IMMOBILIZATION OF Β-GALACTOSIDASE FROM HYPOCREA JECORINA ON HYDROXYAPATIDE AND ALUMINA

Yıl 2022, Cilt: 5 Sayı: 2, 99 - 104, 30.06.2022

Fulya Aytaç Türkan , Ayşegül Peksel

https://doi.org/10.26650/JARHS2022-1105097

Öz

Objective: β-galactosidase obtained from the Hypocrea jecorina QM9414 mold fungus was immobilized on alumina and hydroxyapatite (HA) using the adsorption method, and the specific activity and protein values of the immobilized enzyme relative to the free enzyme were measured and evaluated.
Materials and Methods: Partially purified β-galactosidase enzyme obtained from the mold fungus Hypocrea jecorina QM9414 was physically bound to alumina and hydroxyapatite carriers with the adsorption method. For this, filtering, washing, and drying processes were carried out. A UV-spectrophotometer device was used for the measurements. Results: The results of the adsorption on HA showed the efficiency of the immobilized enzyme to be 88% as specific activity unit (U/mg), while it was 18.6% when calculated considering the protein amounts (mg/ml). As the result of the adsorption on alumina, the efficiency of the immobilized enzyme was found to be 36.8% as specific activity unit (U/mg), while it was 22.4% when calculated considering the protein amounts (mg/ml). Conclusion: It has been shown that β-galactosidase can be efficiently immobilized on biocompatible HA via a very simple immobilization protocol, offering a promising strategy to perform repeated enzymatic hydrolysis reactions. The fact that β-galactosidase, which is partially purified from Hypocrea jecorina QM9414 mold fungus, is immobilized on alumina and hydroxyapatite with the adsorption method, makes our study unique as this is the first time it appears in the literature.

Anahtar Kelimeler

β-galactosidase, Immobilization, adsorption, alumina, hydroxyapatite, Hypocrea jecorina

HYPOCREA JECORİNA’DAN ELDE EDİLEN Β-GALAKTOZİDAZ’IN HİDROKSİAPATİT VE ALÜMİNA ÜZERİNE İMMOBİLİZASYONU

Öz

Amaç: Hypocrea jecorina QM9414 küf mantarından elde edilen, kısmi olarak saflaştırılan β-galaktozidaz’ın alümina ve hidroksiapatit (HA) üzerine adsorbsiyon yöntemiyle immobilize edilmesi ve bu şekilde hareketsizleştirilmiş olan enzimin serbest enzime göre spesifik aktivite ve protein değerlerinin ölçülerek değerlendirilmesidir.
Gereç ve Yöntem: Hypocrea jecorina QM9414 küf mantarından elde edilen, kısmi olarak saflaştırılan β-galaktozidaz enzimi alümina ve hidroksiapatit taşıyıcıları üzerine adsorsiyon yöntemiyle fiziksel olarak bağlandı. Bunun için süzme, yıkama ve kurutma işlemleri yapıldı. Ölçümler için UV-spektrofotometre cihazından yararlanıldı.
Bulgular: HA üzerine gerçekleştirilen adsorbsiyon sonucunda immobilize olan enzimin verimi spesifik aktivite birimi (U/mg) açısından %88 bulunurken, protein miktarları (mg/ml) göz önüne alınarak hesaplandığında %18,6 sonucunu göstermiştir. Alümina üzerine gerçekleştirilen adsorbsiyon sonucunda immobilize olan enzimin verimi spesifik aktivite birimi (U/mg) açısından %36,8 bulunurken, protein miktarları (mg/ml) göz önüne alınarak hesaplandığında %22,4 sonucunu göstermiştir.
Sonuç: β-galaktozidazın, çok basit bir immobilizasyon protokolü aracılığıyla biyouyumlu HA üzerinde verimli bir şekilde immobilize edilebileceği, tekrarlanan enzimatik hidroliz reaksiyonlarını gerçekleştirmek için umut verici bir strateji sunduğu gösterildi. Hypocrea jecorina QM9414 küf mantarından kısmi olarak saflaştırılan β-galaktozidaz’ın alümina ve hidroksiapatit üzerine adsorbsiyon yöntemiyle immobilize edilmesi literatürde ilk defa yer alacak olması çalışmamızı özgün kılmaktadır.

Anahtar Kelimeler

β-galaktozidaz, İmmobilizasyon, adsorbsiyon, alümina, hidroksiapatit, Hypocrea jecorina

Kaynakça

• 1. Telefoncu A. Enzimoloji, Yüksek Lisans Yaz Okulu Ders Notları. Kuşadası: Aydın; 1997. google scholar
• 2. Özçömlekçi E. Proteaz Enziminin Glutaraldehit Kullanarak Kovalent Bağlanma ile İmmobilizasyonunda Optimum şartların Belirlenmesi. Yüksek Lisans Tezi. İstanbul: İTÜ Fen Bilimleri Enstitüsü. 2006. google scholar
• 3. Peter J, Yamini S, Sandra V, Wim D, Ludo D, Winnie D. Characterization and Optimization of P-Galactosidase Immobilization Process on A Mixed-matrix Membrane. Enzyme Microb Technol 2011;49(6-7):580-8. google scholar
• 4. Kasavi C. Kovalent Bağlanma ve Fiziksel Adsorpsiyon Metotları ile Proteaz Enziminin İmmobilizasyonu. Yüksek Lisans Tezi. İstanbul: İstanbul Teknik Üniversitesi Fen Bilimleri Enstitüsü. 2006. google scholar
• 5. Lima JS, Boemo APFI, Araujo PHH, Oliveira D. Immobilization of endoglucanase on kaolin by adsorption and covalent bonding. Bioprocess Biosyst Eng 2021;44(8):1627-37. google scholar
• 6. Jia J, Zhang W, Yang Z, Yang X, Wang N, Yu X. Novel magnetic cross-linked cellulase aggregates with a potential application in lignocellulosic biomass bioconversion. Molecules 2017; 22(2):269. google scholar
• 7. Husain Q. Nanomaterials immobilized cellulolytic enzymes and their industrial applications: a literature review. JSM Biochem Mol 2017; 4(3):1029. google scholar
• 8. Califano V, Costantini A. Immobilization of cellulolytic enzymes in mesostructured silica materials. Catalysts 2020;10(6):706. google scholar
• 9. Khoshnevisan K, Vakhshiteh F, Barkhi M, Baharifar H, Poor-Akbar E, Zari N, et al. Immobilization of cellulase enzyme onto magnetic nanoparticles: applications and recent advances. Mol Catal 2017;442:66-73. google scholar
• 10. Pâmela CL, Isadora G, Alexandra MGC ,Daniela B, Darlene C, Deborade O, et al. P—galactosidase from Kluyveromyces lactis in genipin-activated chitosan: An investigation on immobilization, stability, and application in diluted UHT milk. Food Chem 2021;349:129050. doi: 10.1016/j.foodchem.2021.129050. google scholar
• 11. Francesc C, Anna A, Maite C, Purificacian R, Juan R.M. Subjective Perception of Lactose Intolerance Does Not Always İndicate Lactose Malabsorption. Clin Gastroenterel Hepatol 2010;8(7):581-6. google scholar
• 12. Uyanık A. Beta-galaktosidaz Enziminin Mikrobiyal Hücrelerden İzolasyonu. Yüksek Lisans Tezi. Ankara: Ankara Üniversitesi Fen Bilimleri Enstitüsü. 2008. google scholar
• 13. Kermasha S, Eskin MNA. Enzymes: Novel Biotechnological Approaches for the Food Industry, Academic Press, London; 2021. google scholar
• 14. Ureta MM, Martins GN, Figueira O, Pires PF, Castilho PC, Gomez-Zavaglia A. Recent advances in P-galactosidase and fructosyltransferase immobilization technology. Crit Rev Food Sci Nutr 2021;61(16):2659-90. google scholar
• 15. Panesar PS, Kumari S, Panesar R. Potential applications of immobilized P-galactosidase in food processing industries. Enzyme Res 2010;2010:473137. doi:10.4061/2010/473137. google scholar
• 16. Datta S, Christena LR, Rajaram YRS. Enzyme immobilization: an overview on techniques and support material.3 Biotech 2013;3(1):1-9. google scholar
• 17. Jesionowski T, Zdarta J, Krajewska B. Enzyme immobilization by adsorption: a review. Adsorption 2014;20(5-6):801-21. google scholar
• 18. Zdarta J, Meyer AS, Jesionowski T, Pinelo M. A general overview of support materials for enzyme immobilization: characteristics, properties, practical utility. Catalysts 2018;8(2):92. google scholar
• 19. Atyaksheva LF, Dobryakova IV, Pilipenko OS. Adsorption of P-galactosidase on silica and aluminosilicate adsorbents. Russ J Phys Chem A 2015;89(3):497-501. google scholar
• 20. Bulut B. Ticari İnert Cam Katkılı Hidroksiapatit-alümina Ve Hidroksiapatit-zirkonya Kompozitlerinin Üretimi Ve Karakterizasyonu. Yüksek Lisans Tezi. İstanbul: İstanbul Teknik Üniversitesi Fen Bilimleri Enstitüsü.2014. google scholar
• 21. Coutinho TC,Tardioli PW,Farinas CS. Phytase Immobilization on Hydroxyapatite Nanoparticles Improves Its Properties for Use in Animal Feed. Appl Biochem Biotechnol 2020;190(1):270-92. google scholar
• 22. Akça SG. Doğal hidroksiapatit toz üretimi, karakterizasyonu ve antibakteriyel özelliklerinin belirlenmesi. Yüksek Lisans Tezi. Sakarya: Sakarya Üniversitesi Fen Bilimleri Enstitüsü.2016. google scholar
• 23. Martins ML, Pinto TS, Gomes AM, Jr Franchi GC, Zambuzzi W, Rodrigues CG, et al. Immobilization of Paclitaxel on Hydroxyapatite for Breast Cancer Investigations. Langmuir 2020;36(30):8723-32. google scholar
• 24. Denes E, Barriere G, Poli E, Leveque G. Alumina Biocompatibility. J Long Term Eff Med Implants 2018;28(1):9-13. google scholar
• 25. Maryam R, Masoud M, Biocompatibility of alumina-based biomaterials-A review. J Cell Physiol 2019;234(4):3321-35. google scholar
• 26. Neupane S, Patnode K, Li H, Baryeh K, Liu G, Hu J, et al. Enhancing Enzyme Immobilization on Carbon Nanotubes via Metal-Organic Frameworks for Large-Substrate Biocatalysis. ACS Appl Mater Interfaces 2019;27;11(12):12133-41. google scholar
• 27. Souza Lima J, Boemo APSI, Araûjo PHH , Oliveira D. Immobilization of endoglucanase on kaolin by adsorption and covalent bonding. Bioprocess Biosyst Eng 2021;44(8):1627-37. google scholar
• 28. Kujawa J, Gtodek M, Koter I, Osmiatowski B, Knozowska K, Al-Gharabli S, et al. Molecular Decoration of Ceramic Supports for Highly Effective Enzyme Immobilization-Material Approach. Materials (Basel) 2021;3;14(1):201. google scholar
• 29. Li S, Zhong L, Wang H, Li J, Cheng H, Ma Q. Process optimization of polyphenol oxidase immobilization: Isotherm, kinetic, thermodynamic and removal of phenolic compounds. Int J Biol Macromol 2021;185:792-803. google scholar
• 30. Zhong L, Li J, Tian D, Cai J, Wang H, Ma Q. Immobilization of polyphenol oxidase on chitosan/organic rectorite composites for phenolic compounds removal. Water Sci Technol 2021;83(4):906-21. google scholar
• 31. Coutinho CT, Tardioli PW, Farinas CS. Hydroxyapatite nanoparticles modified with metal ions for xylanase immobilization. Int J Biol Macromol 2020;150:344-53. google scholar
• 32. Saire-Saire S, Garcia-Segura S, Luyo C, Andrade LH, Alarcon H. Magnetic bio-nanocomposite catalysts of CoFe2O4/hydroxyapatite-lipase for enantioselective synthesis provide a framework for enzyme recovery and reuse. Int J Biol Macromol 2020;148:284-91. google scholar