IMMOBILIZATION OF Bacillus subtilis E6-5 PROTEASE AND COMMERCIAL PROTEASE IN NANOFIBRILS CONTAINING DIFFERENT AMINO ACIDS

Year 2020, , 39 - 45, 15.04.2020

Baran Enes Guler Elif Demirkan , Tuba Sevgi

https://doi.org/10.23902/trkjnat.633788

Abstract

In this study, polyamide 6 polymer surfaces that have a high surface area were produced by electrospinning method with the participation of Glycine, Tyrosine and Glutamic acid amino acids, and lyophilized Bacillus subtilis E6-5 protease and commercial protease enzymes were immobilized on nanofibrils. Enzyme reusability were investigated. The immobilization efficiencies of the enzymes were approximately between 50-55 %. In studies with lyophilized Bacillus protease, glutaraldehyde activated PA6 nanofibrils and glutaraldehyde unactivated PA6 nanofibrils were found to be more immobilized in the presence of Glutamic acid. Although the lyophilized protease enzyme immobilized on non-glutaraldehyde activated and activated surfaces has been used 4 times, the best functional stability has been achieved with 2 times use. In pure PA6/Glutamic acid nanofibrils, the immobilization yield of the two times used enzymes was found to be 38 %. In glutaraldehyde-activated PA6 nanofibrils, the PA6/Glutamic acid nanofibril surfaces were found to have 65 % immobilization yield of the two repetitive used enzymes. The enzyme immobilization efficiency has been doubled by glutaraldehyde activation of the nanofibrils. In studies with commercial protease, the most functional stability was obtained for 3 repeated uses, although the enzyme was used 6 times on the non-glutaraldehyde activated nanofibril surfaces. The most successful immobilization was found in 58 % of PA6 nanofibrils. In glutaraldehyde-activated PA6 nanofibrils, the enzyme was found to be used 6 times, but the functional stability was maintained as much as 4 times of repeated use.

Keywords

immobilization, elektrospinnig, protease, reusability

References

• 1. Al-Zuhair, S., Ling, F.W. & Jun, L.S. 2007. Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Process Biochemistry, 42(6): 951-960.
• 2. Aykut, Y., Sevgi, T. & Demirkan, E. 2017. Cellulose monoacetate/polycaprolactone and cellulose monoacetate/polycaprolactam blended nanofibers for protease immobilization. Journal of Applied Polymer Science, 134(44): 45479.
• 3. Butt, K.Y., Altaf, A., Malana, M.A., Ghori, M.I. & Jamil, A. 2018. Optimal production of proteases from Bacillus subtilis using submerged fermentation. Pakistan Journal of Life and Social Sciences, 16(1):15-19.
• 4. Chae, H.J., In, M.J. & Kim, E.Y. 1998. Optimization of protease immobilization by covalent binding using glutaraldehyde. Applied Biochemistry and Biotechnology, 73(2-3): 195-204.
• 5. Chaplin, M.F. & Bucke, C. 1990. Enzyme technology. CUP Archive.
• 6. Chen, H. & Hsieh, Y.L. 2005. Enzyme immobilization on ultrafine cellulose fibres via poly (acrylic acid) electrolyte grafts. Biotechnology and Bioengineering, 90(4): 405-413.
• 7. Demirkan, E., Avci, T. & Aykut, Y. 2018. Protease immobilization on cellulose monoacetate/chitosan-blended nanofibers. Journal of Industrial Textiles, 47(8): 2092-2111.
• 8. Gupta, R., Beg, Q., & Lorenz, P. 2002. Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59(1): 15-32.
• 9. Habeeb, A.F.S.A. & R. Hiramoto. 1968. Reaction of proteins with glutaraldehyde. Archives of Biochemistry and Biophysics 126:16-26.
• 10. Hwang, S., Lee, K.T., Park, J.W., Min, B.R., Haam, S., Ahn, I.S. & Jung, J.K. 2004. Stability analysis of Bacillus stearothermophilus L1 lipase immobilized on surface-modified silica gels. Biochemical Engineering Journal, 17(2): 85-90.
• 11. Keay, L. & Wildi, B.S. 1970. Proteases of the genus Bacillus. I. Neutral proteases. Biotechnology and Bioengineering, 12(2): 179-212.
• 12. Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y. & Lee, S. 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Applied and Environmental Microbiology, 62(7): 2482-2488.
• 13. Kumar, R. & Vats, R. 2010. Protease production by Bacillus subtilis immobilized on different matrices. New York Science Journal, 3(7): 20-24.
• 14. Kumar, V., Singh, D., Sangwan, P. & Gill, P.K. 2014. Global market scenario of industrial enzymes. Industrial enzymes: Trends, scope and relevance. Nova Science Publishers, New York, 173-196.
• 15. Migneault, I., Dartiguenave, C., Bertrand, M.J. & Waldron, K.C. 2004. Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques, 37(5): 790-802.
• 16. Monsan, P., Puzo, G. & Mazarguil, H. 1975. Étude du mécanisme d’établissement des liaisons glutaraldehyde protéines. Biochimie, 57, 1281-1292.
• 17. Qadar, S.A.U., Shireen, E., Iqbal, S. & Anwar, A. 2009. Optimization of protease production from newly isolated strain of Bacillus sp. PCSIR EA-3. Indian Journal of Biotechnology, 8(3): 286-290.
• 18. Reshmi, R., Sanjay, G. & Sugunan, S. 2006. Enhanced activity and stability of α-amylase immobilized on alumina. Catalysis Communications, 7(7): 460-465.
• 19. Rao, M.B., Tanksale, A.M., Ghatge, M.S. & Deshpande, V.V. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews, 62(3): 597-635.
• 20. Saallah, S., Naim, M.N., Lenggoro, I.W., Mokhtar, M.N., Bakar, N.F.A. & Gen, M. 2016. Immobilisation of cyclodextrin glucanotransferase into polyvinyl alcohol (PVA) nanofibres via electrospinning. Biotechnology Reports, 10, 44-48.
• 21. Sheldon R. & Rantwijk, F. 2004. Biocatalysis for Sustainable Organic Synthesis. Australian Journal of Chemistry, 57, 281-289.
• 22. Sidhu, G.S., Sharma, P., Chakrabarti, T. & Gupta, J. K. 1997. Strain improvement for the production of a thermostable α-amylase. Enzyme and Microbial Technology, 21(7): 525-530.
• 23. Taher, H., Al-Zuhair, S., Al-Marzouqi, A.H., Haik, Y. & Farid, M.M. 2011. A review of enzymatic transesterification of microalgal oil-based biodiesel using supercritical technology. Enzyme Research, 4, 1-25.
• 24. Tanksale, A., Chandra, P.M., Rao, M. & Deshpande, V. 2001. Immobilization of alkaline protease from Conidiobolus macrosporus for reuse and improved thermal stability. Biotechnology Letters, 23(1): 51-54.
• 25. Wang, Z.G., Wan, L.S., Liu, Z.M., Huang, X.J. & Xu, Z.K. 2009. Enzyme immobilization on electrospun polymer nanofibers: an overview. Journal of Molecular Catalysis B: Enzymatic, 56(4): 189-195.
• 26. Zaborsky, O.R. 1973. Immobilized Enzymes. CRC Press, Cleveland, OH.