بهینه‌سازی تولید آنزیم آمیلاز در باکتری مقاوم به سرمای sp. ATR812 Janthinobacterium با استفاده از روشResponse Surface Methodology

نوع مقاله: مقاله پژوهشی

نویسندگان

1 دانش آموخته کارشناسی ارشد، گروه زیست شناسی، دانشکده علوم، دانشگاه فردوسی مشهد، مشهد، ایران

2 استادیار، گروه زیست شناسی، دانشکده علوم، دانشگاه فردوسی مشهد، مشهد، ایران

3 دانشجوی دکترا، گروه بیوتکنولوژی پزشکی، دانشکده پزشکی، دانشگاه علوم پزشکی مشهد، مشهد، ایران

چکیده

آمیلاز (EC 3.2.1.1) یکی از مهم‌ترین آنزیم های صنعتی است که کاربردهای گسترده‌ای در صنایع مختلف دارد. این آنزیم پیوند گلیکوزیدی (4-1) α را در نشاسته هیدرولیز کرده و محصولاتی با وزن مولکولی کمتر مثل گلوکز، مالتوز و مالتوتریوز تولید می‌کند. هدف از انجام این پژوهش بهینه‌سازی شرایط رشد و تولید آنزیم آمیلاز در باکتری مقاوم به سرمای sp. Janthinobacterium می‌باشد. شرایط رشد و تولید آنزیم آمیلاز در مرحله اول با استفاده از روش تک متغیره برای متغیرهای دما،pH ، منبع کربن، منبع نیتروژن، درصد نشاسته و میزان تلقیح بهینه‌سازی شد. در مرحله بعدی به منظور بررسی اثر برهم کنش بین متغیرهای مختلف بهینه متغیرسازی به روش طراحی روی سطح پاسخ (Response Surface Methodology) صورت گرفت. شناسایی مولکولی جدایه با استفاده از ژن S rRNA16 نشان داد که باکتری به جنس Janthinobacterium تعلق دارند. روش تک متغیره نشان داد که متغیرهای دما، pH و درصد نشاسته بیشترین تاثیر را در تولید آنزیم دارا می‌باشند. مقایسه شرایط قبل و بعد از بهینه سازی نیز مشخص می‌سازد که میزان تولید انزیم از 094/0 به 116/0 واحد آنزیمی افزایش می‌یابد. در نتیجه اعمال شرایط بهینه شده با استفاده از روش RSM، ترشح آنزیم به میزان 434/0 واحد آنزیمی افزایش پیدا می‌کند.

کلیدواژه‌ها

عنوان مقاله [English]

Optimization of amylase enzyme production by a psychrothrophic Janthinobacterium sp. ATR812 with Response Surface Methodology

نویسندگان [English]

  • razieh ghazi birjandi 1
  • bahar shahnavaz 2
  • masome bahreini 2
  • maryam mahjoobin tehran 3

1 MSc student, Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

2 Assistant Professor, Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.

3 PHD student,Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Science, Mashhad, Iran

چکیده [English]

Amylases (EC 3.2.1.1) constitute a class of industrial enzymes having great potential for application in industries. Amylase hydrolyses the internal α (1, 4) glycosidase linkages in starch and produces products with lower molecular weight such as glucose, maltose and maltotriose. This research aims the optimization growth and enzyme production in a psychrotolerant bacterium. In the first step, the enzyme production was optimized by one-factor- at- a time method using different variables including temperature, pH, carbon and nitrogen sources, the level of starch, and inoculation rate. In order to examine the interaction between these variables, optimization was performed by response surface method. Molecular identification of strain using 16S rRNA gene showed that bacteria belonged to the genus Janthinobacterium. One-factor-at-a-time method showed that temperature, pH and the level of starch are the most effective factors in enzyme production, the amount of strain production increased to 0.116 U/ml by the mentioned method. The use of response surface methodology increased amylase production up to 0.434 U/ml.

کلیدواژه‌ها [English]

  • Janthinobacterium sp
  • RSM
  • Amylase
  • psychrotolerant bacteria

Abou-elela, G., El-sersy, N. A. and Wefky, S. (2009) Statistical optimization of cold adapted α-amylase production by free and immobilized cells of Nocardiopsis aegyptia. Journal of Applied Science Research 5(3): 286-292.

 Anto, H., Trivedi, U. and Patel, K. (2006) Alpha Amylase Production by Bacillus cereus MTCC 1305 Using Solid-State Fermentation. Food Technology and Biotechnology 44(2): 241–245.

 Banerjee, R. and Bhattacharyya B. (1992) Extracellular alkaline protease of newly isolatedRhizopus oryzae. Biotechnology letters 14(4): 301-304.

 Box, G. E. and Behnken, D. W. (1960) Some new three level designs for the study of quantitative variables. Technometrics 2(4): 455-475.

 Brenfeld, P. (1995) Amylases, alpha and beta. In: Colowick SP, Kaplan ON, editors.Methods in enzymology Academic Press Inc., New York, 149–58.

 Caf, Y., Valipour, E. and Arikan, B. (2014) Isolation and Characterization of Alkalin,Halotolerant, Detergent-Stable and Cold-Adaptive-Amylase from a Novel Isolate Bacillus sp. Calp12-7. International Journal Current Microbiology and Applied Sciences 3(4): 950-960.

 Carrasco, M., Villarreal, P., Barahona, S., Alcaino, J., Cifuentes, V. and Baeza, M. (2016) Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts. BMC Microbiology 16: 21.

 Cavicchioli, R. (2002) Extremophiles and the search for extraterrestrial life. Astrobiology 2 (3) 281-292.

 Cotarleţ, M. (2013) Medium optimization for the production of cold-active beta amylase by psychrotrophic Streptomyces MIUG 4 alga using response surface methodology. Microbiology 82(2): 147-154.

 Cotarlet, M. and Bahrim, G. E. (2011) Optimization of cold-adapted amylases and protease production by psychrotrophic Streptomyces 4 Alga using response surface methodology. Turkish Journal of Biochemistry/Turk Biyokimya Dergisi 36(2):83-92.

 Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J. P., Claverie, P., Collins, T., D’amico, S.,Dumont, J., Garsoux, G. and Georlette, D. (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends in biotechnology 18(3): 103-107.

 Gouda, M. and Elbahloul, Y. (2008) Statistical optimization and partial characterization of amylases produced by Halotolerant Penicillium sp. World Journal of Agricultural Sciences 4(3): 359-368.

 Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K. and Chauhan, B. (2003)Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 38(11):1599-1616.

 Halder, D., Biswas, E. and Basu, M. (2014) Amylase production by Bacillus cereus strain BRSC-S-A26MB under optimal laboratory condition. International Journal of Current. Microbiology and Applied Sciences 3(6):1035-1047.

 Kuddus, M. (2014) Bio-statistical approach for optimization of cold-active α-amylase production by novel psychrotolerant Microbacterium foliorum GA2 in solid state fermentation. Biocatalysis and Agricultural Biotechnology 3: 175-181.

 Kuddus, M., Roohi., A. and Ahmad, I. Z. (2012) Cold-active extracellular α-amylase production from novel bacteria Microbacterium foliorum GA2 and Bacillus cereus GA6 isolated from Gangotri glacier, Western Himalaya. Journal of Genetic Engineering and Biotechnology 10(1): 151-159.

 Liu, J., Zhang, Z., Liu, Z., Zhu, H, Dang, H., Lu J. and Cui Z. (2011) Production of cold-adapted amylase by marine bacterium Wangia sp. C52: optimization,modeling, and partial characterization. Marine Biotechnology 13(5): 837-844.

 Lu, M. S., Fang, Y., Li, H., Liu, H. and WangS. (2010) Isolation of a novel cold-adapted amylase-producing bacterium and study of its enzyme production conditions.Annals of Microbiology 60(3): 557-563.

 Margesin, R. and Feller, G. (2010) Biotechnological applications of psychrophiles. Environmental Technology 31(8-9): 835-844.

 Margesin, R., Zacke, G. and Schinner, F. (2002) Characterization of heterotrophic microorganisms in alpine glacier cryoconite. Arctic, Antarctic, and Alpine Research 34(1): 88-93.

 Morita, Y., Nakamura, T., Hasan, Q., Murakami, Y., Yokoyama, K. and Tamiya, E. (1997) Cold-active enzymes from cold-adapted bacteria. Journal of the American Oil Chemists› Society 74(4): 441-444.

 Ominyi, M. C. (2013) Optimization of α-amylase and glucoamylase production from three fungal strains isolated from Abakaliki, Ebonyi State. European Journal of Experimental Biology 3(4): 26-34.

 Pandey, A., Nigam, P., Soccol, C., Soccol, V., Singh, D. and Mohan, R. (2000)Advances in microbial amylases. Biotechnology and Applied Biochemistry 31:135-152.

Shabbiri, K., Adnan, A., Noor, B. and Jamil, S. (2012) Optimized production, purification and characterization of alpha amylase by Brevibacterium linens DSM 2015, using bio-statistical approach. Annals of Microbiology 62(2): 523-532.

 Smith, M. R. and Zahnley, J. C. (2005) Production of amylase by Arthrobacter psychrolactophilus. Journal of Industrial Microbiology and Biotechnology 32(7):277-283.

Souza, P. M. D. (2010) Application of microbial α-amylase in industry-A review. Bra286 zilian Journal of Microbiology 41(4): 850-861.

 Tanyildizi, M. S., Elibol, M. and Ozer D. (2006) Optimization of growth medium for the production of α‐amylase from Bacillus amyloliquefaciens using response surface methodology. Journal of Chemical Technology and Biotechnology 81(4):618-622.

 Yu, Y., Li, H. R., Zeng, Y. X. and Chen, B. (2011) Bacterial diversity and bioprospecting for cold-active hydrolytic enzymes from culturable bacteria associated with sediment from Nella Fjord, Eastern Antarctica. Marine Drugs 9(2): 184-195.

 Zou, X., Chen, C. F., Hang, H. F., Chu J., Zhuang, Y. P. and Zhang, S. L. (2010)Response surface methodology for optimization of the erythromycin production by fedbatch fermentation using an inexpensive biological nitrogen source. Chemical and Biochemical Engineering Quarterly 24(1): 95-100