PRODUCTION OF α -AMYLASE FROM ARCHAEAL SOURCE Azade Attar 1 , G urbet Ejder 1 , Y eliz Başaran-Elalmış 1 , S evil Yücel 1 , M eral Birbir 2 1 - Department of Bioengineering , Yıldız Technical University, Istanbul, Turkey 2 - Department of Biology, Marmara University, Istanbul, Turkey 2011 Biochimie Biochemistry Poster 2
<ul><li>Depending on the phylogenic arguments it has been thought that Archaea may have played a key role in the early history of life (Forterre et al., 1992). </li></ul><ul><li>Archaea can adapt their biological systems to different types of habitats. </li></ul><ul><li>Their molecular adaptations are responsible for the ability to survive and grow in extreme environments (Jacquemet et al., 2009). </li></ul><ul><li>Extremely halophilic archaea are obligate halophiles that require more than 15% salt for optimal growth. </li></ul><ul><li>Intracellular and extracellular proteins from halophilic archaea face very saline conditions which must be stable and functional at those extremely salty conditions (Hutcheon et al., 2005). </li></ul><ul><li>Therefore haloarchaeal proteins are subject of interest that contain specific adaptations to prevent aggregation and loss of activity in such conditions. </li></ul>Poster 2 Fig.1 Haloarcula hispanica grown in liquid culture. 1. INTRODUCTION
<ul><li>Enzymes are protein structured molecules which catalyze biochemical reactions on mild conditions. </li></ul><ul><li>Enzymes that found wide and extensive research topic in medicine, biochemistry and biology have got into daily and economical life. Among these enzymes, α-amylase (EC.18.104.22.168) is a commonly used one in industry. </li></ul><ul><li>Leaning to wide microbial biodiversity of Turkey we investigated archaebacterial isolates which produce carbohydrate hydrolyzing enzymes in extremely salty environments, hence have biotechnological importance. </li></ul><ul><li>In this study, α-amylase enzyme of Haloarcula hispanica 2TK2 strain isolat ed in 2004 from Tuzköy Salt Mine of Turkey was used. </li></ul><ul><li>Enzyme production capacity of Haloarcula hispanica 2TK2 strain was determined and the activity of archaeal α-amylase was characterized. </li></ul>Poster 2 Fig.2 Phylogenetic tree of life. 1. INTRODUCTION
<ul><li>2.1 Microbial production </li></ul><ul><li>Haloarcula hispanica 2TK2 isolate was grown in Brown medium at 40°C. </li></ul><ul><li>Samples used in enzyme assay were taken between 2 nd and 6 th weeks of incubation. </li></ul><ul><li>Cultured cells were centrifuged at 10000 rpm for 10 minutes and the supernatant was used in the enzyme activity assays . </li></ul><ul><li>The effect of NaCl concentration (3 - 5 M) to enzyme activity was investigated at different time intervals in the presence and absence of 20% (w/v) starch during microbial production. </li></ul>Poster 2 2. Materials and Methods Fig.3 Brown medium prepared for Haloarcula hispanica .
<ul><li>2.2 Determination of α -amylase activity </li></ul><ul><li>Enzyme activity w as determined by DNS assay using starch as substrate. </li></ul><ul><li>The effects of buffer and salt concentration to the enzyme activity were investigated for Haloarcula hispanica 2TK2 isolate. </li></ul><ul><li>I nvestigated phosphate buffer concentrations were 0.01, 0.02, 0.05, 0.07 and 0.1 M while NaCl concentrations were 0.001, 0.003, 0.006, 0.009, 0.012 and 0.015M. </li></ul>Poster 2 2. Materials and Methods Fig.4 Picture of reaction tubes taken after α -amylase activity assay.
3.1 Optimum buffer and NaCl concentrations for α -amylase isolate d from Haloarcula hispanica 2TK2 strain Poster 2 3. Results Table 1. The effect of sodium phosphate buffer concentration on α-amylase activity. Table 2. The effect of sodium chloride concentration on α-amylase activity. BUFFER (M) ABS (540 nm) 0,01 0,600 0,02 0,651 0,05 0,270 0,07 0,307* (precipitation) 0,1 0,350* ( precipitation ) 0,110 0,012 NaCl (M) ABS (540 nm) 0,001 0,330 0,003 0,340 0,006 0,437 0,009 0,220 0,015 0,090
3.2 Enzyme activity depending on Haloarcula hispanica 2TK2 i ncubation period Samples used in enzyme assay were taken between 2nd and 6th weeks of incubation. Maximum activity values were obtained in the 5th week of incubation. Poster 2 3. Results Fig.5 Time versus relative activity graph. 3.3 The effect NaCl concentration The effect of NaCl concentration (3 - 5 M) to enzyme activity in the presence and absence of 20% (w/v) starch during microbial production. The highest α -amylase activity was obtained from Haloarcula hispanica 2TK2 culture containing 5 M NaCl with 20% starch. Fig.6 Growth medium variables versus relative activity graph.
Poster 2 4. Conclusion <ul><li>Haloarcula hispanica 2TK2 strain grow in high salt concentrations therefore it was expected that α -amylase obtained from this strain may have high activity in the presence of high salt concentration. When the effect of sodium chloride concentration on α-amylase activity was investigated maximum activity values obtained in 0,006M NaCl containing test tubes (Table 2) which is parallel with the literature. </li></ul><ul><li>The highest enzyme activity obtained from growth media containing 5 M NaCl with starch at the 5 th week of microbial incubation (Fig. 5) . </li></ul><ul><li>Haloarcula hispanica will be a source of a microorganism promising in industry due to its new defined halophilic structure and high thermostability. </li></ul>References Forterre, P., Benachenhou-Lahfa, N., Confalonieri, F., Duguet, M., Elie, C., Labedan, B., ( 1992), “The nature of the last universal ancestor and the root of the tree of life, still open questions”, Biosystems , 28 (1-3): 15-32. Jacquemet, A., Barbeau, J., Lemiègre, L., Benvegnu, T., (2009), “Archaeal tetraether bipolar lipids: Structures, functions and applications”, Biochimie , 91 (6): 711-717. Hutcheon, G.W., Vasisht, N., Bolhuis, A., (2005), “Characterisation of a highly stable α -amylase from the halophilic archaeon Haloarcula hispanica ”, Extremophiles, 9: 487–495. Birbir, M., Ogan, A., Calli, B. ve Mertoğlu, B., (2004), “Enzymatic Characteristics of Extremely Halophilic Archaeal Community in Tuzkoy Salt Mine, Turkey”, World Journal of Microbiology and Biotechnology , 20: 613-621.