c3518cb17d976b8

کلونینگ و بیان ژن رمزگذار آنزیم تجزیه‌کننده ترکیبات آلی فسفره (opd) در باکتری Escherichia coli

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه گیاهپزشکی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

2 استاد، گروه گیاهپزشکی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

3 پژوهشگر پسا دکتری، گروه زیست شناسی، دانشگاه مک مستر، همیلتون کانادا

چکیده

کاربرد گستردۀ ترکیب‌های آلی فسفرۀ منجر به بروز اثر زیان‌آور زیست‌محیطی بسیاری شده است. استفاده از میکروارگانیسم­ها در پالایش و اندازه‌گیری این ترکیب‌های ناگوار به‌عنوان یک رویکرد دوستدار محیط­زیست و مناسب شناخته شده است. آنزیم ارگانوفسفروس هیدرولاز از آنزیم‌های هیدرولیزکنندۀ فسفوتری­استری است که در برخی میکروارگانیسم­های خاک به‌ویژه Flavobacterium sp.شناسایی شده و قادر به هیدرولیز تعدادی از ترکیب‌های فسفرۀ آلی است. در این پژوهش بهینه‌سازی کدون‌های توالی رمزگذار این پروتئین برای بیان در باکتری Escherichia coli انجام شد و سپس همراه با پروموتر lac، در وکتور پروموتر-پروب pTH1705 به‌صورت ادغام الگوبرداری کلون شد و ترانسفورماسیون‌ در دو سویۀ‌ باکتری E. coli انجام گرفت. محیط کشت معدنی M9 حاوی غلظت 10و 50 میلی­گرم در لیتر دیازینون، برای سنجش عملکرد باکتری تراریخت بیان‌کنندۀ ژن opd (به‌صورت in trans) استفاده شد. بررسی منحنی رشد استرین E. coli V103 نشان داد، رشد باکتری در غلظت 50 میلی­گرم در لیتر دیازینون تا حد زیادی تحت تأثیر (وجود آفت­کش) قرار می­گیرد درحالی‌که در غلظت 10 میلی‌گرم در لیتر دیازینون این تأثیر کمتر است. نتایج حاصل از کروماتوگرافی مایع با کارایی بالا به‌عنوان یک روش استاندارد نشان داد، غلظت دیازینون در محیط کشت باکتری، در چهار استرین V100، V101، V102 و V103 پس از 24 ساعت از 10 میلی­گرم در لیتر به ترتیب به 38/6، 19/7، 09/7 و 74/5 میلی­گرم در لیتر کاهش یافت. این نتایج بیانگر انتقال موفق و نیز بیان ژن مورد نظر در باکتری است و این باکتری نوترکیب مهندسی‌شده توانایی تجزیۀ ترکیب‌های فسفره را دارد.

کلیدواژه‌ها

عنوان مقاله [English]

Cloning and studing the function of opd gene encoding organophosphorus hydrolase in Escherichia coli

نویسندگان [English]

  • Seyedeh Minoo Sajadian 1
  • Vahid Hosseini naveh 2
  • Khalil Talebi 2
  • Maryam Zamani 3
1 Ph. D. Candidate, Department of Plant Protection, University College of Agriculture & Natural Resoucrs, University of Tehran, Karaj, Iran
2 Professor, Department of Plant Protection, University College of Agriculture & Natural Resoucrs, University of Tehran, Karaj, Iran
3 Postdoc Fellowship, Department of Biology, McMaster University, Hamilton, Canada
چکیده [English]

Organophosphorus compounds are widely used, and their presence in different components of the ecosystem has led to harmful effects on the environment. The use of microorganisms in detoxification of xenobiotic compounds and measurement of pesticide residues is considered as an environmental-friendly and appropriate method. Organophosphorus hydrolase (opd), is a phosphotriester hydrolase enzyme which is discovered in some soil microorganisms such as Flavobacterium sp. and has a wide range of substrates and is able to hydrolyze many organophosphorus compounds. In the present study, codon optimization was done to express the protein in Escherichia coli. Following omission of the signal peptide sequence and substitution of serine by methionine as the start codon, this fragment was cloned under the control of lac promoter in a promoter-probe vector (pTH1705) as a transcriptional fusion and was used for transformation in two Escherichia coli strains DH5α and XL1-blue. To measure the performance of the transgenic bacteria expressing opd gene (as in trans), they were cultured in M9 mineral medium containing 10 and 50 mg/L diazinon and appropriate available carbon and nitrogen sources. Studying the growth curve of V103 strain revealed that bacterial growth in the presence of 50 mg/L diazinon had been affected while this effect in the presence of 10 mg/L diazinon was minor. Results obtained from high-performance liquid chromatography as a standard method to compare with the performance of obtained transgenic strain showed that after 24 hours, diazinon concentration (initially 10mg/L ) in the bacterial culture medium of V100, V101, V102 and V103 strains reached to 6.38, 7.19, 7.09 and 5.74, respectively. These results reflected the successful transfer and expression of target gene in the bacterium and this genetically engineered bacterium was able to degrade organophosphorus compounds directly.

کلیدواژه‌ها [English]

  • degradation
  • HPLC
  • organophosphorus compounds
  • transgenic bacterium

مراجع

  1. Baumgartner, P., Raemaekers, R. J., Durieux, A., Gatehouse, A., Davies, H. & Taylor, M. (2002). Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification, 26, 394-405.
  2. Cai, Z., Ma, J., Wang, J., Rong, Y., Chen, J., Li, S., Zhang, W. & Zhao, X. (2016) Aerobic biodegradation kinetics and pathway of the novel cis-nitromethylene neonicotinoid insecticide Paichongding in yellow loam and Huangshi soils. Applied Soil Ecology, 98, 150-158.
  3. Chae, M. Y., Postula, J. F. & Raushel, F. M. (1994) Stereospecific enzymatic hydrolysis of phosphorus–sulfur bonds in chiral organophosphate triesters. Bioorganic and Medicinal Chemistry Letters, 4, 1473-1478.
  4. Chen, H., He, X., Rong, X., Cai, W. C. P., Liang, W., Li, S. & Huang, Q. (2009) Adsorption and biodegradation of carbaryl on montmorillonite, kaolinite and goethite. Applied clay science, 46: 102-108.
  5. Chen, W. & Mulchandani, A. (1998). The use of live biocatalysts for pesticide detoxification. Trends in Biotechnology, 16(2), 71-76.
  6. Chu, X. Y., Wu, N. F., Deng, M. J., Tian, J., Yao, B., Fan, Y. L. (2006) Expression of organophosphorus hydrolase OPHC2 in Pichia pastoris: purification and characterization. Protein Expression and Purification, 49(1), 9-14.
  7. Cowie, A., Cheng, J., Sibley, C. D., Fong, Y., Zaheer, R., Patten, C. L., Morton, R. M., Golding, G. B. & Finan, T. M. (2006) An integrated approach to functional genomics: construction of a novel reporter gene fusion library for Sinorhizobium meliloti. Applied and Environmental Microbiology, 72(11), 7156-7167.
  8. Cullington, J. E. & Walker, A. (1999). Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biology and Biochemistry, 31(5), 677-686.
  9. Daneshvar, N., Aber, S., Khani, A. & Rasoulifard, M. H. (2007) Investigation of adsorption kinetics and isotherms of imidacloprid as a pollutant from aqueous solution by adsorption onto industrial granular activated carbon. Journal of Food, Agriculture and Environment, 5, 425-429.
  10. Diez, M. C. (2010) Biological aspects involved in the degradation of organic pollutants. Journal of Plant Nutrition and Soil Science, 10(3), 244-267.
  11. Donarski, W. J., Dumas, D. P., Heitmeyer, D. P., Lewis, V. E. & Raushel, F. M. (1989) Structure–activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. Biochemistry, 28, 4650-4655.
  12. Ekkhunnatham, A., Jongsareejit, B., Yamkunthong, W. & Wichitwechkarn, J. (2012) Purification and characterization of methyl parathion hydrolase from Burkholderia cepacia capable of degrading organophosphate insecticides. World Journal of Microbiology and Biotechnology, 28(4), 1739-1746.
  13. Ewida, A. Y. (2014) Biodegradation of alachlor and endosulfan using environmental bacterial strains. World Applied Sciences Journal, 32, 540-547.
  14. Harper, L. L., Mcdaniel, C. S., Miller, C. E. & Wild, J. R. (2006). Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC27551) contain identical opd genes. Applied and Environmental Microbiology, 54, 2586-2589.
  15. Hofman, J., Beerendonk, E. F., Flolmer, H. C. & Kruithof, J. C. (1997) Removal of pesticides and other micro pollutants with cellulose-acetate, polyamide and ultra-low pressure reverse osmosis membranes. Desalination, 113, 209-214.
  16. Horne, I., Sutherland, T. D., Harcourt, R. L., Russell, R. J. & Oakeshott, J. G. (2002) Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate. Applied and Environmental Microbiology, 68(7), 3371-3376.
  17. Hsu, T. S. & Bartha, R. (1979) Accelerated mineralization of two organophosphate insecticides in the rhizosphere. Applied and Environmental Microbiology, 37, 36-41.
  18. Hsu, Y. T., Su, C. Y., Du, H. C., Jao, S. C., Li, W. S. (2008) Evaluation of organophosphorus chemicals-degrading enzymes: a comparison of Escherichia coli and human cytosolic aminopeptidase P. Chemistry and Biodiversity, 5, 1401-11.
  19. Hua, W., Bennett, E. R. & Letcher, R. J. (2006) Ozone treatment and the depletion of detectable pharmaceuticals and atrazine herbicide in drinking water sourced from the upper Detroit River, Ontario, Canada. Water Research, 40, 2259-2266.
  20. Jabeen, H., Iqbal, S., Anwar, S. (2014) Biodegradation of chlorpyrifos and 3, 5, 6‐trichloro‐2‐pyridinol by a novel rhizobial strain Mesorhizobium sp. HN3. Water and Environment Journal, 15, 300-306.
  21. Kazemi, M., Tahmasbi, A. M., Valizadeh, R., Naserian, A. A. & Soni, A. (2012) Organophosphate pesticides: a general review. Agricultural Science Research Journal, 2, 512-522.
  22. Keprasertsup, C., Upatham, E. S., Sukhapanth, N. & Prempree, P. (2001) Degradation of methyl parathion in an aqueous medium by soil bacteria. Scienceasia, 27, 261-270.
  23. Kolakowski, J. E., DeFrank, J. J., Harvey, S. P., Szafraniec, L. L., Beaudry, W. T., Lai, K. & Wild, J. R. (1997) Enzymatic hydrolysis of the chemical warfare agent VX and its neurotoxic analogues by organophosphorus hydrolase. Biocatalysis and Biotransformation, 15, 297-312.
  24. Lai, K., Stolowich, N. J. & Wild, J. R. (1995) Characterization of P–S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase. Archives of Biochemistry and Biophysics, 318, 59-64.
  25. Lan, W. S., Gu, J. D., Zhang, J. L., Shen, B. C., Jiang, H., Mulchandani, A., ... & Qiao, C. L. (2006) Coexpression of two detoxifying pesticide-degrading enzymes in a genetically engineered bacterium. International Biodeterioration & Biodegradation, 58(2), 70-76.
  26. Li, Q., Wang, P., Chen, R., Li, W., Wu, Y.J. (2014) Construction of genetically engineered bacteria that degrades organophosphorus pesticide residues and can be easily detected by the fluorescence. Environmental Technology, 35(5), 556-561.
  27. Li, Q. & Wu, Y. J. (2014) A safety type genetically engineered bacterium with red fluorescence which can be used to degrade organophosphorus pesticides. International Journal of Environmental Science and Technology, 11, 891-898.
  28. Mulbry, W. W., Karns, J. S. (1989) Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein. Journal of bacteriology, 171: 6740-6746.
  29. Mulchandani, A. Rajesh. (2011) Microbial biosensors for organophosphate pesticides. Applied Biochemistry and Biotechnology, 165, 687-699.
  30. Mulchandani, A., Kaneva, I. & Chen, W. (1999) Detoxification of organophosphate nerve agents by immobilized Escherichia coli with surface-expressed organophosphorus hydrolase. Biotechnology and Bioengineering, 63(2), 216-223.
  31. Munnecke, D. M. (1980). Enzymatic detoxification of waste organophosphate pesticides. Journal of Agricultural and Food Chemistry, 28, 105-111.
  32. Musa, S., Gichuki, J. W., Raburu, P. O. & Aura, C. M. (2011) Risk assessment for organochlorines and organophosphates pesticide residues in water and sediments from lower Nyando/Sondu-Miriu river within Lake Victoria Basin, Kenya. Lakes and Reservoirs: Reserarch and Management, 16, 273-280.
  33. Rogers, K. R., Wang, Y., Mulchandani, A., Mulchandani, P. & Chen, W. (1999) Organophosphorus hydrolase-based fluorescence assay for organophosphate pesticides. Biotechnology Progress, 15, 517-522.
  34. Rowland, S. S., Speedie, M. K. & Pogell, B. M. (1991) Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans. Applied and Environmental Microbiology, 57(2), 440-444.
  35. Schofield, D. A., Westwater, C., Barth, J. L. & DiNovo, A. A. (2007) Development of a yeast biosensor–biocatalyst for the detection and biodegradation of the organophosphate paraoxon. Applied Microbiology and Biotechnology, 76, 1383-1394.
  36. Serdar, C. M., Gibson, D. T., Munnecke, D. M. & Lancaster, J. H. (1982) Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta. Applied and Environmental Microbiology, 44, 246-249.
  37. Shimazu, M., Mulchandani, A. & Chen, W. (2001) Simultaneous degradation of organophosphorus pesticides and p‐nitrophenol by a genetically engineered Moraxella sp. with surface‐expressed organophosphorus hydrolase. Biotechnology and Bioengineering, 76(4), 318-324.
  38. Singh, B. K. & Walker, A. (2006) Microbial degradation of organophosphorus compounds. FEMS Microbiology Reviews, 30, 428-471.
  39. Somara, S., Manavathi, B., Tebbe, C. C. & Siddavatam, D. (2002) Over-expression of parathion hydrolase of Flavobacterium balustinum in E. coli: Purification and characterization of His-tagged parathion hydrolase. Indian Journal of Biochemistry & Biophysics, 39(2), 82-6.
  40. Stoytcheva M. Enzyme vs. bacterial electrochemical sensors for organophosphorus pesticides quantification, Universidad Autónoma de Baja California, Instituto de Ingeniería México. Source: Intelligent and Biosensors, Book edited by: Vernon S. Somerset, pp. 386, January 2010, InTech, Croatia. Fownloaded from SCIYO.COM.
  41. Takayama, K., Suye, S. I., Kuroda, K., Ueda, M., Kitaguchi, T., Tsuchiyama, K., ... & Mulchandani, A. (2006) Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae. Biotechnology Progress, 22(4), 939-943.
  42. Talebi-jahromi, Kh. (2008). Pesticides toxicology (2nd ed.) University of Tehran press. 492 pp.
  43. Wang, H., Gong, Y., Xie, W., Xiao, W., Wang, J., Zheng, Y., Hu, J. & Liu, Z. (2011) Identification and characterization of a novel thermostable gh-57gene from metagenomic fosmid library of the Juan de Fuca Ridge hydrothermal vent. Applied Biochemistry and Biotechnology, 164, 1323-1338.
  44. Wang, J., Chatrathi, M. P., Mulchandani, A. & Chen, W. (2001) Capillary electrophoresis microchips for separation and detection of organophosphate nerve agents. Analytical Chemistry, 73, 1804-1808.
  45. Wong, D. (2006) The ABCs of Gene Cloning (2nd Edition). New York, NY: Springer. Springer, 196 pp.
  46. Wu, C. F., Cha, H. J., Rao, G., Valdes, J. J. & Bentley, W. E. (2000) A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase. Applied Microbiology and Biotechnology, 54(1), 78-83.
  47. Wu, C. F., Valdes, J. J., Rao, G. & Bentley, W. E. (2001) Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions. Biotechnology and Bioengineering, 75(1), 100-103.
  48. Wu, N. F., Deng, M. J., Liang, G. Y., Chu, X. Y., Yao, B. & Fan, Y. L. (2004) Cloning and expression of ophc2, a new organphosphorus hydrolase gene. Chinese Science Bulletin, 49(12), 1245-1249.
  49. Zertal, A., Jacquet, M., Lavedrine, B. & Sehili, T. (2005) Photodegradation of chlorinated pesticides dispersed on sand. Chemosphere, 58, 1431-1437.
  50. Zhao, J. H. & Zhao, D. G. (2009) Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos. Journal of Zhejiang University Science B, 10(2), 142-146.
  51. Zheng, Y., Long, L., Fan, Y., Gan, J., Fang, J., Jin, W. (2013) A review on the detoxification of organophosphorus compounds by microorganisms. African Journal of Microbiology Research, 7, 2127-2134.
  52. Zhongli, C., Shunpeng, L., Guoping, F. (2001) Isolation of methyl parathion-degrading strain M6 and cloning of the methyl parathion hydrolase gene. Applied and Environmental Microbiology, 67(10), 4922-4925.

فایل ها

سابقه مقاله

  • تاریخ دریافت: 17 اردیبهشت 1396
  • تاریخ بازنگری: 02 مرداد 1396
  • تاریخ پذیرش: 03 بهمن 1396

هم رسانی

ارجاع به این مقاله

آمار