پایدارسازی آنزیم‌های صنعتی با استفاده از ترکیبات هتروسیکلیک: بررسی اثر ایندول و ترانس چالکون بر پایداری حرارتی و فرم آمورف آنزیم آلفا آمیلاز باسیلوس آمیلو لیکوئی فاشینس
محورهای موضوعی : بیوشیمیلیلا ادیبی 1 , پریچهر یغمایی 2 , پروانه مقامی 3 , آزاده ابراهیم حبیبی 4 *
1 - گروه زیست‌شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
2 - گروه زیست‌شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران.
3 - گروه زیست‌شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
4 - متابولیسم، پژوهشکده علوم بالینی غدد و متابولیسم، دانشگاه علوم پزشکی تهران، تهران، ایران
کلید واژه: کلیدواژه‌ها: آنزیم‌های صنعتی, ترکیبات هتروسیکلیک, ایندول, ترانس چالکون, پایداری حرارتی, آلفا آمیلاز, باسیلوس آمیلو لیکوئی فاشینس.,
چکیده مقاله :
هدف: پروتئین‌ها می‌توانند با تغییر ساختار طبیعی، به سمت تجمعات آمیلوئیدی یا بدون شکل (آمورف) پیش روند. تجمع‌های بی‌شکل، به خصوص در مورد آنزیم‌های با کاربرد صنعتی یا درمانی، با اهمیت هستند و می‌توانند موجب بروز واکنش‌های نامطلوب در بدن شوند. آنزیم آلفا آمیلاز ازجمله آنزیم‌های صنعتی بوده و در پژوهش حاضر تاثیر ترکیبات ایندولی و ترانس- چالکون بر پایداری و تشکیل تجمعات آمورف آن بررسی شده است.
مواد و روش‌ها: پایداری دمایی آنزیم با بررسی فعالیت باقیمانده آن در طول زمان (20 دقیقه) در 60 درجه سانتیگراد مطالعه شده است. بررسی تجمع آمورف آنزیم با استفاده از اسپکتروفوتومتر و با اندازه‌گیری کدورت محلول به صورت کینتیکی انجام شد. آزمون‌های میزان دآمیداسیون آنزیم و تست کنگو رد به ترتیب جهت بررسی تغییر کووالان و عدم تشکیل تجمع‌های آمیلوئیدی انجام شد. این آزمایش‌ها در غیاب و حضور ترانس چالکون و مشتقات ایندول انجام شدند و وضعیت تجمع آمورف آنزیم با استفاده از میکروسکوپ الکترونی عبوری مطالعه شد.
یافته‌ها: ایندول، که یک ترکیب هتروسیکلیک حاوی نیتروژن است، نسبت به ترانس چالکون که ساختار پیش‌ساز فلاونوئیدهاست، تاثیر بهتری بر پایدارسازی آنزیم و مهار تجمع آن دارد. میزان دآمیداسیون آنزیم در حضور ایندول کمتر از ترانس چالکون بود. بررسی تصاویر میکروسکوپ الکترونی عبوری نیز نشان داد که در حضور ایندول، ذرات تجمع آمورف میزان کمتری دارند.
نتیجه‌گیری: ساختار ایندولی به عنوان کاندیدای مناسبی برای طراحی ترکیبات مهارکننده تجمع آمورف پیشنهاد می‌شود.
Objective: Proteins can undergo structural changes leading to amyloid or amorphous aggregates. Amorphous aggregates are particularly significant for industrial or therapeutic enzymes, as they may trigger adverse reactions in biological systems.
Alpha-amylase, a widely used industrial enzyme, was studied here to evaluate the stabilizing effects of indole and trans-chalcone on its thermal stability and amorphous aggregation.
Materials and Methods: The thermal stability of the enzyme was assessed by measuring its residual activity over 20 minutes at 60°C. Amorphous aggregation
was kinetically monitored via turbidity measurements using a spectrophotometer. Deamidation assays and Congo red tests were performed to evaluate covalent changes and amyloid formation, respectively. These experiments were conducted in the presence and absence of trans-chalcone and indole derivatives. The amorphous aggregation state was further analyzed using transmission electron microscopy (TEM).
Findings: Indole, a nitrogen-containing heterocyclic compound, demonstrated superior stabilization and anti-aggregation effects compared to trans-chalcone, a flavonoid precursor. Enzyme deamidation was significantly lower in the presence of indole. TEM images confirmed reduced amorphous particle aggregation in indole-treated samples.
Conclusion: Indole-based structures are proposed as promising candidates for designing compounds that inhibit amorphous aggregation in industrial enzymes.
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