مطالعات سینتیکی و پاسخ کمی لیپواکسیژناز در توت¬فرنگی در طول رشد و پس از انبارمانی در برابر اثرات محیطی
محورهای موضوعی : بیوشیمی
شهریار سعیدیان
1
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نبی خلیلی اقدم
2
,
ستار قادری
3
1 - گروه زیست شناسی دانشگاه پیام نور، تهران، ایران،
2 - گروه کشاورزی دانشگاه پیام نور، تهران، ایران،
3 - گروه زیست شناسی، دانشگاه پیام نور، تهران، ایران،
کلید واژه: آنزیم , سوبسترا , لیپواکسیژناز, لینولئیک اسید , مدل ,
چکیده مقاله :
لیپواکسیژناز یکی از آنزیمهای مورد توجه شیمیدانها است. این آنزیم یک دیاکسیژناز بوده و اکسیداسیون اسیدهای چرب غیر اشباع چندگانه را برعهده دارد. این تحقیق با هدف مطالعه اثردما (در 8 سطح)، اسیدهای آلی (در 4 سطح)، نمکهای معدنی (در3 سطح) و تعیین غلظت سوبسترای لینولئیک (در 13 سطح) روی سه نوع توتفرنگی نارس، رسیده و در شرایط انبارمانی انجام شد. آزمایش به صورت فاکتوریل در قالب طرح کاملا تصادفی در سه تکرار انجام شد. نتایج تحقیق نشان داد که بیشترین پاسخ آنزیم به لینولئیک اسید روی هر سه نوع توتفرنگی نارس، رسیده وانبارمانی به ترتیب برابر42/9، 49/9 و10 میلیمولار لینولئیک اسید بود. نتایج اثر دما بر میزان فعالیت آنزیم لیپوکسیژناز نیز نشان داد که برای هر سه نوع توتفرنگی نارس، رسیده و انبارمانی، بیشینه فعالیت آنزیم در دمای 35 درجه سانتیگراد بود. درحالیکه کمینه فعالیت آنزیم در دمای 80 درجه سانتیگراد برای هر سه نوع توتفرنگی اتفاق افتاد. نتایج اثر اسیدهای آلی بر میزان فعالیت آنزیم لیپوکسیژناز نیزحاکی از معنیدار بودن این اثر بود. بیشترین فعالیت آنزیم در حضور ترکیب آلی، توکروفرول در هر سه نوع توتفرنگی و کمترین فعالیت آنزیم درحضور آسکوربیک اسید بود. نتایج همچنین موید اثر معنیدار نمکهای معدنی روی فعالیت لیپواکسیژناز بود. در بین نمکها، بیشترین فعالیت آنزیم در حضور کلرید کلسیم با غلظت1/0 میلی مولار و کمترین در حضور کلرید سدیم با غلظت 1 میلی مولار بود. نتایج بطور کلی بیانگر اثر القائی بالای نمک کلرید کلسیم، دمای 35 درجه و ترکیب توکروفرول با حضور سوبسترای با غلظت 10 میلیمولار لینولئیک اسید بود.
Lipoxygenase is one of the enzymes of interest to chemists. This enzyme is a dioxygenase and is responsible for the oxidation of polyunsaturated fatty acids. With special attention to the functioning of this enzyme and its role, this research is to study the effect of temperature (on 8 levels), organic acids (on 4 levels), inorganic salts (on 3 levels) and determining the concentration of linoleic substrate (on 13 levels) on three types Strawberries were unripe, ripe and stored. The experiment was conducted as a factorial in the form of a completely random design in three replications, and for data analysis, nonlinear regression analysis and interaction effect cutting were used. The results of the research showed that the highest enzyme response to linoleic acid on all three types of unripe, ripe Anbarmani strawberries was 9.42, 9.49 and 10 mM linoleic acid respectively, and based on these results, the level of 10 mM linoleic acid as the level A suitable concentration was considered as the substrate of the enzyme. The results of the effect of temperature on the activity of lipoxygenase enzyme also showed that for all three types of unripe, ripe and stored strawberries, the maximum activity of the enzyme was at 35 degrees Celsius. While the minimum enzyme activity occurred at 80 degrees Celsius for all three types of strawberries. The results of the effect of organic acids on the activity of lipoxygenase enzyme also indicate the significance of this effect. The highest enzyme activity was in the presence of organic compound, tocroferol in all three types of strawberries and the lowest enzyme activity was in the presence of ascorbic acid. Among other two organic acids, benzoic acid had more induction effect on lipoxygenase enzyme than nicotiamine acid.
Introduction:Lipid oxidation is a common metabolic reaction in biological systems and plays an important role in plant growth, development, senescence, and responses to biotic and abiotic stresses. The products of lipid oxidation are collectively known as oxylipins, many of which are formed through enzymatic pathways. In plants, lipoxygenases (LOXs) are key enzymes that catalyze the dioxygenation of polyunsaturated fatty acids such as linoleic and linolenic acids. Although the precise physiological role of plant LOXs is not fully understood, extensive evidence suggests their involvement in stress responses, wound signaling, membrane degradation, and the formation of volatile flavor compounds in fruits and vegetables. Strawberry is a highly perishable fruit in which LOX activity contributes to aroma development as well as quality deterioration during ripening and storage. The present study aimed to characterize crude lipoxygenase activity in strawberry fruit at immature, ripe, and post-storage stages, with emphasis on substrate specificity, kinetic properties, thermal stability, buffer systems, inhibitors, and the effects of metal ions.
Materials and Methods:Strawberry fruits were harvested at immature and ripe stages, and a portion of ripe fruits was stored at room temperature for seven days. Crude LOX was extracted from fruit tissues using phosphate buffer, followed by ammonium sulfate precipitation and dialysis. Enzyme activity was measured spectrophotometrically at 234 nm using linoleic acid as the substrate. The effects of substrate concentration, pH, temperature, buffer type, thermal stability, organic acid inhibitors, and metal ions were evaluated. Kinetic parameters were determined using the Michaelis–Menten model. All experiments were conducted in triplicate and analyzed using ANOVA and Duncan’s multiple range test.
Results and Discussion:LOX activity in all developmental stages showed a biphasic response to increasing linoleic acid concentration, with maximum activity at approximately 10 mM, followed by substrate inhibition at higher concentrations. The optimal temperature for LOX activity was 35 °C in immature, ripe, and stored fruits, while enzyme activity sharply declined above 70 °C, indicating thermal denaturation. Tris-HCl buffer (pH 7.5) provided the highest specific activity. Storage significantly reduced LOX activity, although catalytic efficiency increased due to lower Km values. Ascorbic acid exhibited the strongest inhibitory effect on LOX activity, acting as a non-competitive inhibitor, whereas α-tocopherol showed the weakest inhibition. Among metal ions, Ca²⁺ significantly activated LOX at low concentrations, while Na⁺ and Fe³⁺ strongly inhibited enzyme activity. These results highlight the sensitivity of LOX activity to physiological stage, environmental conditions, and chemical modulators.
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