Three-Phase Partitioning of α-Galactosidase from Aspergillus lentulus: Optimization of System and Characterization of Enzyme

Yıl 2020, Cilt: 48 Sayı: 1, 83 - 98, 17.04.2020

Derya Çamurlu Hasan Bayraktar , Sennur Çalışkan Ataç Uzel , Seçil Önal

https://doi.org/10.15671/hjbc.511731

Cited By: 1

Öz

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^oC’de maksimum aktivite gözlendi. α-Galaktozidaz 25-60°C sıcaklık aralığında ve pH 2.6-5.5 aralığında
oldukça iyi bir kararlılık gösterdi. K_M ve V_max değerleri
sırasıyla 0.365 mM ve 0.093 U olarak belirlendi. Metal iyonları ve şekerler
arasında Na₂CO₃ ve
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. 

Anahtar Kelimeler

α-galactosidase, bioseparation, three-phase partitioning (TPP), enzyme purification

Kaynakça

• [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,Three-phase 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. Ultrasonics-Sonochemistry. 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 protease-resistant 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 -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 three-phase partitioning. Sep. Purif. Technol. 83 (2011) 130-136.
• [30] A., Sharma, M.N. Gupta, Purification of pectinases by three-phase 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.