نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی ارشد، گروه بیوتکنولوژی، پژوهشگاه علوم و تکنولوژی پیشرفته و علوم محیطی، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته، کرمان، ایران.

2 دانشیار، گروه زیست شناسی سلولی-مولکولی، دانشکده علوم پایه، دانشگاه کوثر بجنورد، بجنورد، ایران.)

3 استادیار، گروه بیوتکنولوژی، پژوهشگاه علوم و تکنولوژی پیشرفته و علوم محیطی، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته، کرمان، ایران.

4 استادیار، مرکز تحقیقات بیماری‌های غیر واگیر‌دار، دانشگاه علوم پزشکی و خدمات بهداشتی و درمانی بم، بم، ایران

چکیده

مقدمه: پراکسیداز لپیدیوم درابا (LDP) آنزیمی از کلاس III پراکسیداز‌های گیاهی می‌باشد که توالی اسیدآمینه‌ای آن با پراکسیداز ترب کوهی (HRP) بیش از 90 درصد تشابه دارد.

روش‌ها: در این پژوهش، به‌منظور اتصال محکم LDP به چارچوب آلی-فلزی روی (Zn-MOF)، بعد از بیان و تخلیص آنزیم، شرایط تثبیت با استفاده از لینکر گلوتارآلدئید بهینه‌سازی شد و سپس ویژگی‌های فیزیکوشیمیایی، سینتیکی و پایداری آن با آنزیم آزاد مقایسه شدند.

نتایج و بحث: بهترین شرایط تثبیت با بازده‌ 67%، در غلظت‌های؛ Zn-MOF 02/0 گرم، آنزیم mg/ml 75/0 و گلوتارآلدئید 2/1 دسی‌مولار به مدت سه ساعت انکوبه‌شدن بدست آمد. نتایج نشان داد که فعالیت ویژه‌ آنزیم تثبیت شده بیش از دو برابر آنزیم آزاد افزایش یافته است و Km آن به سوبسترای TMB نسبت به آنزیم آزاد، 49 درصد کاهش یافته است. همچنین پایداری سینتیکی آنزیم تثبیت شده در برابر pH و دما نسبت به آنزیم آزاد کاهش یافت

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Covalent Immobilization of recombinant Lepidium draba peroxidase on zinc metal-organic framework using glutaraldehyde

نویسندگان [English]

  • Soudabeh Farhadi 1
  • Ali Riahi-Madvar 2
  • Mojtaba Mortazavi 3
  • Ghasem Sargazi 4

1 Master's student, Department of Biotechnology, Research Institute of Advanced Science and Technology and Environmental Sciences, Post Graduate University of Industrial and Advanced Technology, Kerman, Iran.

2 Associate Professor, Department of Cell-Molecular Biology, Faculty of Basic Sciences, Kausar University, Bojnord, Iran.)

3 Assistant Professor, Department of Biotechnology, Research Institute of Advanced Science and Technology and Environmental Sciences, Postgraduate University of Industrial and Advanced Technology, Kerman, Iran

4 Assistant Professor, Non-Communicable Diseases Research Center, Bam University of Medical Sciences and Health Services, Bam, Iran

چکیده [English]

Introduction: Lepidium draba peroxidase (LDP) belonging to the class III plant peroxidases that its amino acid sequence shows over 90% similarity with horseradish peroxidase (HRP).

Methods: In this study, after expression and purification of LDP, its immobilization conditions were optimized on the Zinc metal-organic framework (Zn-MOF) using glutaraldehyde as a cross-linking agent for firm binding. Then physicochemical properties, kinetic parameters and stability of the immobilized enzyme were compared with the free one.

Results and discussion: The best conditions for enzyme immobilization with 67% yield were optimized at concentration of 0.02 g of Zn-MOF, 0.75mg/ml and 1.2 dM of glutaraldehyde, after 3h incubation. The results showed that the specific activity of the immobilized enzyme increased more than doubled that of free enzyme and its Km was reduced by 49% compared to the free one for TMB substrate. Also its kinetic stability reduced against pH and temperature in compared to the free enzyme.

کلیدواژه‌ها [English]

  • Kinetics
  • Optimization
  • Specific Activity
  • Stability
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