Zymomonas mobilis LEVANSUKRAZ ENZİMİNİN LEVAN ÜRETİMİNDE KULLANILMASI

Öz

Levan gıda, kozmetik ve ilaç sanayi gibi birbirinden farklı endüstrilerde kullanım alanına sahip bir fruktoz polimeridir. Levansukrazlar sakkarozu substrat olarak kullanarak fruktoz polimerleri oluşumunu katalizleyen enzimlerdir. Bakteriler tarafından hücre dışına salgılanan levansukrazlar bakterinin yüksek sakkaroz konsantrasyonunda levan ve/veya fruktooligosakkarit polimerlerini sentezlemesini sağlar. Bu çalışmada Zymomonas mobilis NRRL B-14023 kullanılarak levansukraz enziminin üretilmesi ve levan polimeri üretme koşulları incelenmiştir. Yapılan çalışmada Z. mobilis levansukrazı üretilmiş ve ham enzimin levan aktivitesi gösterilmiştir. Levansukraz, Z. mobilis B-14023 hücreleri tarafından 36 saat boyunca 30.3 °C’de 159 g/L sakkaroz, pH 4.91 ortamında statik kültürde üretilmiştir. Levansukrazın levan üretimi için optimum inkübasyon süresi ve sıcaklığı 24 saat ve 25 °C olarak bulunduktan sonra aktiviteye etki eden faktörler araştırılmıştır. Artan NaCl konsantrasyonunda levan üretiminde azalma olduğu görülmüştür. Ayrıca 10 mM EDTA ve MgCl₂ varlığında enzimin levan üretim aktivitesinde azalma olduğu bulunmuştur. Sonuç olarak Z. mobilis NRRL B-14023 levansukrazı ile ortalama 62.42 g/L levan üretilmiştir.

Anahtar Kelimeler

• Zymomonas mobilis,Levansukraz,Levan

USE of Zymomonas mobilis LEVANSUCRASE IN LEVAN PRODUCTION

Öz

Levan has a wide range of uses in different industries such as food, cosmetics and pharmaceutical. Levansucrases are enzymes, which catalyze the formation of fructose polymers using sucrose as substrate. Levansucrases are produced and secreted by bacteria and enable them to synthesize levan/fructooligosaccharides in medium containing high sucrose concentrations. In this study, Zymomonas mobilis levansucrase was produced and its levan activity was investigated. Levansucrase was produced by Z. mobilis NRRL B-14023 cells in static culture in medium with 159 g/L sucrose, pH 4.91 at 30.3 °C for 36 h. The optimum incubation time, temperature of crude enzyme for levan production were found as 24 h and 25 °C. The levan production of levansucrase decreased with increasing NaCl concentrations. Furthermore the presence of 10 mM EDTA and MgCl₂ showed an inhibitory effect on levan production. As a result, on average 62.42 g/L levan was produced using Z. mobilis NRRL B-1423 levansucrase.

Anahtar Kelimeler

• Zymomonas mobilis,Levansucrase,Levan

Kaynakça

1. Abdel-Fattah, A. F., Mahmoud, D. A. R., & Esawy, M. A. T. (2005). Production of levansucrase from Bacillus subtilis NRC 33a and enzymic synthesis of levan and fructo-oligosaccharides. Current Microbiology, 51(6), 402–407. https://doi.org/10.1007/s00284-005-0111-1
2. Arvidson, S. A., Rinehart, B. T., & Gadala-Maria, F. (2006). Concentration regimes of solutions of levan polysaccharide from Bacillus sp. Carbohydrate Polymers, 65(2), 144–149. https://doi.org/10.1016/j.carbpol.2005.12.039
3. AU - Lawrence, A.-M., & AU - Besir, H. (2009). Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid. JoVE, (30), e1350. https://doi.org/doi:10.3791/1350
4. Bakar, B., & Kaplan-Türköz, B. (2017). Structural Modelling and Structure-Function Analysis of Zymomonas mobilis Levansucrase. Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 21(1), 279. https://doi.org/10.19113/sdufbed.81065
5. Bekers, M., Upite, D., Kaminska, E., Laukevics, J., Grube, M., Vigants, A., & Linde, R. (2005). Stability of levan produced by Zymomonas mobilis. Process Biochemistry, 40(5), 1535–1539. https://doi.org/10.1016/j.procbio.2004.01.052
6. Belghith, H. (1996). Optimal conditions for levan formation by an overexpressed recombinant levansucrase. Biotechnology Letters, 18(4), 467–472. https://doi.org/10.1007/BF00143472
7. Ben Ammar, Y., Matsubara, T., Ito, K., Iizuka, M., Limpaseni, T., Pongsawasdi, P., & Minamiura, N. (2002). Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature. Journal of Biotechnology, 99(2), 111–119. https://doi.org/10.1016/S0168-1656(02)00160-8
8. Bersaneti, G. T., Pan, N. C., Baldo, C., & Celligoi, M. A. P. C. (2017). Co-production of Fructooligosaccharides and Levan by Levansucrase from Bacillus subtilis natto with Potential Application in the Food Industry. Applied Biochemistry and Biotechnology, 1–14. https://doi.org/10.1007/s12010-017-2587-0

9. Borsari, R. R. J., Celligoi, M. A. P. C., Buzato, J. B., & Silva, R. S. D. S. F. Da. (2006). Influence of carbon source and the fermentation process on levan production by Zymomonas mobilis analized by the surface response method. Ciência e Tecnologia de Alimentos, 26(3), 604–609. https://doi.org/10.1590/S0101-20612006000300019
10. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3
11. Caputi, L., Nepogodiev, S. A., Malnoy, M., Rejzek, M., Field, R. A., & Benini, S. (2013). Biomolecular characterization of the levansucrase of erwinia amylovora, a promising biocatalyst for the synthesis of fructooligosaccharides. Journal of Agricultural and Food Chemistry, 61(50), 12265–12273. https://doi.org/10.1021/jf4023178
12. Chambert, R., Gonzy-Treboul, G., & Dedonder, R. (1974). Kinetic Studies of Levanosucrase of Bacillus subtilis. European Journal of Biochemistry, 41, 285–300. https://doi.org/10.1111/j.1432-1033.1974.tb03269.x
13. Chiang, C. J., Wang, J. Y., Chen, P. T., & Chao, Y. P. (2009). Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads. Applied Microbiology and Biotechnology, 82(3), 445–451. https://doi.org/10.1007/s00253-008-1772-z
14. Crittenden, R. G., & Doelle, H. W. (1994). Identification and Characterization of the Extracellular Sucrases of Zymomonas-Mobilis Uqm-2716 (Atcc-39676). Applied Microbiology and Biotechnology, 41(3), 302–308. https://doi.org/10.1007/BF00221223
15. Erdal, Ö., Kaplan-Türköz, B., Taştan, Ö., & Göksungur, Y. (2017). Levansucrase production by Zymomonas mobilis: Optimization of process parameters and fructooligosaccharide production. Journal of Food Biochemistry, 41(3), 1–9. https://doi.org/10.1111/jfbc.12361
16. Ergene, E., & Avcı, A. (2016). Mikrobiyel ekzopolisakkaritler. SAÜ Fen Bil Der, 20. Cilt(2.Sayı), 193–202.
17. Hernandez, L., Arrieta, J., Menendez, C., Vazquez, R., Coego, A., Suarez, V., … Chambert, R. (1995). Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochemical Journal, 309(1), 113–118. https://doi.org/10.1042/bj3090113
18. Hettwer, U., Gross, M., & Rudolph, K. (1995). Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola. Journal of Bacteriology, 177(10), 2834–2839. https://doi.org/10.1128/jb.177.10.2834-2839.1995
19. Jakob, F., Meißner, D., & Vogel, R. F. (2012). Comparison of novel GH 68 levansucrases of levan-overproducing Gluconobacter species. Acetic Acid Bacteria, 1(1), 2. https://doi.org/10.4081/aab.2012.e2
20. Kekez, B. D., Gojgic-Cvijovic, G. D., Jakovljevic, D. M., Stefanovic Kojic, J. R., Markovic, M. D., Beskoski, V. P., & Vrvic, M. M. (2015). High Levan Production by Bacillus licheniformis NS032 Using Ammonium Chloride as the Sole Nitrogen Source. Applied Biochemistry and Biotechnology, 175(6), 3068–3083. https://doi.org/10.1007/s12010-015-1475-8
21. Ki-hyo, J., Kang, S. A., Cho, Y., Kim, Y., Lee, Y.-J., Hong, K., … Choue, R. W. (2003). Prebiotic Properties of Levan in Rats
22. Laemmli, U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227, 680. Retrieved from http://dx.doi.org/10.1038/227680a0
23. Menéndez, C., Hernández, L., Selman, G., Mendoza, M. F., Hevia, P., Sotolongo, M., & Arrieta, J. G. (2002). Molecular cloning and expression in Escherichia coli of an exo-levanase gene from the endophytic bacterium Gluconacetobacter diazotrophicus SRT4. Current Microbiology, 45(1), 5–12. https://doi.org/10.1007/s00284-001-0044-2
24. Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3), 426–428. https://doi.org/10.1021/ac60147a030
25. Öner, E. T., Hernández, L., & Combie, J. (2016). Review of Levan polysaccharide: From a century of past experiences to future prospects. Biotechnology Advances, 34(5), 827–844. https://doi.org/10.1016/j.biotechadv.2016.05.002
26. P. O’Mullan, M. S.-D. and D. E. E. (1991). Identification of Saccharolytic Enzymes of Zymomonas Mobilis CP4, 13(2), 137–142.
27. Ritsema, T., & Smeekens, S. (2003). Fructans: Beneficial for plants and humans. Current Opinion in Plant Biology, 6(3), 223–230. https://doi.org/10.1016/S1369-5266(03)00034-7
28. Roberfroid, M. B. (2000). Prebiotics and probiotics: are they functional foods? The American Journal of Clinical Nutrition, 71(6 Suppl), 1682S–7S; discussion 1688S–90S. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10837317
29. Roberts, E.J., Garegg, P. J. (1998). Levan derivatives, their preparation, composition and applications includingmedical and food applications. World patent appl.WO9803184.
30. Santos-Moriano, P., Fernandez-Arrojo, L., Poveda, A., Jimenez-Barbero, J., Ballesteros, A. O., & Plou, F. J. (2015). Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: Effect of reaction conditions. Journal of Molecular Catalysis B: Enzymatic, 119, 18–25. https://doi.org/10.1016/j.molcatb.2015.05.011
31. Senthilkumar, V., & Gunasekaran, P. (2005). Influence of fermentation conditions on levan production by Zymomonas mobilis CT2. Indian Journal of Biotechnology, 4(4), 491–496.
32. Shaheen, S., Aman, A., & Siddiqui, N. N. (2017). Influence of Metal ions , Surfactants and Organic Solvents on the Catalytic Performance of Levansucrase from Zymomonas mobilis, 41–46.
33. Shih, I. L., Chen, L. D., Wang, T. C., Wu, J. Y., & Liaw, K. S. (2010). Tandem production of levan and ethanol by microbial fermentation. Green Chemistry, 12(7), 1242–1247. https://doi.org/10.1039/b924765c
34. Shih, I. L., Yu, Y. T., Shieh, C. J., & Hsieh, C. Y. (2005). Selective production and characterization of levan by Bacillus subtilis (Natto) Takahashi. Journal of Agricultural and Food Chemistry, 53(21), 8211–8215. https://doi.org/10.1021/jf058084o
35. Silbir, S., Dagbagli, S., Yegin, S., Baysal, T., & Goksungur, Y. (2014). Levan production by Zymomonas mobilis in batch and continuous fermentation systems. Carbohydrate Polymers, 99, 454–461. https://doi.org/10.1016/j.carbpol.2013.08.031
36. Srikanth, R., Reddy, C. H. S. S. S., Siddartha, G., Ramaiah, M. J., & Uppuluri, K. B. (2015). Review on production, characterization and applications of microbial levan. Carbohydrate Polymers, 120, 102–114. https://doi.org/10.1016/j.carbpol.2014.12.003
37. Srikanth, R., Siddartha, G., Sundhar Reddy, C. H. S. S., Harish, B. S., Janaki Ramaiah, M., & Uppuluri, K. B. (2015). Antioxidant and anti-inflammatory levan produced from Acetobacter xylinum NCIM2526 and its statistical optimization. Carbohydrate Polymers, 123, 8–16. https://doi.org/10.1016/j.carbpol.2014.12.079
38. Srikanth, S., Swathi, M., Tejaswini, M., Sharmila, G., Muthukumaran, C., Jaganathan, M. K., & Tamilarasan, K. (2014). Statistical optimization of molasses based exopolysaccharide and biomass production by Aureobasidium pullulans MTCC 2195. Biocatalysis and Agricultural Biotechnology, 3(3), 7–12. https://doi.org/10.1016/j.bcab.2013.11.011
39. Trujillo Toledo, L. E., Gómez Riera, R., Banguela Castillo, A., Soto Romero, M., Arrieta Sosa, J. G., & Hernández García, L. (2004). Catalytical properties of N-glycosylated Gluconacetobacter diazotrophicus levansucrase produced in yeast. Electronic Journal of Biotechnology, 7(2), 0–0. https://doi.org/10.2225/vol7-issue2-fulltext-4
40. Tuncer, M. (2008). Protein Saflaştırma 1 Kromatografik Teknikler.
41. Vigants, A., Kruce, R., Bekers, M., & Zikmanis, P. (1998). Response of Zymomonas mobilis levansucrase activity to sodium chloride. Biotechnology Letters, 20(11), 1017–1019. https://doi.org/10.1023/A:1005454921301
42. Viikari, L. (1984). Applied o . Microbiology Biotechnology Formation o f levan and sorbitol from sucrose by Zymomonas mobilis, 252–255.
43. Vijayendra, S. V. N., & Shamala, T. R. (2014). Film forming microbial biopolymers for commercial applications-A review. Critical Reviews in Biotechnology, 34(4), 338–357. https://doi.org/10.3109/07388551.2013.798254
44. Wuerges, J., Caputi, L., Cianci, M., Boivin, S., Meijers, R., & Benini, S. (2015). The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site. Journal of Structural Biology, 191(3), 290–298. https://doi.org/10.1016/j.jsb.2015.07.010
45. Xiao, M., Feng, F., Lu, L. (2014). Preparation method of levan-contained yogurt. Chinese patent CN103190478B.
46. Zhang, T., Li, R., Qian, H., Mu, W., Miao, M., & Jiang, B. (2014). Biosynthesis of levan by levansucrase from Bacillus methylotrophicus SK 21.002. Carbohydrate Polymers, 101(1), 975–981. https://doi.org/10.1016/j.carbpol.2013.10.045