IJE TRANSACTIONS B: Applications Vol. 32, No. 2 (February 2019) 205-211    Article in Press

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H. Heydarzadeh, S. Gilani, M. Farrokhi, Seyed Mohammad Mahdi Nouri and G. Karimi
( Received: October 06, 2018 – Accepted: January 03, 2019 )

Abstract    In the present study, textural and structural characterizations of chitosan bead for immobilization of alpha amylase were studied in detail by N2 adsorption–desorption, MP, BJH plots and FESEM observations. Pore structure observation revealed chemical activation of chitosan bead by glutaraldehyde can change both the isotherm type of adsorption and pores shape. In consistence with textural analysis the high value of pore volume distribution with range of mesopores region indicates the porosity of activated chitosan bead was uniformly increased. Intra-particle diffusion model indicate 97.6% of amylase was adsorbed inside the mesopores of activated chitosan bead owing to increase in kid (rate constant) and reduce of boundary layer effect on diffusion process. In addition, the stability experiments (pH, storage and thermal stability), enzyme leakage, Ca2+ and salt concentration effects were evaluated for immobilized amylase and compared with its free form. Ca2+ concentration of 1 mM shows an excellent impact on relative activity of amylase in its free and immobilized form. NaCl experiments indicate 84% of amylase was covalently immobilized on activated chitosan beads. Further, the Michaelis–Menten kinetic coefficients, Km (~0.4mg/ml) and, Vmax(~227 U/mg Enzyme), point out strong affinity and high activity of immobilized enzyme.


Keywords    Structural characterization; Chitosan bead; Alpha Amylase; Covalent immobilization


چکیده    در این تحقیق خواص بافتی و ساختاری دانه های کیتوزان به منظور تثبیت آنزیم آلفا آمیلاز به وسیله ی جذب و واجذب نیتروژن، MP، BJH و مشاهدات FESEM مورد مطالعه قرار گرفت. مشاهدات ساختاری حفرات نشان داد که فعال سازی شیمیایی دانه کیتوزان توسط گلوتارآلدئید می تواند هم نوع ایزوترم جذب و هم شکل حفرات را تغییر دهد. به طور موافق با تحلیل بافت، میزان بالاي توزیع حجمی حفرات در محدوده mesopores نشان می دهد که تخلخل دانه کیتوزان فعال شده به طور یکنواخت افزایش یافته است. مدل نفوذ درون ذره ای نشان می دهد که به دلیل افزایش kid (ثابت سرعت) و کاهش اثر لایه مرزی بر پدیده نفوذ، 6/97٪ آمیلاز در داخل حفرات دانه فعال شده کیتوزان جذب شده است. همچنین آزمایش های پایداری ( pH، ذخیره سازی و پایداری حرارتی)، نشت آنزیم، اثرات غلظت نمک و یون کلسیم برای آمیلاز تثبیت شده بررسی و با آنزیم آزاد مقایسه گردید. غلظت 1 میلی مولار یون کلسیم تاثیر بسیار خوبی بر فعالیت نسبی آمیلاز در شکل آزاد و تثبیت شده نشان داده است. آزمایشات تاثیر نمک کلرید سدیم نشان می دهد که 84٪ از آمیلاز به صورت کوالانسی بر روی دانه های فعال کیتوزان تثبیت شده است. علاوه بر این ضرایب سینتیکی میکائیلیس- منتنKm (~ 0.4mg / ml) و Vmax (~ 227 U /mg Enzyme)، نشان دهنده تمایل قوی و فعالیت بالای آنزیم تثبیت شده است.

References    1.      Klapiszewski, Ł., Zdarta, J. and Jesionowski, T., "Titania/lignin hybrid materials as a novel support for α-amylase immobilization: A comprehensive study", Colloids and Surfaces B: Biointerfaces,  Vol. 162, No., (2018), 90-97. 2.      Velmurugan, R. and Incharoensakdi, A., "Immobilization of α-amylase on metal nanoparticles mediated by xylan aldehyde improves hydrolysis of glycogen from synechocystis sp. Pcc 6803", Fuel,  Vol. 210, No., (2017), 334-342. 3.      Nwagu, T.N., Okolo, B., Aoyagi, H. and Yoshida, S., "Chemical modification with phthalic anhydride and chitosan: Viable options for the stabilization of raw starch digesting amylase from aspergillus carbonarius", International Journal of biological macromolecules,  Vol. 99, No., (2017), 641-647. 4.      Pandey, G., Munguambe, D.M., Tharmavaram, M., Rawtani, D. and Agrawal, Y., "Halloysite nanotubes-an efficient ‘nano-support’for the immobilization of α-amylase", Applied Clay Science,  Vol. 136, No., (2017), 184-191. 5.      Singh, K., Srivastava, G., Talat, M., Srivastava, O.N. and Kayastha, A.M., "Α-amylase immobilization onto functionalized graphene nanosheets as scaffolds: Its characterization, kinetics and potential applications in starch based industries", Biochemistry and Biophysics Reports,  Vol. 3, No., (2015), 18-25. 6.      Kar, S., Swain, M.R. and Ray, R.C., "Statistical optimization of alpha-amylase production with immobilized cells of streptomyces erumpens mtcc 7317 in luffa cylindrica l. Sponge discs", Applied Biochemistry and Biotechnology,  Vol. 152, No. 2, (2009), 177. 7.      Talekar, S., Joshi, A., Kambale, S., Jadhav, S., Nadar, S. and Ladole, M., "A tri-enzyme magnetic nanobiocatalyst with one pot starch hydrolytic activity", Chemical Engineering Journal,  Vol. 325, No., (2017), 80-90. 8.      Konovalova, V., Guzikevich, K., Burban, A., Kujawski, W., Jarzynka, K. and Kujawa, J., "Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration affinity membrane", Carbohydrate Polymers,  Vol. 152, No., (2016), 710-717. 9.      Božić, N., Ruiz, J., López-Santín, J. and Vujčić, Z., "Production and properties of the highly efficient raw starch digesting α-amylase from a bacillus licheniformis atcc 9945a", Biochemical Engineering Journal,  Vol. 53, No. 2, (2011), 203-209. 10.    Agrawal, M., Pradeep, S., Chandraraj, K. and Gummadi, S.N., "Hydrolysis of starch by amylase from bacillus sp. Kca102: A statistical approach", Process Biochemistry,  Vol. 40, No. 7, (2005), 2499-2507. 11.    Goyal, N., Gupta, J. and Soni, S., "A novel raw starch digesting thermostable α-amylase from bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch", Enzyme and Microbial Technology,  Vol. 37, No. 7, (2005), 723-734. 12.    Nwagu, T.N., Okolo, B., Aoyagi, H. and Yoshida, S., "Improved yield and stability of amylase by multipoint covalent binding on polyglutaraldehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions", Process Biochemistry,  Vol. 48, No. 7, (2013), 1031-1038. 13.    Yang, L., Lei, M., Zhao, M., Yang, H., Zhang, H., Li, Y., Zhang, K. and Lei, Z., "Synthesis of the light/ph responsive polymer for immobilization of α-amylase", Materials Science and Engineering: C,  Vol. 71, No., (2017), 75-83. 14.    Wahba, M.I., "Porous chitosan beads of superior mechanical properties for the covalent immobilization of enzymes", International Journal of Biological Macromolecules,  Vol. 105, No., (2017), 894-904. 15.    Guo, H., Tang, Y., Yu, Y., Xue, L. and Qian, J.-q., "Covalent immobilization of α-amylase on magnetic particles as catalyst for hydrolysis of high-amylose starch", International Journal of Biological Macromolecules,  Vol. 87, No., (2016), 537-544. 16.    Swarnalatha, V., Esther, R.A. and Dhamodharan, R., "Immobilization of α-amylase on gum acacia stabilized magnetite nanoparticles, an easily recoverable and reusable support", Journal of Molecular Catalysis B: Enzymatic,  Vol. 96, No., (2013), 6-13. 17.    Gilani, S.L., Najafpour, G.D., Heydarzadeh, H.D. and Moghadamnia, A., "Enantioselective synthesis of (s)‐naproxen using immobilized lipase on chitosan beads", Chirality,  Vol. 29, No. 6, (2017), 304-314. 18.    Gilani, S.L., Najafpour, G.D., Moghadamnia, A. and Kamaruddin, A.H., "Stability of immobilized porcine pancreas lipase on mesoporous chitosan beads: A comparative study", Journal of Molecular Catalysis B: Enzymatic,  Vol. 133, No., (2016), 144-153. 19.    Gilani, S., Najafpour, G., Moghadamnia, A. and Kamaruddin, A., "Kinetics and isotherm studies of the immobilized lipase on chitosan support", International Journal of Engineering Trans A. 2016b,  Vol. 29, No. 10, (2016), 1402-1414. 20.    Tripathi, P., Kumari, A., Rath, P. and Kayastha, A.M., "Immobilization of α-amylase from mung beans (vigna radiata) on amberlite mb 150 and chitosan beads: A comparative study", Journal of Molecular Catalysis B: Enzymatic,  Vol. 49, No. 1-4, (2007), 69-74. 21.    Gong, W., Ran, Z., Ye, F. and Zhao, G., "Lignin from bamboo shoot shells as an activator and novel immobilizing support for α-amylase", Food Chemistry,  Vol. 228, No., (2017), 455-462. 22.    Donohue, M. and Aranovich, G., "Classification of gibbs adsorption isotherms", Advances in Colloid and Interface Science,  Vol. 76, No., (1998), 137-152. 23.    Yilmaz, E., Can, K., Sezgin, M. and Yilmaz, M., "Immobilization of candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic naproxen methyl ester", Bioresource Technology,  Vol. 102, No. 2, (2011), 499-506. 24.    Akceylan, E., Akoz, E., Sahin, O. and Yilmaz, M., "Enantioselective hydrolysis of (r, s)-naproxen methyl ester using candida rugosa lipase with calix [4] arene derivatives", Journal of Inclusion Phenomena and Macrocyclic Chemistry,  Vol. 81, No. 1-2, (2015), 237-243. 25.    Yilmaz, E., Sezgin, M. and Yilmaz, M., "Enantioselective hydrolysis of rasemic naproxen methyl ester with sol–gel encapsulated lipase in the presence of sporopollenin", Journal of Molecular Catalysis B: Enzymatic,  Vol. 62, No. 2, (2010), 162-168. 26.    Straksys, A., Kochane, T. and Budriene, S., "Catalytic properties of maltogenic α-amylase from bacillus stearothermophilus immobilized onto poly (urethane urea) microparticles", Food Chemistry,  Vol. 211, No., (2016), 294-299. 27.    KHAN, M.J., KHAN, F.H. and HUSAIN, Q., "Application of immobilized ipomoea batata β amylase in the saccharification of starch", Journal of Applied Biological Sciences,  Vol. 5, No. 2, (2011).

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