, S.A.U., Shireen, E., Iqbal, S. & Anwar, A. 2009. Optimization of protease production from newly isolated strain of Bacillus sp. Indian Journal of Biotechnology, 8: 286-290.
53. Rao, C.S., Sathish, T., Mahalaxmi, M., Laxmi, G.S., Rao, R.S. & Prakasham, R.S. 2007. Modelling and optimization of fermentation factors for enhancement of alkaline protease production by isolated Bacillus circulans using feed-forward neural network and genetic algorithm. Journal of Applied Microbiology, 104: 889-898.
54. Rawlings, N.D., Morton, F.R. & Barrett, A.J. 2007. An introduction to peptidases and the Merops database, Pp. 161-179. In: Polaina, J. & MacCabe, A. (eds) Industrial enzymes structure, function and applications. Springer, The Netherlands, xii + 641 pp.
55. Razak, C.N.A., Tang, S.W., Basri, M. & Salleh, A.B. 1997. Preliminary study on the production of extracellular protease from a newly isolated Bacillus sp. (No.1) and the physical factors affecting its production. Pertanika Journal of Science and Technology, 5(2): 169-177.
56. Reddy, L.V.A., Wee, Y.J., Yun, J.S. & Ryu, H.W. 2008. Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches. Bioresource Technology, 99: 2242-2249.
57. Rosenkvist, H. & Hansen, A. 1995. Contamination profiles and characterization of Bacillus species in wheat bread and raw materials for bread production. International Journal of Food Microbiology, 26: 353-363.
58. Sandhya, C., Nampoothiri, K.M. & Pandey, A. 2005. Microbial proteases, Pp. 165-179. In: Barredo, J.L. (ed) Microbial enzymes and biotransformations, Humana Press, Totowa, xi+319 pp.
59. Schallmey, M., Singh, A. & Ward, O.P. 2004. Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 50: 1-17.
60. Shafee, N., Aris, S.N., Rahman, R.N.Z.A., Basri, M. & Salleh, A.B. 2005. Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacterium Bacillus cereus strain 146. Journal of Applied Science Research, 1(1): 1-8.
61. Simonen, M. & Palva, I. 1993. Protein secretion in Bacillus species. Microbiological Reviews, 57(1): 109-137.
62. Singh, J., Batra, N. & Sobti, R.C. 2001. Serine alkali protease from a newly isolated Bacillus sp. SSR1. Process Biochemistry, 36: 781-785.
63. Suganthi, C., Mageswari, A., Karthikeyan, S., Anbalagan, M., Sivakumar, A. & Gothandam, K.M. 2013. Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments. Journal of Genetic Engineering and Biotechnology, 11: 47-52.
64. Tari, C., Genckal, H. & Tokatlı, F. 2006. Optimization of a growth medium using a statistical approach for the production of an alkaline protease from a newly isolated Bacillus sp. L21. Process Biochemistry, 41: 659-665.
65. Thompson, J.M., Dodd, C.E.R. & Waites, W.M. 1993. Spoilage of bread by Bacillus. International Biodeterioration and Biodegradation, 32: 55-66.
66. Thompson, J.M., Waites, W.M. & Dodd, C.E.R. 1998. Detection of rope spoilage in bread caused by Bacillus species. Journal of Applied Microbiology, 85: 481-486.
67. Tunail, N. 2009. Mikrobiyoloji, 1st edition, Pelin Ofset, Ankara, 448 pp.
68. Uttatree, S. & Charoenpanich, J. 2016. Isolation and characterization of a broad pH- and temperature-active, solvent and surfactant stable protease from a new strain of Bacillus subtilis. Biocatalysis and Agricultural Biotechnology, 8: 32-38.
69. Uttatree, S., Kobtrakool, K., Ketsuk, A., Kaenngam, W., Thakolprajak, P. & Charoenpanich, J. 2017. A novel metal-tolerant, solvent and surfactant stable protease from a new strain of Bacillus megaterium. Biocatalysis and Agricultural Biotechnology, 12: 228-235.
70. van Dijl, J.M. & Hecker, M. 2013. Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microbial Cell Factories, 12(3): 1-6.
71. Vetter, R., Wilke, D., Moeller, B., Mueller, M., Muecke, I., Takenberg, M. & Konieczny-Janda, G. 1995. Alkaline proteases from Bacillus pumilus. US Patent No. 5478742.
72. Volavsek, P.J.A., Kirschner, L.A.M. & von Holy, A. 1992. Accelerated methods to predict the rope-inducing potential of bread raw materials. South African Journal of Science, 88: 99-102.
73. Voysey, P.A. 1989. Rope: a problem for bakers. Journal of Applied Bacteriology, 67: xxv–xxvi.
74. Yang, J.K., Shih, I.L., Tzeng, Y.M. & Wang, S.L. 2000. Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzyme and Microbial Technology, 26: 406-413.