1. ORGANIC SOLVENT TOLERANT Β-AMYLASE FROM LACTABACILLUS FERMENTUM Samet KOCABAY1, Serap ÇETINKAYA2, Birnur AKKAYA2, Ali Fazıl YENIDUNYA2
1 Inonu University, Faculty of Science and Litrature, Department of Molecular Biology and Genetic, Malatya, Turkey 2 Cumhuriyet University, Faculty of Science, Deparment of Molecular Biology and Genetic, Sivas, Turkey .
Introduction
For a long time, the role of enzymes have been indispensible for many factories from food industries to drug. Their using derive from ancient Greek that used to make bread, beer, cheese… Many of bacterial enzymes are commercially present. Amylase and proteaz account for 85% enzyme sales in world[1]. Β-amylase hydrolyses the starch as a substrate and occurs the maltose end of the reactions. The maltose are often used in food industries such as sweetening power, baking goods, caramel like-taste[2].
Currently, our better knowledge and biotechnological developments in purification and isolation of enzymes have contributed to increasing of their application manyfold [3]. Although enzymes are used in many industries, there is a major problem that is enzyme instability . Many scientists try to increase enzyme stability. Enzyme immobilization, organic solvent using, manipulation of amino acid sequencing, for example are some strategies for the enzyme stabilities. However organic solvents often inactive the enzyme activities. So scientists devoted to handle this problems and increase the enzyme lifetime[4]. Instead of using water in media, organic solvent has numerous advantages for enzyme or aqueous solutions including organic solvents have increased the solubility of nonpolar substrate and decreased the microbial contamination in the reaction solutions. Especially, In a organic solvent environment favor for halophilic enzymes because of salt concentration has a negative effect on water activity [1]. Developments of biotechnological tools and our better understanding of enzyme activity in organic solvents are thank that they will contribute the economic potential of existing enzymatic processes and will also contribute new area that nobody know about that, but they just estimated[4].
3. Results
Figure 1: Shows that the enzyme relative activity increased in the pH 5 solution, including 50%, 25%, 12.5% concentration of toluene.
Figure 2: Shows that the enzyme relative activity increased in the pH 5 solution, including 50%, 25%, 12.5% concentration of isoamyl alcohol
2. Materials and Methods
Bacterial species were isolated from fecal samples in newborn via pour plate method. Then they were stocked at ultra low temperature. The bacterial species were growth in MRS broth medium including 10 g peptone, 20 g glucose, 10 g meat extract, 5 g yeast extract, 2 g ammonium three basics, 2 g potassium dihydrogen, 5 g sodium acetate, 200 mg magnesium sulphate, 50 mg mangane per 1 liter. The bacteria secreted the extracellular β-amylase enzyme during the the overnight. This MRS broth culture was shared to falcon tubes and then they were centrifuged at 5000 g for 10 minutes at +4 ºC temperatures. The supernatants were poured in glass bottle and was added absolute ethanol (1:3 v/v). Then waited at -20 ºC temperature. After that were shared to falcon tubes and were centrifuged at 5000 g for 15 minutes at +4 ºC. The supernatants were discarded and air dry step was carried out. They were used to be enzyme source. Different organic solvent reagents that are methanol, butanol, isoamyl alcohol, toluene, dichloromethane, 2-praponol, glycerol, ethyl methyl ketone, N-N dimethylformamide, chloroform were obtained. Enzyme substrate that is starch (1% g/v) was prepared in 50%, 25%, 12.5 % concentrations of the organic solvents. The enzyme solution in pH 5 and pH 10 buffer was prepared and reacted with its substrate for ten minutes at 45 ºC and 37 ºC temperatures ,respectively. After that, the enzyme activity was measured via 3,5-dinitrosalicylic acid (DNS) method including 2 g phenol, 0.5 g sodium sulphit, 10 g sodium hydroxide, 10 g DNS, 40 % sodium potassium tartarate per 1 liter. One unit (U) of amylase activity was defined as the amount of enzyme necessary to produce 1 mg of maltose per minute under the optimum conditions. All studies were made triplicate.
Figure 3: Shows the enzyme thermostability that the enzyme relative activity decreased in the pH 5 solution, including 50% concentration of isoamyl alcohol at different temperatures .
Figure 4: Shows the enzyme thermo stability that the enzyme relative activity decreased in the pH 5 solution, including 50% concentration of toluene at different temperatures .
The enzyme relative activity was found the most biggest at 50% concentration of toluene and isoamyl alcohol in figure 1 and figure 2, respectively. The enzyme relative activity continued 33 hours at 90 ºC and 75 ºC in figure 3 and figure 4, respectively.
5.References
1-Xin Li & Hui-Ying Yu (2012) Purification and characterization of novel organic-solventtolerant β-amylase and serine protease from a newly isolated Salimicrobium halophilum strain LY20, FEMS Microbiology letters
2-Tadessa Daba, Kenji Kojima, Kuniyo Inouye (2012) Characterization and solvent engineering of wheat -amylase for enhancing its activity and stability, Enzyme and Microbial Technology
3-G.D. Haki, S.K. Rakshit (2003) Developments in industrially important thermostable enzymes: a review, Bioresource Technology
4-Veronika Stepankova, Sarka Bidmanova,Tana Koudelakova,Zbynek Prokop,Radka Chaloupkova, and Jiri Damborsky (2014) Strategies for Stabilization of Enzymes in Organic Solvents, Catalaysis
ProtStab 2016
11th International Conference on Protein Stabilisation
Istanbul Harbiye Military Museum 9-11 May, 2016