Hasan BAYRAKTAR, Ataç UZEL, Derya ÇAMURLU, Semanur ÇALIŞKAN ÖZDEMİR, Seçil ÖNAL

α-Galaktozidazin Aspergillus lentulus’dan Üçlü-Faz Ayırımı: Sistemin Optimizasyonu ve Enzimin Karakterizasyonu

Üçlü-faz ayırma (TPP) tekniği α-galaktozidazın Aspergillus lentulus’dan tek adımda kısmi saflaştırılması için ilk kez başarıyla kullanıldı. Yüksek aktivite ve saflaştırma katı elde etmek için enzimin ekstraksiyon etkinliğine amonyum sülfat konsantrasyonu, ekstrakt t-butanol oranı ve pH etkisi araştırıldı. Sistemin optimum saflaştırma parametreleri %55(w/v) amonyum sülfat konsantrasyonu, 1:1 (v/v) ham ekstrakt t-butanol oranı ve pH 5.5 olarak belirlendi. Bu optimize TPP sistemi α-galaktosidaz için 5.3 saflaştırma katı ile %178 aktivite verimi oluşturdu. pH 6.5 ve 50°C’de maksimum aktivite gözlendi. α-Galaktozidaz 25-60°C sıcaklık aralığında ve pH 2.6-5.5 aralığında oldukça iyi bir kararlılık gösterdi. KM ve Vmax değerleri sırasıyla 0.365 mM ve 0.093 U olarak belirlendi. Metal iyonları ve şekerler arasında Na2 CO3ve galaktoz enzim aktivitesi üzerinde kuvvetli inhibitor etkisi gösterdi. TPP ile A. lentulus’dan farklı biyokimyasal özelliklere sahip yeni bir α-galaktozidazın elde edilmesi onun çeşitli biyoteknolojik uygulamaları açısından ilgi çekici olacaktır.

Three-Phase Partitioning OF α-Galactosidase From Aspergillus Lentulus: Optimization of System And Characterization of Enzyme

Three-phase partitioning (TPP) technique is successfully used for the first time for partial purification of α-galactosidasefrom Aspergillus lentulus in single step. The influence of variations in ammonium sulfate concentration, extract tot-butanol ratio and pH on extraction efficiency of the enzyme are investigated to achieve the highest yield and purity fold.Optimal purification parameters of the system are determined as 55% (w/v) ammonium sulfate concentration with 1:1 (v/v)ratio of crude extract to t-butanol at pH 5.5. This optimized TPP system gave 5.3 purification fold with 178% recovery ofα-galactosidase. The maximum activity was registered at pH 6.5 and 50°C. α-Galactosidase showed a good stability withinthe temperature range of 25-60°C and pH range of 2.6-5.5. The KM and Vmax values were determined as 0.365 mM and0.093 U, respectively. Among the metals and sugars Na2CO3 and galactose showed strong inhibitory effect on the activity ofenzyme. The availability of a new α-galactosidase with different biochemical properties from A. lentulus by using TPP maybe of interest for its various biotechnological applications

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1. J.K., Yan, Y.Y., Wang, W.Y., Ma, H., Qiu, Z.B., Wang, J.Y. Wu, Three-phase partitioning as an elegant and versatile platform applied to non-chromatographic bioseparation processes. Crit. Rev. Food Sci. Nutr., 13 (2017) 1-16.
2. C.R., Rachana, J.V. Lyju, Three-phase partitioning-A novel protein purification method. Int. J. Chem Tech Res., 6 (2014) 3467-3472.
3. C., Dennison, R. Lovrien,Three-phase partitioning: Concentration and purification of proteins. Protein Exp. Purif., 11(1997) 149-161.
4. C., Dennison, R.N. Pike, Protein fractionation by three-phase partititioning in aqueous/t-butanol mixtures. Biotechnol. Bioeng., 33 (1989) 221-228.
5. R.E., Lovrein, C., Goldensoph, P., Anderson, B. Odegard,Threephase partitioning (TPP) via butanol: Enzyme separation from crudes, in: R. Burgess (Ed.), Protein purification: Micro to macro, A.R., Liss, Inc., New York, 1987, pp.131-148.
6. R., Dutta, U., Sarkar, A. Mukherjee,Process optimization for the extraction of oil from Crotalaria juncea using three phase partitioning. Ind. Crops Prod., 71 (2015) 89-96.
7. D.C., Panadare, V.K. Rathod, Three phase partitioning for extraction of oil: A review, Trends Food Sci. Technol., 68 (2017) 145-151.
8. J.K., Yan, Y.Y., Wang, W.Y., Qui, Z.B., M., H. Wang, Ultrasound synergized with three-phase partitioning for extraction and separation of Corbicula fluminea polysaccharides and possible relevant mechanisms. Ultrasonics-Sonochemistry, 40 (2018) 128-134.
9. Z.J., Tan, C.Y., Wang, Y.J., Yi, H.Y., Wang, W.L., Zhou, S.Y., Tan, F.F. Li, Three-phase partitioning for simultaneous purification of aloe polysaccharide and protein using a single-step extraction. Process Biochem., 50 (2015) 482-486.
10. V.M., Kulkarni, V.K. Rathod, Extraction of magniferin from Magnifera indica leaves using three phase partitioning coupled with ultrasound. Ind. Crops Prod., 52 (2014) 292- 297.
11. L., Saxena, B.K, Iyer, L. Ananthanarayan, Three phase partitioning as a novel method for purification of ragi(Eleusine coracana) bifunctionalamylase/protease inhibitor. Process Biochem., 42 (2007) 491-495.
12. I., Roy, M.N. Gupta, Three-phase affinity partitioning of proteins. Anal. Biochem. 300 (2002) 11-14.
13. C., Jampani, K.S.M.S. Raghavarao, Differential partitioning for purification of anthocyanins from Brassica oleracea L. Sep. Purif. Technol. 151 (2015) 57-65.
14. D., Panadare, V.K. Rathod, Extraction of peroxidase from bitter gourd (Momordica charantia) by three phase partitioning with dimethyl carbonate (DMC) as organic phase. Process Biochem. 61 (2017) 195-201.15. S.V., Pakhale, S.S. Bhagwat, Purification of serratiopeptidase from Serratio macescens NRRL B 23112 using ultrasound assisted three phase partitioning. UltrasonicsSonochemistry. 31 (2016) 532-538.
16. P.D., Patil, G.D. Yadav, Application of microwave assisted three phase partitioning method for purification of laccase from Trametes hirsuta. Process Biochem., 65 (2018) 220-227.
17. S.T., Sagu, E.J., Nso, T., Homann, C., Kapseu, H.M. Rawel, Extraction and purification of beta-amylase from stems of Abrus precatorius by three phase partitioning. Food Chem., 183 (2015) 144-153.
18. H.S., Choonia, S.S. Lele, Three-phase partitioning of β-galactosidase produced by an indigous Lactobacillus acidophilus isolate. Sep. Purif. Technol. 110 (2013) 144-150.
19. M., Shanmugaprakash, V., Vinothkumar, J., Ragupathy, D.A. Reddy, Biochemical characterization of three phase partitioned naringinase from Aspergillus brasiliensis MTCC 1344. Int. J. Biol.Macromol. 80 (2015) 418-423.
20. L., Morales-Quintana, C., Faundez, R., Herrera, V., Zavaleta, M.C., Ravanal, J., Eyzaguirre, M.A., Moya-Leon, Biochemical and structural characterization of Penicillium purpurogenum α-D-galactosidase: Binding of galactose to an alternative pocket may explain enzyme inhibition. Carbohyd. Res., 448 (2017) 57-66.
21. V.V., Daitx, J., Mezzalira, V., da Costa Moraes, A.C., Breirer, J., Ce, J.C. Coelho, Comparing the alpha-galactosidase A biochemical properties from healthy individuals and Fabry disease patients. Clin. Chim. Acta, 445 (2015) 60-64.
22. C., Wang, H., Wang, R., Ma, P., Shi, C., Niu, H., Luo, P., Yang, B. Yao, Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity. J. Biosci. Bioeng., 121 (2016) 7-12.
23. A., Maruta, M., Yamane, M., Matsubara, S., Suzuki, M., Nakazawa, M., Ueda, T. Sakamoto, A novel α-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain. Enzyme Microb. Technol., 103 (2017) 25-33.
24. Y., Hu, G., Tian, L., Zhao, H., Wang, T.B. Ng, A proteaseresistant α-galactosidase from Pleurotus djamor with broad pH-stability and good hydrolytic activity toward raffinose family oligosaccharides. Int. J. Biol.Macromol. 94 (2017) 122-130.
25. J., Zhou, Q., Lu, R., Zhang, Y., Wang, Q., Wu, J. Li,, X., Tang, B., Xu, J., Ding, Z. Huang, Characterization of two glycoside hydrolyse family 36- α-galactosidases: Novel transglycosylation activity, lead-zinc tolerance, alkaline and multiple pH optima and low-temperature activity. Food Chem. 194 (2016) 156-166.
26. S. Önal, A. Telefoncu, Preparation and properties of α-galactosidase chemically attached to the activated chitin. Art. Cells Blood Subst. Biotechnol. 31 (2003) 339-355.
27. M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Chem. 72 (1976) 248-254.
28. B. Özer, E., Akardere, E.B., Çelem, S. Önal, Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato. Biochem. Eng. J., 50 (2010) 110- 115.
29. A., Şen, M., Eryılmaz, H., Bayraktar, S., Önal, Purification of α-galactosidase from pepino (Solanum muricatum) by threephase partitioning. Sep. Purif. Technol. 83 (2011) 130-136.
30. A., Sharma, M.N. Gupta, Purification of pectinases by threephase partitioning, Biotechnol. Lett. 23 (2001) 1625-1627.
31. A.V., Narayan, M.C., Madhusudhan, K.S.M.S. Raghavarao, Extraction and purification of Ipomoea peroxidase employing three-phase partitioning. Appl. Biochem. Biotechnol. 151 (2008) 263-272.
32. J., Szames, E. Kiss, Three-phase partitioning of crude protein extract. J. Colloid Interface Sci., 170 (1995) 290-292.
33. R., Garg, N. Thorat, Nattokinase purification by three-phase partitioning and impact of t-butanol on freeze drying. Sep. Purif. Technol. 131 (2014) 19-26.
34. C., Dennison, L., Moolman, C.S., Pillay, R.E. Meinesz, t-Butanol: nature’s gift for protein isolation. S. Afr. J. Sci. 96 (2000) 159-160.
35. D., Martinez-Maqueda, B., Hernandez-Ledesma, I., Amigo, B., Mirales, J.A. Gomez-Ruiz, Extraction/fractionation techniques for proteins and peptides and protein digestion. Proteomics in Foods, Springer, US, Boston, MA, 2013, pp. 21-50..
36. G.M., Rather, M.N., Gupta, Three phase partitioning leads to subtle structural changes in proteins. Int. J. Biol. Macromol. 60 (2013) 134-140.
37. C., Wang, H., Wang, R., Ma, P., Shi, C., Niu, H., Luo, P. Yao, Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity. J. Biosci. Bioeng. 121 (2016) 7-12.
38. J.G., Ferreira, A.P., Reis, V.M., Guimaraes, D.L., Falkoski, S. F., Fialho, S.T., Rezende, Purification and characterization of Aspergillus terreus α-galactosidases and their use for hydrolysis of soymilk oligosaccharides. App. Biochem. Biotechnol. 164 (2011) 1111-1125.
39. G.S.,. Anisha, R.P., John, P. Prema, Biochemical and hydrolytic properties of multiple thermostable α-galactosidases from Streptomyces griseoloalbus: Obvious existence of a novel galactose-tolerant enzyme. Process Biochem. 44 (2009) 327-333.
40. L.D., Fialho, V.M., Guimaraes, C.M., Callegari, A.P., Reis, D.S., Barbosa, E.E., de L. Borges, M.A., Moreira, S.T. de Rezende, Characterization and biotechnological application of an acidic α-galactosidase from Tachigali multijuga Benth. Seeds. Phytochem. 69 (2008) 2579-2585.
41. M.M., Gote, M.I., Khan, D.V., Gokhale, K.B., Bastawde, J.M. Khire, Purification, characterization and substrate specificity of thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146). Process Biochem. 41 (2006) 1311-1317.