IJE TRANSACTIONS B: Applications Vol. 32, No. 2 (February 2019) 207-216    Article in Press

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H. Heydarzadeh Darzi, S. Gilani, M. Farrokhi, S. M. M. Nouri and G. Karimi
( Received: October 06, 2018 – Accepted in Revised Form: January 03, 2019 )

Abstract    In the present study, textural and structural characterizations of chitosan bead for immobilization of alpha amylase in detail by N2 adsorption–desorption, Microspore Analysis (MP), Barrett–Joyner–Halenda (BJH) plots and Field Emission Scanning Electron Microscope (FESEM) observations were investigated. 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 indicated the porosity of activated chitosan bead was uniformly increased. Intra-particle diffusion model designated that 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 activities. Ca2+ concentration of 1 mM shows an excellent impact on relative activity of amylase on its free and immobilized forms. NaCl experiments indicated that 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 enzym


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



در این تحقیق خواص بافتی و ساختاری دانه های کیتوزان به منظور تثبیت آنزیم آلفا آمیلاز به وسیله ی جذب و واجذب نیتروژن،نمودارهای انالیز میکروحفرات، نمودارهای بارت-جوینر-هالندا و مشاهدات میکروسکوپ الکترونی روبشی نشر میدانی مورد مطالعه قرار گرفت. مشاهدات ساختاری حفرات نشان داد که فعال سازی شیمیایی دانه کیتوزان توسط گلوتارآلدئید می تواند نوع ایزوترم جذب و همچنین شکل حفرات را تغییر دهد. در تایید نتایج تحلیل بافت، توزیع حجمی حفرات به مقدار زیادی در محدوده مزوحفره ها می باشد که همچنین نشان می دهد، 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 ثابت معادله میکائیلیس- منتن (~ 0.4mg / ml) و بیشترین سرعت در معادله میکائیلیس- منتن و (~ 227 U /mg Enzyme) به دست امده است که نشان دهنده تمایل قوی و فعالیت بالای آنزیم تثبیت شده می باشد.


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