ردیابی ژن اندوگلوکاناز در باکتری‌های تجزیه کننده سلولز غربال شده از خاک‌های جنگلی مازندران، ایران

نوع مقاله : مقالات پژوهشی

نویسندگان

1 گروه میکروبیولوژی، واحد فلاورجان، دانشگاه آزاد اسلامی، اصفهان، ایران

2 گروه زیست شناسی، دانشکده علوم، دانشگاه اصفهان، اصفهان، ایران

چکیده

هدف این پژوهش، غربال­گری باکتری­های تجزیه کننده سلولز از خاک مناطق جنگلی مازندران و ردیابی ژن رمز کننده آنزیم اندوگلوکاناز در جدایه واجد بالاترین فعالیت سلولازی بوده است. جدایه­های مولد سلولاز با استفاده از رنگ کنگورد در محیط کشت کربوکسی متیل سلولز انتخاب شدند و میزان فعالیت اندوگلوکانازی آنها با روش سنجش میزان قند احیای آزاد شده با معرف دی نیتروسالیسیلیک اسید اندازه­گیری شد. شناسایی گونه باکتری­ها از طریق تکثیر و توالی یابی 16S rDNA انجام شد. همچنین، تولید آنزیم در جدایه منتخب در شرایط رشدی مختلف مورد ارزیابی قرار گرفت. سپس در بانک ژنی توالی­های ژن اندوگلوکاناز در سویه­های مختلف گونه باکتریایی که واجد بالاترین فعالیت اندوگلوکانازی بود جستجو شد و بر اساس اطلاعات به دست آمده اقدام به طراحی پرایمر جهت تکثیر ژن گردید. توالی قطعه تکثیر شده در بانک ژنی با توالی­های موجود مقایسه شد. سنجش فعالیت آنزیم نشان داد که بالاترین فعالیت اندوگلوکانازی به ترتیب متعلق به جدایه­های باسیلوس سوبتیلیس A2 (U/min.ml 92/1)، باسیلوس سوبتیلیس B2 (U/min.ml 65/1) و باسیلوس سرئوسH3 (U/min.ml 51/1) بود. ارزیابی مولکولی ژن اندوگلوکاناز در باسیلوس سوبتیلیس B2 مشابهت 77 درصدی با ژن اندوگلوکاناز (elgS)  باسیلوس سوبتیلیس زیر گونه سوبتیلیس را نشان داد. همچنین بیشترین تولید اندوگلوکاناز جدایه باسیلوس سوبتیلیس B2 در غلظت 8 گرم در لیتر کربوکسی متیل سلولز، pH برابر با 7 و فقدان نمک کلرید سدیم بود. توالی ژن اندوگلوکاناز این جدایه توانمند به عنوان یک توالی جدید در این مطالعه تکثیر و خالص شد و می­تواند به منظور اهداف کلونینگ مورد استفاده قرار گیرد.

کلیدواژه‌ها


عنوان مقاله [English]

Detection of Endoglucanase Gene in Cellulose Degrading Bacteria Screened from Forest Soils of Mazandaran, Iran

نویسندگان [English]

  • E. Ghadiri 1
  • N.S. Naghavi 1
  • K. Ghaedi 2
1 Department of Microbiology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
2 Department of Biology, Faculty of Sciences, Isfahan University, Isfahan, Iran
چکیده [English]

Introduction: Cellulase enzymes are the second largest group of the enzymes with many industrial applications such as in textile industries, production of detergents, animal and human food processing, paper industries and biofuel production. Many microorganisms are capable for production cellulases, but only a small number of them produce significant amounts of this enzyme. The main sources of cellulases production are microorganisms including fungi and bacteria. Among the cellulose degrading aerobic and anaerobic bacteria, most of the studies have been done on Cellulomonas spp. and Clostridium spp., respectively. Also Bacillus spp. has been used for production of cellulase in a homologous manner. Expression of cellulases in some bacterial genera such as Bacillus, Pseudomonas, Ralstonia and Zymomonas, as well as some yeast species such as Saccharomyces cerevisiae and some fungal genera such as Aspergillus and Trichoderma has been reported too. Low levels of cellulase production has always been a major concern that leads to researches for finding of highly active microorganisms strains and employing biological technologies for identification of their enzyme coding genes suitable for probable transformation other organisms. The purpose of this study was the screening of the cellulose degrading bacteria in Mazandaran forest soils and detection of the enzyme coding gene in the isolate with the highest cellulase activity.
Materials and Methods: In order to isolate cellulase producing bacteria, soil samples were obtained from different regions of Mazandaran province forests including Nanoacla (A), Siaocla (B), Someesara (C), Namakabroud (D), Noor (E), Izadshahr (F), Sisanghan (G) and Sehezar (H) forests. Cellulase producing isolates were selected on carboxymethyl cellulose agar using congo red dye and the amount of their endoglucanase activities was measured by assessment of released glucose using dinitrosalicylic acid reagent. Each micromole of released glucose in 1 ml of enzyme solution per minute was considered as an enzyme activity unit (U/min.ml). Identification of bacterial species was performed by amplification and sequencing of a 1500 bp length fragment in 16S rDNA by using 1492R and 27F universal primers. Enzyme production by the selected isolates was also detected in different growth conditions. In order to investigate the effect of carbon source concentration, the amounts of 2-10 g/L of carboxymethyl cellulose were added to bacterial growth culture media. The effect of growth pH values in the range of 4 to 10 and sodium chloride at concentrations of 0 to 10 g/L were studied on endoglucanase production by the selected isolates in carboxymethyl cellulose media. Then the endoglucanase coding sequences in different strains of the bacterial sp. with the highest endoglucanase activity were investigated in Gen Bank and the primers were designed based on the obtained data for the gene amplification.
Results and Discussion: The results of heterotrophic bacteria counting showed the highest number at station B (Siakla forest). Subsequently, station A (Nanocla forest) and station G (Sisangan forest) had the highest number of heterotrophic bacteria, respectively. From the eight selected forest stations, eight top cellulase producing isolates were selected in carboxy methyl cellulose broth medium. The highest endoglucanase activities were belonged to the isolates A2 (1.92 U/min.ml), B2 (1.65 U/min.ml), and H3 (1.51 U/min.ml), respectively. The amplification of the 16SrRNA gene resulted in the formation of a 1500 bp band after electrophoresis in agarose gel electrophoresis. Sequencing results of the purified PCR products showed that B2 and A2 isolates belonged to Bacillus subtilis with 99% similarity. H3 isolate also belonged to Bacillus cereus with 99% similarity. In other studies in the forest soils of Iran for isolation of cellulase producing bacteria, Bacillus had been one of the most active cellulase enzyme producers. The present study showed that Noor forests are among the best places to isolate bacterial cellulase-producing strains. PCR amplification protocol was designed and the total sequence of endoglucanase with 1072 bp length was amplified. Molecular evaluation of endoglucanase gene in Bacillus subtilis (B2) showed 77% similarity to the endoglucanase gene (elgS) in Bacillus subtilis subsp. subtilis. Since the strain B2 had the highest production of endoglucanase among the isolated bacteria, it was attempted to enhance the production of the enzyme using this strain by changing the growth conditions. The isolate B2 was able to grow at different concentrations of carboxymethyl cellulose as a carbon source, but the highest endoglucanase activity was observed at the concentration of 8 g/L with a significant difference compared to other concentrations. The pH equal to 7 and the absence of sodium chloride salt was also led to significant highest endoglucanase production by this isolate.
Conclusion: The endoglucanase gene obtained in this study was reported for the first time with a new sequence. The enzyme showed more sustainable activity than other aerobic bacterial endoglucanases which had previously been studied. This sequence can be introduced into high expressional bacterial strains and used to produce high amounts of endoglucanase for bio-energy industries applications.

کلیدواژه‌ها [English]

  • Endoglucanase
  • Bacillus subtilis
  • Soil bacteria
  • elgS
1- Assareh R., Zahiri H.S., and Eshghi S. 2014. Isolation and identification of native cellulose-degrading bacteria from soil. Journal of Cell and Molecular Research 27(1): 99-110. (In Persian)
2- Azizi M., and Hemmat J. 2016. Isolation of thermotolerant Isoptericola variabilis IDAH9 and optimization of its exoglucananse activity. Modares Journal of Biotechnology 7(2): 70-80. (In Persian)
3- Belda E., Sekowska A., Le Fèvre F., Morgat A., Mornico D., Ouzounis C., Vallenet D., Medigue C., and Danchin A. 2013. An updated metabolic view of the Bacillus subtilis 168 genome. Microbiology 159(4): 757-70.
4- Brooks G.F., Butel J.S., and Morse S.A. 2010. Jawets Melnick and Adelbergs medical microbiology, McGraw Hill companies, New York.
5- Ho S.H., Li P.J., Liu C.C., and Chang J.S. 2013. Bioprocess development on microalgae-based CO2 fixation and bioethanol production using Scenedesmus obliquus CNW-N. Bioresource Technology 145: 142-149.
6- Liang Y.L., Zhang Z., Wu M., Wu Y., and Feng J.X. 2014. Isolation, screening, and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1. BioMedical Research International 512497.
7- Lynd L.R., Weimer P.J., and VanZyl W.H. 2002. Microbial cellulose utilization: Fundamentals and biotechnology. Microbiology and Molecular Biology Reviews 66: 506-577.
8- Mandel M., and Weber J. 1969. Exoglucanase activity by microorganisms. Advances in Chemistry 95: 391-414.
9- Menendez E., Garcia-Fraile P., and Rivas R. 2015. Biotechnological applications of bacterial cellulases. AIMS Bioengineering 2(3): 163-182.
10- Naghavi N.S., Saffari S., Zia M.A., and Ghalamkari G.R. 2012. Isolation and identification of the fungus Caecomyces from sheep rumen and optimization of its cellulolytic activity. Journal of Veterinary Research 12: 27-34. (In Persian)
11- Pandey S., Kushwah J., Tiwari R., Kumar R., Somvanshi V.S. Nain, L., and Saxena A.K. 2014. Cloning and expression of β-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill. Microbiology Research 169(9-10): 693-698.
12- Past S., Nazemi A., Khataminejad M.R., Mirinargesi M.S., Mousavi S., and Salehi A. 2012. Isolation and molecular identification of cellulase producing Bacillus strains from Mazandaran forests soil. Microbial Biotechnology 4(12): 1-6. (In Persian)
13- Rastogi G., Aditya B., Adhikari A., Bischoff K., Hughes S., Christopher L., and Sani R. 2010. Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresource Technology 101: 8798-8806.
14- Rasul F., Afroz A., Rashid U., Mehmood S., Sughra K., and Zeeshan N. 2015. Screening and characterization of cellulase producing bacteria from soil and waste (molasses) of sugar industry. International Journal of Bioscience 6(3): 230-236.‏
15- Sethi S., Datta A., Gupta B.L., and Gupta S. 2013. Optimization of cellulase production from bacteria isolated from soil. ISRN Biotechnology, 2013:985685.
16- Singhania R.R., Adsul M., Pandey A., and Patel A.K. 2017. Cellulases In: S. Dubey et al. (ed.) Current developments in biotechnology and bioengineering. Elsevier, London.‏
17- Wang CY., Hsieh YR., Ng CC., Chan H., Lin HT., Tzeng WS., and Shyu YT. 2009. Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05. Enzyme and Microbial Technology 44: 373-9.
18- Ye M., Sun L., Yang R., Wang Z., and Qi K. 2017. The optimization of fermentation conditions for producing cellulase of Bacillus amyloliquefaciens and its application to goose feed. Royal Society Open Science 4(10): 171012.
CAPTCHA Image