غربالگری و کلونینگ ژن کد کننده آنزیم دکستراناز از استرپتومایسس¬های خاک جهت استفاده در موارد دندان پزشکی
محورهای موضوعی :
اشکان گودرزی
1
,
کیومرث امینی
2
,
محدثه لاری پور
3
1 - دانشگاه آزاد اسلامی
2 - دانشیار، گروه میکروبیولوژی، دانشکده علوم پایه، واحد ساوه، دانشگاه آزاد اسلامی، ساوه، ایران
3 - .دانشگاه آزاد اسلامی واحد تهران شمال
کلید واژه: واژگان کلیدی: استرپتومایسس خاک, دکستراناز, کلونینگ ,
چکیده مقاله :
مقدمه: دکستراناز یکی از مهم‌ترین آنزیم¬های درمانی و صنعتی است. به دلیل اهمیت و کاربرد گسترده این آنزیم، هدف از این مطالعه تعیین مولکولی ،کلونینگ و کینتیک آنزیم دکستراناز از استرپتومایسس¬های خاک جهت استفاده در دندان پزشکی بود. روش کار: پس از شناسایی و انجام تست¬های بیوشیمیایی و مولکولی، استرپتومایسس¬های خاک جداسازی شدند. از طریق واکنش PCR سویه¬های استرپتومایسس دارای ژن آنزیم دکستراناز مشخص شدند. اسیدیته و دمای بهینه به منظور تولید دکستراناز نیز تعیین گردید. ژن دکستراناز سویه¬های مثبت از طریق وکتور به باکتری میزبان اشرشیا کلی الحاق و از طریق تکنیک TA کلونینگ کلون شد. در پایان از طریق تکنیک Real Time PCR، بیان ژن دکستراناز در اشرشیا کلی XL1blue سنجیده شد. نتایج: از غربالگری نمونه های خاک که بر اساس خصوصیات مورفولوژیک و بیوشیمیایی تعیین هویت شدند، در مجموع 12 ایزوله استرپتومایسس جداسازی شد. از 12 ایزوله تنها 3 ایزوله استرپتومایسس کویلی¬کالر دارای ژن دکستراناز بودند. اسیدیته بهینه دکستراناز 2/5 و دمای بهینه در محدوده 65-55 درجه سانتی¬گراد تعیین شد. صحت کلونینگ از طریق کلنی سلکشن، PCR و توالی¬یابی محصول PCR تایید شد. نتایج Real time PCR نیز بیان ژن دکستراناز را در باکتری اشریشیا کلی XL1blue تایید نمود. نتیجه¬گیری: نتایج این تحقیق نشان داد بررسی استرپتومایسس خاک بدلیل پتانسیل بالا در توليد دکستراناز، این سویه می-تواند زمینه¬ساز مطالعات آینده باشد و می¬توان این ایزوله¬ها را به عنوان کاندیدای مناسبی در طراحی و ساخت سویه¬های تولید کننده¬ی آنزیم معرفی نمود.
Introduction: This study aimed to explore the molecular, cloning, and kinetic properties of the dextranase enzyme produced by Streptomyces isolated from soil, for potential applications in dentistry. Dextranase is an enzyme that breaks down dextran, a polysaccharide, and is important in both therapeutic and industrial fields, including use in blood substitutes, vasodilators, and the sugar industry. Methods: Microbial enzymes, such as those from soil bacteria, are preferred over plant and animal-derived enzymes due to their variety, lower cost, and greater stability. Soil bacteria, in particular, are abundant sources of biologically active compounds with minimal toxicity, making them suitable for human treatment. Results: The study isolated 12 Streptomyces strains from soil samples. After biochemical and molecular analysis, three strains, identified as Streptomyces quilicolor, were found to contain the dextranase gene. The optimal conditions for dextranase production were determined to be a pH of 5.2 and a temperature range of 55-65°C. The dextranase gene was successfully cloned into Escherichia coli using a TA cloning technique, and its expression was confirmed through Real-Time PCR. Conclusion: The results suggest that Streptomyces from soil, with its high potential for dextranase production, could serve as a valuable resource for future industrial and therapeutic applications. The isolated strains are promising candidates for the development of efficient dextranase-producing strains, particularly for use in dental treatments and other clinical fields.
1. Himmel ME, Xu Q, Luo Y, Ding S-y, Lamed R, Bayer EA. Microbial enzyme systems for biomass conversion: emerging paradigms. Biofuels. 2010;1(2):323-41.
2. Bashari M, Eibaid A, Wang J, Tian Y, Xu X, Jin Z. Influence of low ultrasound intensity on the degradation of dextran catalyzed by dextranase. Ultrasonics Sonochemistry. 2013;20(1):155-61.
3. Ren W, Cai R, Yan W, Lyu M, Fang Y, Wang S. Purification and characterization of a biofilm-degradable dextranase from a marine bacterium. Marine drugs. 2018;16(2):51.
4. Shahid F, Aman A, Nawaz MA, Karim A, Ul Qader SA. Chitosan hydrogel microspheres: an effective covalent matrix for crosslinking of soluble dextranase to increase stability and recycling efficiency. Bioprocess and biosystems engineering. 2017;40(3):451-61.
5. Chan YJ, Chong MF, Law CL. An integrated anaerobic–aerobic bioreactor (IAAB) for the treatment of palm oil mill effluent (POME): Start-up and steady state performance. Process Biochemistry. 2012;47(3):485-95.
6. Khalikova E, Susi P, Korpela T. Microbial dextran-hydrolyzing enzymes: fundamentals and applications. Microbiology and molecular biology reviews. 2005;69(2):306-25.
7. Ren W, Wang S, Lü M, Wang X, Fang Y, Jiao Y, et al. Optimization of four types of antimicrobial agents to increase the inhibitory ability of marine Arthrobacter oxydans KQ11 dextranase mouthwash. Chinese Journal of Oceanology and Limnology. 2016;34(2):354-66.
8. Wang D, Lu M, Wang S, Jiao Y, Li W, Zhu Q, et al. Purification and characterization of a novel marine Arthrobacter oxydans KQ11 dextranase. Carbohydrate Polymers. 2014;106:71-6.
9. Walker GV, Heng NC, Carne A, Tagg JR, Wescombe PA. Salivaricin E and abundant dextranase activity may contribute to the anti-cariogenic potential of the probiotic candidate Streptococcus salivarius JH. Microbiology. 2016;162(3):476-86.
10. Qiu Y-x, Mao M-y, Jiang D, Hong X, Yang Y-m, Hu T. Co-operative effect of exogenous dextranase and sodium fluoride on multispecies biofilms. Journal of Dental Sciences. 2016;11(1):41-7.
11. Marotta M, Martino A, De Rosa A, Farina E, Cartenı M, De Rosa M. Degradation of dental plaque glucans and prevention of glucan formation using commercial enzymes. Process Biochemistry. 2002;38(1):101-8.
12. Eggleston G, Monge A. Optimization of sugarcane factory application of commercial dextranases. Process Biochemistry. 2005;40(5):1881-94.
13. Bowler G, Wones S. Application of dextranase in UK sugar beet factories. Zuckerindustrie. 2011;136(12):780-3.
14. Park T-S, Jeong H-J, Ko J-A, Ryu Y-B, Park S-J, Kim D-M, et al. Biochemical characterization of thermophilic dextranase from a thermophilic bacterium, Thermoanaerobacter pseudethanolicus. Journal of microbiology and biotechnology. 2012;22(5):637-41.
15. Bertrand E, Pierre G, Delattre C, Gardarin C, Bridiau N, Maugard T, et al. Dextranase immobilization on epoxy CIM® disk for the production of isomaltooligosaccharides from dextran. Carbohydrate polymers. 2014;111:707-13.
16. Purushe S, Prakash D, Nawani NN, Dhakephalkar P, Kapadnis B. Biocatalytic potential of an alkalophilic and thermophilic dextranase as a remedial measure for dextran removal during sugar manufacture. Bioresource technology. 2012;115:2-7.
17. Goulas AK, Cooper JM, Grandison AS, Rastall RA. Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase. Biotechnology and bioengineering. 2004;88(6):778-87.
18. Majeed A, Grobler SR, Moola MH. The pH of various tooth-whitening products on the South African market: scientific. South African Dental Journal. 2011;66(6):278-81.
19. Wang X, Lu M, Wang S, Fang Y, Wang D, Ren W, et al. The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure. International journal of biological macromolecules. 2014;70:284-91.
20. Dehnad A, Esmaili E, Solouki M. Isolation and molecular identification chitinase-producing Streptomyces strains and examination of their in-vitro antagonistic effects. Biological Journal of Microorganism. 2015;4(15):123-34.
21. Hayakawa M, Nonomura H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomyces. J Ferment Tech. 1987;65:501–9.
22. Murray PR, Rosenthal KS, Pfaller MA. Medical microbiology E-book: Elsevier Health Sciences; 2020.
23. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and environmental microbiology. 2008;74(8):2461-70.
24. Erhardt FA, Stammen S, Jördening H-J. Production, characterization and (co-) immobilization of dextranase from Penicillium aculeatum. Biotechnology letters. 2008;30(6):1069-73.
25. Kang HK, Kim SH, Park JY, Jin XJ, Oh DK, Soo Kang S, et al. Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae. Yeast. 2005;22(15):1239-48.
26. García B, Rodríguez E. Carbon source regulation of a dextranase gene from the filamentous fungus Penicillium minioluteum. Current Genetics. 2000;37(6):396-402.
27. Cai R, Lu M, Fang Y, Jiao Y, Zhu Q, Liu Z, et al. Screening, production, and characterization of dextranase from Catenovulum sp. Annals of microbiology. 2014;64(1):147-55.
28. Crawford B, Kasmidi M, Korompis F, Pollnac RB. Factors influencing progress in establishing community-based marine protected areas in Indonesia. Coastal Management. 2006;34(1):39-64.
29. Kim Y-M, Kim D. Characterization of novel thermostable dextranase from Thermotoga lettingae TMO. Applied microbiology and biotechnology. 2010;85(3):581-7.
30. Li K, Lu H, Hang F, Li S, Liu J. Improved dextranase production by Chaetomium gracile through optimization of carbon source and fermentation parameters. Sugar Tech. 2017;19(4):432-7.
31. Zohra RR, Aman A, Zohra RR, Ansari A, Ghani M, Qader SAU. Dextranase: hyper production of dextran degrading enzyme from newly isolated strain of Bacillus licheniformis. Carbohydrate polymers. 2013;92(2):2149-53.
32. Smith KV, Loughran J, Berry A, Dimitrakopoulos C. Developing scientific literacy in a primary school. International Journal of Science Education. 2012;34(1):127-52.
33. Sufiate BL, Soares FEdF, Moreira SS, Gouveia AdS, Cardoso EF, Braga FR, et al. In vitro and in silico characterization of a novel dextranase from Pochonia chlamydosporia. 3 Biotech. 2018;8(3):1-9.
34. Matt C, Hess T, Benlian A. Digital transformation strategies. Business & information systems engineering. 2015;57(5):339-43.
35. Park TS, Jeong HJ, Ko JA, Ryu YB, Park SJ, Kim D, et al. Biochemical characterization of thermophilic dextranase from a thermophilic bacterium, Thermoanaerobacter pseudethanolicus. J Microbiol Biotechnol. 2012;22(5):637-41.
36. KOENIG D, DAY D. The purification and characterization of a dextranase from Lipomyces starkeyi. European journal of biochemistry. 1989;183(1):161-7.
37. Abdel-Naby MA, Ismail A-MS, Abdel-Fattah AM, Abdel-Fattah AF. Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase. Process Biochemistry. 1999;34(4):391-8
