Characterization of Immobilized β-Amylase Enzyme Isolated from Sweet Potato and prepared by Entrapment Method
DOI:
https://doi.org/10.37285/ijpsn.2022.15.6.2Abstract
Aim: This study attempted to isolate β-amylase from sweet potato and enzyme immobilized by encapsulation method, and characterized with various parameters.
Methods: The enzyme β-amylase was isolated with phosphate-buffered saline and purified by centrifugation with ammonium sulfate. The purified enzyme was immobilized on chitosan (0.25 g) and sodium alginate (0.25 g) polymers by entrapment method in the presence of calcium chloride (0.5 M). The immobilized enzyme was characterized by a starch hydrolysis test, the optimal pH and temperature were studied and the stability of the immobilized enzyme was also determined. SEM analysis was performed and Vm and Km were also found.
Results: The starch hydrolysis test showed positive results on the starch agar plates for immobilized enzymes. The thermal inactivation showed a severe loss in the activity of the free enzymes (49.3 %) while the temperature profile of the immobilized enzymes was much broader (84.55 %) at higher temperatures (80° C). The optimal pH and stability indicated that the immobilized enzyme has higher stability in the pH range of 5-8. The Km and Vmax value of free and immobilized enzyme was 7.67 mmol, 21.15 µmol (R2 0.8880), and 4.72 mmol,16.79 µmol (R2 0.8446) respectively. The storage of free and immobilized enzymes for one month showed that 83.5 % and 40 % of free enzymes and 11.6 % and 8.6 % of immobilized enzymes lost activity at 25° C and 4° C, respectively. SEM analysis shows the smooth, porous surface.
Conclusion: Immobilized enzymes (natural polymers) exhibit higher thermal stability the optimal pH and stability indicate immobilized enzyme has higher stability in the pH range of 5-8, and achieves a relative activity of 69.7 %. After 6 uses, the reuse efficiency of the immobilized enzyme decreased from 99.8 % to 52.3 %. The storage of the immobilized enzyme showed much higher stability than the found-free enzyme.
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Keywords:
Beta-amylase, Immobilization, Enzymes, Alginate, Entrapment method
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Aksoy S, Tumturk H, Hasirci NE. Stability of α-amylase immobilized on poly (methyl methacrylate-acrylic acid) microspheres. Journal of Biotechnology. 1998 Feb 5;60(1-2):37-46.
Bharadwaj PS. Extraction and purification of peroxidase enzyme from sweet potato. Pharm. Innov. J. 2019;8:418-22.
Bornscheuer UT. Immobilizing enzymes: how to create more suitable biocatalysts. Angewandte Chemie International Edition. 2003 Jul 28;42(29):3336-7.
Brena B, González-Pombo P, Batista-Viera F. Immobilization of enzymes: a literature survey. Immobilization of enzymes and cells. 2013:15-31.
Cao L. Introduction: Immobilized enzymes: Past, present and prospects. Carrier-bound immobilized enzymes: principles, application and design. 2005;1:1-52.
Chalse MN, Suryawanshi DK, Kharat RB, Mundhe TS, Shinde PP, Jogdand KB, Rathod RV. Extraction and partial purification and kinetic study of beta-amylase from sweet potato. J. Glob. Biosci. 2016;5(4):3892-901.
Chui WK, Wan LS. Prolonged retention of cross-linked trypsin in calcium alginate microspheres. Journal of microencapsulation. 1997 Jan 1;14(1):51-61.
Datta S, Christena LR, Rajaram YR. Enzyme immobilization: an overview on techniques and support materials. 3 Biotech. 2013 Feb;3(1):1-9.
Demirkan E, Dincbas S, Sevinc N, Ertan F. Immobilization of B. amyloliquefaciens α-amylase and comparison of some of its enzymatic properties with the free form. Romanian Biotechnological Letters. 2011 Nov 1;16(6):6690-701.
Eisenmesser EZ, Bosco DA, Akke M, Kern D. Enzyme dynamics during catalysis. Science. 2002 Feb 22;295(5559):1520-3.
Eisenmesser EZ, Millet O, Labeikovsky W, Korzhnev DM, Wolf-Watz M, Bosco DA, Skalicky JJ, Kay LE, Kern D. Intrinsic dynamics of an enzyme underlies catalysis. Nature. 2005 Nov;438(7064):117-21.
Elçin YM. Encapsulation of urease enzyme in xanthan-alginate spheres. Biomaterials. 1995 Oct 1;16(15):1157-61.
Eş I, Vieira JD, Amaral AC. Principles, techniques, and applications of biocatalyst immobilization for industrial application. Applied microbiology and biotechnology. 2015 Mar;99(5):2065-82.
Gawande PV, Kamat MY. Preparation, characterization and application of Aspergillus sp. xylanase immobilized on Eudragit S-100. Journal of Biotechnology. 1998 Dec 11;66(2-3):165-75.
Geethanjali S, Subash A. Optimization and immobilization of purified Labeo rohita visceral protease by entrapment method. Enzyme research. 2013;2013.
Gómez L, Ramírez HL, Villalonga ML, Hernández J, Villalonga R. Immobilization of chitosan-modified invertase on alginate-coated chitin support via polyelectrolyte complex formation. Enzyme and Microbial Technology. 2006 Jan 3;38(1-2):22-7.
Hakkoymaz O, Mazi H. An immobilized invertase enzyme for the selective determination of sucrose in fruit juices. Analytical biochemistry. 2020 Dec 15;611:114000.
Hassan ME, Yang Q, Xiao Z, Liu L, Wang N, Cui X, Yang L. Impact of immobilization technology in industrial and pharmaceutical applications. 3 Biotech. 2019 Dec;9(12):1-6.
Homaei AA, Sariri R, Vianello F, Stevanato R. Enzyme immobilization: an update. Journal of chemical biology. 2013 Oct;6(4):185-205.
Kainuma K, French D. Action of pancreatic alpha-amylase and sweet potato beta-amylase on 62-and 63-α-glucosylmalto-oligosaccharides. FEBS letters. 1970 Feb 16;6(3):182-6.
Krajewska B. Application of chitin-and chitosan-based materials for enzyme immobilizations: a review. Enzyme and microbial technology. 2004 Aug 5;35(2-3):126-39.
Li X. The use of chitosan to increase the stability of calcium alginate beads with entrapped yeast cells. Biotechnology and applied biochemistry. 1996 Jun 1;23(3):269-72.
Mahajan R, Gupta VK, Sharma J. Comparison and suitability of gel matrix for entrapping higher content of enzymes for commercial applications. Indian journal of pharmaceutical sciences. 2010 Mar;72(2):223.
Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology & Biotechnological Equipment. 2015 Mar 4;29(2):205-20.
Mogharabi M, Nassiri-Koopaei N, Bozorgi-Koushalshahi M, Nafissi-Varcheh N, Bagherzadeh G, Faramarzi MA. Immobilization of laccase in alginate-gelatin mixed gel and decolorization of synthetic dyes. Bioinorganic Chemistry and Applications. 2012 Jul 31;2012.
Mudugamuwa Arachchige MP, Mu T, and Ma M. Effect of high hydrostatic pressure- assisted pectinase modification on the Pb2+ adsorption capacity of pectin isolated from sweet potato residue. Chemosphere 2021 Jan 1;262:128102.
Murray RK, Granner DK, Mayes PA, Rodwell VW. Harper’s illustrated biochemistry, 2018, 28 th ed. McGraw-Hill Education New York.
Nascimento RP, Coelho RR, Marques S, Alves L, Gırio FM, Bon EP, Amaral-Collaço MT. Production and partial characterisation of xylanase from Streptomyces sp. strain AMT-3 isolated from Brazilian cerrado soil. Enzyme and Microbial Technology. 2002 Sep 2;31(4):549-55.
Nelson DL, Cox MM. Principles of bioenergetics. Lehninger, Principles of Biochemistry (4th ed., pp. 489-520). New York, USA: WH Freeman and company. 2004.
Pencreac'h G, Leullier M, Baratti JC. Properties of free and immobilized lipase from Pseudomonas cepacia. Biotechnology and bioengineering. 1997 Oct 20;56(2):181-9.
Raghu HS, Rajeshwara NA. Immobilization of [alpha]-Amylase (1, 4-[alpha]-D-Glucanglucano-hydralase) by calcium alginate encapsulation. International Food Research Journal. 2015 Mar 1;22(2):869.
Saleem M, Rashid MH, Jabbar A, Perveen R, Khalid AM, Rajoka MI. Kinetic and thermodynamic properties of an immobilized endoglucanase from Arachniotus citrinus. Process Biochemistry. 2005 Feb 1;40(2):849-55.
Sirisha VL, Jain A, Jain A. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Adv Food Nutr Res. 2016;79:179-211.
Suribabu K, Hemalatha KP. Effect of Inhibitors on partially purified alpha-amylase produced by Brevibacillus borstelensis R1. History. 2014;12(48):63-7.
Thakur K, Attri C, Seth A. Nanocarriers-based immobilization of enzymes for industrial application. 3 Biotech. 2021 Oct;11(10):1-2.
Tousignant A, Pelletier JN. Protein motions promote catalysis. Chemistry & biology. 2004 Aug 1;11(8):1037-42.
Warshel A, Sharma PK, Kato M, Xiang Y, Liu H and Olsson MH. Electrostatic basis for enzyme catalysis. Chemical reviews 2006 (106)8: 3210-3235.
Won K, Kim S, Kim KJ, Park HW, Moon SJ. Optimization of lipase entrapment in Ca-alginate gel beads. Process biochemistry. 2005 May 1;40(6):2149-54.
Yang C, Chen SJ, Chen BW, Zhang KW, Zhang JJ, Xiao R, Li PG. Gene Expression Profile of the Human Colorectal Carcinoma LoVo Cells Treated With Sporamin and Thapsigargin. Frontiers in oncology. 2021 May 25;11:621462.
Yang G, Wu J, Xu G, Yang L. Improvement of catalytic properties of lipase from Arthrobacter sp. by encapsulation in hydrophobic sol–gel materials. Bioresource Technology. 2009 Oct 1;100(19):4311-6.
Yang LW, Bahar I. Coupling between catalytic site and collective dynamics: a requirement for mechanochemical activity of enzymes. Structure. 2005 Jun 1;13(6):893-904.
Yang YM, Wang JW, Tan RX. Immobilization of glucose oxidase on chitosan–SiO2 gel. Enzyme and Microbial Technology. 2004 Feb 5;34(2):126-31.
Yoshida M, Oishi K, Kimura T, Ogata M, Nakakuki T. Continuous production of maltose using a dual immobilized enzyme system. Agricultural and biological chemistry. 1989 Dec 1;53(12):3139-42.
Zhao T, Xiong J, Chen W, Xu A, Zhu D, Liu J. Purification and Characterization of a Novel Fibrinolytic Enzyme from Cipangopaludina Cahayensis. Iranian Journal of Biotechnology. 2021 Jan;19(1):e2805.
