ارزیابی فعالیت آنزیم‌ تریپسین استخراج شده از ضمائم پیلوریک و روده تاسماهی سیبری (Acipenser baerii) تحت تأثیر برخی عوامل فیزیکی و شیمیایی

نوع مقاله : مقاله پژوهشی

نویسنده

گروه علوم و مهندسی شیلات، دانشکده منابع طبیعی و محیط زیست، دانشگاه ملایر، ملایر، همدان

10.22124/janb.2025.31285.1296

چکیده

در ماهیان دارای معده، پپسین، تریپسین و کیموتریپسین مهمترین آنزیم‌های پروتئازی هستند، جایی­ که تریپسین همراه با دیگر پروتئازها و پپتیدازهای قلیایی، هضم اسیدی پروتئین انجام شده در معده را تکمیل می‌کند. بنابراین، فعالیت آنزیم تریپسین می‌تواند به عنوان یک شاخص ارزشمند برای بررسی وضعیت تغذیه‌ای ماهی تلقی شود. هدف از این مطالعه، بررسی اثر برخی عوامل فیزیکی و شیمیایی بر فعالیت آنزیم تریپسین استخراج شده از زوائد باب‌المعدی و روده تاسماهی سیبری (Acipenser baerii) بود. امعاء و احشای ماهی بعد از انتقال به آزمایشگاه و جداسازی زوائد باب‌المعدی و روده، در بافر mM50 تریس HCl همگن شده و در دمای 4 درجه سانتی‌گراد سانتریفیوژ شد. سپس، مایع رویی به دست آمده برای بررسی فراسنجه‌های فیزیکی و شیمیایی شامل دمای بهینه، پایداری دمایی، pH بهینه، پایداری pH، بازدارنده‌ها و یون‌های فلزی بر فعالیت آنزیم استخراج شده استفاده شد. یافته‌ها نشان داد که دما و pH بهینه فعالیت آنزیم استخراج شده از زوائد باب‌المعدی و روده به ترتیب 55 درجه سانتی گراد و 8 بود و در دمای50-10درجه سانتی‌گراد و pH 11-7 نیز پایدار بود. بازدارنده‌های SBTI، TLCK،PMSF ، TPCK، پپاستاتین A، یدواستیک اسید و EDTA به طور معنی‌دار از فعالیت آنزیم جلوگیری کردند، جایی ­که SBTI و TLCK اثر مهارکنندگی کاملی بر فعالیت آنزیم داشتند (05/0>P). فعالیت آنزیم تریپسین استخراج شده از زوائد باب‌المعدی و روده در حضور یون‌هایCa2+ وMg2+ افزایش معنی‌دار و در حضور یون‌هایCu2+،Ba2+،  Zn2+وAl3+ کاهش معنی‌دار داشت (05/0>P). بر اساس نتایج به دست آمده، فعالیت آنزیم‌ تریپسین استخراج شده از زوائد باب‌المعدی و روده تاسماهی سیبری به طور معنی‌دار تحت تأثیر فراسنجه‌های فیزیکی و شیمیایی مورد مطالعه قرار داشت که ممکن است بر فعالیت بهینه این آنزیم در فیزیولوژی گوارش تاسماهی سیبری مؤثر باشد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Evaluation of the trypsin activity extracted from the pyloric caeca and intestine of Siberian sturgeon (Acipenser baerii) under some physical and chemical factors

نویسنده [English]

  • Abbas Zamani
Department of Fisheries, Faculty of Natural Resources and Environment, Malayer University, Malayer, Hamadan, Iran
چکیده [English]

Introduction: The ability of fish to digest food particles depends on the presence of digestive enzymes in the gastrointestinal tract. In gastric fish species, proteolytic enzymes especially pepsin, trypsin, and chymotrypsin appear to play a very important role in catalyzing hydrolysis process of certain peptide bonds. Trypsin, as a serineprotease, complete the acid predigestion of protein delivered to the stomach in combination with other alkaline proteases and peptidases where specifically cleaves at the carboxylic side of lysine and arginine residues. Therefore, trypsin activity could be a sensitive and reliable indicator of nutritional and physiological status of fish at different stages of growth. The aim of this work was to study the effect of some physicochemical factors on the activity of extracted trypsin from pyloric caeca and intestine of Siberian sturgeon (Acipenser baerii).
Materials and Methods: The collected fish viscera from 10 samples (5 ± 0.4 kg) were transported to the laboratory and after separating the pyloric caeca and intestine, those samples were washed with cold distilled water (4 °C), pooled and then homogenized in 50 mM Tris-HCl buffer. After centrifugation at 4 °C, the resulting supernatant from each sample was collected and finally, the physicochemical factors including optimum temperature, thermostability, optimum pH, pH stability, inhibitors and metal ions were evaluated on the extracted trypsin according to the standard protocols. Data obtained from the experimental assessments was analyses by a One-Way ANOVA and the comparison of means was carried out by Duncan’s multiple range tests with a statistical significance at P<0.05.
Results and Discussion: According to the results, optimum temperature of the trypsin from pyloric caeca and intestine was recorded at 55 °C. Similar optimum temperature was recorded for trypsins in silver mojarra, unicorn leatherjacket, beluga and sevruga. The optimal temperature of trypsin is in the range of 30-60 °C and the differences could be attributed to the temperature of fish habitat or experimental conditions used in assessments. The stability of the trypsin was well preserved at temperatures of up to 50 °C. The results of thermostability were in accordance with those of mandarin fish, mrigal carp, common dolphin fish, beluga and sevruga. In general, thermostability of the trypsin enzyme might vary by some factors such as fish species and experimental conditions. The optimum pH of trypsin from both samples was recorded at 8.0 and the results were similar to data reported from trypsin in other species such as mrigal carp and common dolphinfish. The pH stability of trypsin from both samples was highly preserved at pH values comprised between 7.0 and 11.0. Similar results were reported for trypsins from grey triggerfish, zebra blenny, catfish, common dolphin fish, beluga and sevruga. The high ranges of pH may change the net charge and conformation of an enzyme and inhibit to bind to substrate properly, resulting in the abrupt loss of enzymatic activity. Trypsins are mainly known to be more activity within a range of pH values comprised between 7.5 and 10.5. The inhibitors SBTI, TLCK, PMSF, TPCK, pepstatin A, iodoacetic acid, and EDTA significantly inhibited the enzyme activity, whereas SBTI and TLCK had a completely inhibitory effect on the enzymatic activity (P<0.05). The sensitivity of protease enzymes to various inhibitors is a valuable tool for their proper functional characterization. Our findings were in agreement with data reported in the brownstripe red snapper, silver mojarra, zebra blenny, mrigral carp, Asian seabass, beluga and sevruga. The enzyme activity was significantly increased in the presence of Ca+2 and Mg+2 and decreased by Cu+2, Ba+2, Zn+2, and Al+3 (P<0.05). Our results were in agreement with data reported in mandarin fish, zebra blenny, common dolphin fish, beluga and sevruga. Metal ions can affect enzyme-catalyzed reactions by changing the electron flow in a substrate or enzyme and play a key role in binding to the substrate, depending on the functional groups present in the active site of enzyme.
Conclusion: Based on the obtained results, the extracted trypsin activity from Siberian sturgeon pyloric caeca and intestine was significantly affected by the evaluated physicochemical factors that can be effective on the optimal activity of the enzyme in the digestive physiology of the fish.

کلیدواژه‌ها [English]

  • Intestine
  • Pyloric caeca
  • Siberian sturgeon
  • Trypsin

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