مطالعۀ آنزیمی و بیوانفورماتیکی باکتری های بومی کوتینازی

نویسندگان

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

چکیده
کوتین پلیمری است که از تراکم و اکسیدشدن اسیدهای چرب در گیاهان به ­وجود آمده و نقش کلیدی در حفاظت از گیاهان در برابر عوامل بیماری­زا ایفا می­ کند. کوتیناز یک آنزیم هیدرولازی است که کوتین را تجزیه می­ کند. هدف این پژوهش استخراج کوتین سیب قرمز، بررسی فعالیت باکتری­ های تولید­کنندۀ آنزیم کوتیناز در محیط LB و تحلیل­ های بیوانفورماتیکی است. بدین منظور، از پوست میوه­ سیب قرمز و به کمک بافر اگزالات، کوتین جداسازی شد. سپس سویه­ های تولیدکنندۀ آنزیم که قبلاً جداسازی شده ­اند، در داخل پلیت­ های حاوی محیط کشت کوتین تلقیح داده شدند. پس از کشت اولیه، باکتری ها را در محیط LB کشت داده و به کمک سوبسترای اختصاصی پارانیتروفنول بوتیرات فعالیت کوتینازی نمونه­ ها سنجیده شد. به منظور انجام تحلیل­ های بیوانفورماتیکی، توالی جداسازی­ شده شش نمونۀ باکتریایی تولیدکنندۀ آنزیم کوتیناز در پایگاه ­های محاسباتی تحلیل شد و درخت فیلوژنتیکی هر توالی تهیه شد. سپس، توالی آنزیمی نمونه­ های تولیدکنندۀ کوتیناز بررسی و با رسم درخت فیلوژنتیکی میزان شباهت این توالی­ ها تحلیل شد. نتایج نشان داد که کوتینازهای پروکاریوتی از یوکاریوتی جدا شده ­اند. در ادامه، توالی ناحیه 16S rDNA این نمونه­ها به همراه توالی نمونه­های­ جدید، ارزیابی شده و روابط فیلوژنتیکی این گونه­ها تعیین شد. این تحلیل نشان داد که توالی جدید در کنار نمونه ­های باکتریایی قرار می ­گیرد؛ بنابراین، احتمالاً آنزیم کوتیناز نمونه ­های شناسایی ­شده، از نظر ساختار و کارکرد با آنزیم کوتیناز این باکتری ها، ممکن است شباهت زیادی داشته باشد.




کلیدواژه‌ها


عنوان مقاله English

An enzymatic and bioinformatics study of native cutinase bacteria

نویسندگان English

Mojtaba Mortazavi
Nasrin Parvaresh
Masoud Torkzadeh
چکیده English

Cutin is a polymer that is constructed in plants by the condensation and oxidation of fatty acids and plays a key role in the protection of plants against pathogens. Cutinase is a hydrolase enzyme that breaks down the cutin. The purpose of this study was to extract cutin from red apples with oxalate buffer, cutinase enzyme activity assay in LB culture, and bioinformatic analysis. To attain these purposes the cutinase-producing strains that had previously been isolated were inoculated in culture medium containing cutin. After initial culture, the bacteria were cultured in LB medium and cutinase activity was measured using the p-Nitrophenyl butyrate. In order to execute bioinformatic analysis, the isolated sequences of six cutinase-producing bacteria were analyzed based on computational data bases and their phylogenetic trees were prepared. Then, the similarities in the sequences of a large number of cutinase-producing samples were analyzed by drawing the phylogenetic tree. The results showed the separation of cutinase-producing prokaryotes from cutinase-producing eukaryotes. Then, the sequence of 16S rDNA of these cutinase-producing samples as well as the samples we had prepared were evaluated and their phylogenetic relationships were determined. This analysis showed that the new sequence stood alongside the bacterial samples. Thus, our cutinases may be similar with these bacterial cutinases in structure and function.






کلیدواژه‌ها English

cutinase enzyme
enterobacter
Klebsiella
phylogenetic tree
SplitsTree4
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  • تاریخ دریافت 18 خرداد 1405
  • تاریخ انتشار 18 خرداد 1405