Aktinobakteri İzolatlarının Transglutaminaz, Levansukraz ve Beta Galaktozidaz Üretim Yetenekleri

Yıl 2022, , 30 - 39, 03.04.2022

Elif Gülşen Karabacak , Ali Osman Adıgüzel Hayrettin Saygın Ahmet Hilmi Çon

https://doi.org/10.24323/akademik-gida.1097824

Öz

Aktinobakteriler ekstrem şartlarda gelişme, büyük miktarlarda enzim üretme potansiyeli, biyokimyasal çeşitlik ve genetik manipülasyonlara uygunluk özellikleriyle alternatif enzim kaynakları arasında önemli bir konumdadır. Çalışmada, endüstriyel alanda kullanımı fazla olan transglutaminaz, β-galaktozidaz ve levansukraz enzimleri için uygun bir üretici Aktinobakteri cinsi mikroorganizmanın seçilmesi hedeflenmiştir. Bu amaçla ekstrem koşullara sahip habitatlardan farklı araştırmacılar tarafından izole edilmiş 46 aktinobakteri izolatının hedeflenen enzimleri üretim yetenekleri araştırılmıştır. Aktinobakteri izolatlarının ilgili enzimler açısından üretici olup olmadıkları, önce veri tabanlarında kayıtlı olan genom dizilerinin “Rapid Annotation using Subsystem Technology Version 2.0” kullanılarak taranmış, devamında ilgili gene sahip olanların transglutaminaz için Hidroksimat Yöntemi (Kağıt Disk Yöntemiyle), β-galaktozidaz için ONPG yöntemi, levansukraz için ise mukoid yapı oluşturma fenotipinin belirlenmesi şeklinde enzim üretme yetenekleri belirlenmiştir. Biyoinformatik taramada tüm izolatların “transglutaminaz benzeri enzim” kodlayan gen bölgesi içerdiği, kalitatif tarama sonucunda farklı türe sahip ve besiyerinde daha hızlı gelişim gösteren 9 adet bakteri izolatının potansiyel olduğu belirlenmiştir. Levansukraz enzim genine ise sadece Micromonospora sp. KC721 ve Micromonospora sp. KC213 izolatlarının sahip olduğu ancak hiçbir izolat ne katı ne de sıvı besiyerinde aktivite göstermemiştir. ß-Galaktozidaz enzim üretim geni varlığı 38 izolatta saptanmıştır. Enzim üretim genine sahip izolatlara uygulanan kalitatif test sonucunda, daha yoğun renk oluşturan, farklı türe sahip olan ve besiyerinde diğerlerine göre hızlı gelişim gösteren 17 izolat potansiyel β-galaktozidaz üreticisi olarak seçilmiş ve farklı biyoteknolojik uygulamalar için endüstriyel ölçekli enzim üretiminde kullanım potansiyeline sahip aktinobakter izolatları olarak belirlenmiştir.

Anahtar Kelimeler

Enzim üretim yeteneği, Transglutaminaz, Levansukraz, ß-Galaktozidaz, Aktinobakteri

Production Capabilities of Transglutaminase, Levansucrase and Beta Galactosidase of Actinobacteria Isolates

Öz

Actinobacteria occupy an important position among alternative enzyme sources with their ability to growth in extreme conditions, their potential to produce large amounts of enzymes, biochemical diversity and compatibility with genetic manipulations. In this study, the selection of suitable producer microorganisms for transglutaminase, ß-galactosidase and levansucrase enzymes was aimed, and the enzyme production capabilities of 46 actinobacterial isolates were determined. The ability of these actinobacterial isolates to produce related enzymes was determined by scanning the genome sequences deposited in “GenBank using Rapid Annotation using Subsystem Technology Version 2.0”. Subsequently, the enzyme production capabilities of these isolates with relevant gene were determined by Hydroximate Method for transglutaminase (with the Paper Disc Method), the ONPG method for ß- galactosidase and the mucoid structure formation phenotype for levansucrase. By bioinformatics scanning, it was determined that all isolates contained a “transglutaminase-like enzyme” gene region and by qualitative screening, 9 isolates with different species and that can grow rapidly in medium were identified as potential isolates. A levansucrase-production gene was determined in only 2 isolates (Micromonospora sp. KC 721 and Micromonospora sp. KC213) but none of these showed activity in agar and broth medium. The presence of ß-galactosidase enzyme production gene was detected in 38 isolates. As a result of the qualitative test, 17 isolates with more intense color, different species and that can grow rapidly in medium were selected as potential ß-galactosidase-producers and determined as actinobacterial isolates with a potential to be used in industrial scale enzyme production for different biotechnological applications.

Anahtar Kelimeler

Enzyme production capability, Transglutaminase, Levansucrase, ß-Galactosidase, Actinobacteria

Kaynakça

• [1] Ningthoujam, D.S., Sanasam, S., Nimaichand, S. (2009). Screening of Actinomycete isolates from niche habitats in Manipur for antibiotic activity, American Journal of Biochemistry and Biotechnology, 5, 221-225.
• [2] Mitra, A., Santra, S.C., Mukherjee, J. (2008). Distribution of actinomycetes, their antagonistic behaviour and the physico-chemical characteristics of the world’s largest tidal mangrove forest, Applied Microbiology and Biotechnology, 80, 685-695.
• [3] Vaijayanthi, G., Vijayakumar, R., Dhanasekaran, D. (2016). Actinobacteria-A Biofactory of Novel Enzymes. Actinobacteria Basics and Biotechnological Applications. https://www.intechopen.com/books/actinobacteria-basics-and-biotechnological-applications/actinobacteria-a-biofactory-of-novel-enzymes (05.07.2021)
• [4] Barka, E.A., Vatsa, P., Sanchez, L., Gaveau-Vaillant, N., Jacquard, C., Klenk, H., Clement, C., Ouhdouch, Y., Wezel, G. (2015). Taxonomoy, physiology and natural products of Actinobacteria. Microbiology and Molecular Biology Reviews, 80(1), 1-43.
• [5] Ul-Hassan, A., Wellington, E.M. (2009). Actinobacteria. In Desk Encyclopedia of Microbiology, Edited by Schaechter, M, Academic Press, USA, 1-19.
• [6] Berdy, J. (2005). Bioactive microbial metabolites. Journal of Antibiotics, 58, 1-26.
• [7] Berdy, J. (2012). Thoughs and facts about antibiotics: where we are now and where we are heading. Journal of Antibiotics, 65, 385-395.
• [8] Prakash, D., Nawani, N., Prakash, M., Bodas, M., Mandal, A., Khetmalas, M., Kapadnis, B. (2013). Actinomycetes: A repertory of green catalysts with a potential revenue resource. BioMed Research International, (Article ID: 264020), 1-8.
• [9] Zhu, D., Wu, Q., Wang, N. (2011). Industrial Enzymes. in Comprehensive Biotechnology. Edited by Moo-Young, M, Pergamon Press, Oxford, 3-13.
• [10] Gaspar, A.L.C., de Góes-Favoni, S.P. (2015). Action of microbial transglutaminase (MTGase) in the modification of food proteins: A review. Food Chemistry, 171, 315-322.
• [11] Yasir, S., Sutton, K., Newbeery, M., Andrews, N., Gerrard, J. (2007). The impact of transglutaminase on soy proteins and tofu texture. Food Chemistry, 104(4), 1491-1501.
• [12] Huang, W., Li, L., Wang, F., Wan, J., Tilley, M., Ren, C., Wu, S. (2010). Effects of transglutaminase on the rheological and Mixolab thermomechanical characteristics of oat dough. Food Chemistry, 121(4), 934-939.
• [13] Şanlı, T., Sezgin, E., Deveci, O., Şenel, E., Benli, M. (2011). Effect of using transglutaminase on physical, chemical and sensory properties of set-type yoghurt. Food Hydrocolloids, 25(6), 1477-1481.
• [14] Kieliszek, M., Misiewicz, A. (2014). Microbial transglutaminase and its application in the food industry. A review. Folia Microbiologica, 59, 241-250.
• [15] Tarapatskyy, M., Domagała, J., Zaguła, G. (2019). The effect of transglutaminase on colloidal stability of milk proteins. Food Measure, 13, 2339-2346.
• [16] Merenkova, S., Zinina, O., Loretz, O., Neverova, O., Sharaviev, P. (2019). Effects of transglutaminase and bacterial concentrates on the development of functional and technological properties of minced meat. Polish Journal of Food and Nutrition Sciences, 69(4), 387-396.
• [17] Li, W., Yu, S., Zhang, T., Jiang, B., Mu, W. (2015). Recent novel applications of levansucrases. Applied Microbiology and Biotechnology, 99(17), 6959-6969.
• [18] Arvidson, S., Rinehart, B., Gadala-Maria, F. (2006). Concentration regimes of solutions of levan polysaccharide from Bacillus sp. Carbohydrate Polymers, 65(2), 144-149.
• [19] Xiao, M., Feng, F., Lu, L. (2014). Preparation method of levan-contained yogurt. https://patentimages.storage.googleapis.com/56/d9/9c/7a577f198ab4fe/ US3816259.pdf (05.07.2020)
• [20] Yıldız, S. (2011). The Metabolism of fructooligosaccharides and fructooligosaccharide-related compounds in plants. Food Reviews International, 27(1), 16-50.
• [21] Kumar, D.J.M., Jayanthisiddhuraj, Amutha, B., Devi, D.M., Kumaran, M.D.B., Kalaichelvan, P.T. (2012). Purification and characterization of α-amylase and β-galactosidase from Bacillus sp. MNJ23 produced in a concomitant medium. American-Eurasian Journal of Agricultural & Environmental Sciences, 12(5), 566-573.
• [22] Ray, B. (1996). Fundamental Food Microbiology. New York: CRC press. 169-180.
• [23] Husain, Q. (2010). β Galactosidases and their potential applications: a review. Critical Reviews in Biotechnology, 30(1), 41-62.
• [24] Gosling, A., Stevens, G.W., Barber, A.R., Kentish, S.E., Gras, S.L. (2010). Recent advances refining galactooligosaccharide production from lactose. Food Chemistry, 121(5), 307-318.
• [25] Gupte, A.M., Nair, J.S. (2010). β-Galactosidase production and ethanol fermentation from whey using Kluyveromyces marxianus NCIM 3551. Journal of Scientific & Industrial Research, 69, 855-859.
• [26] Klein, M.P., Jong, E.V. de, Révillion, J.P.P. (2010). Utilização da β-galactosidase para prevenção da cristalização em doce de leite. Ciência e Agrotecnologia, 34(6), 1530-1535.
• [27] Akın, N., Gündüz, A., Konak, Ç. (2012). Teknolojik açıdan süt ürünlerinde laktoz dönüşümleri ve intoleransı. Academic Food Journal/Akademik GIDA, 10(4), 77-84.
• [28] Geiger, B., Nguyen, H.M., Wenig, S., Nguyen, H.A., Lorenz, C., Kittl, R., Nguyen, T.H. (2016). From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus. Biochemical Engineering Journal, 116, 45-53.
• [29] Skryplonek, K., Henriques, M., Gomes, D., Viegas, J., Fonseca, C., Pereira, C., Mituniewicz-Małek, A. (2019). Characteristics of lactose-free frozen yogurt with κ-carrageenan and corn starch as stabilizers. Journal of Dairy Science, 102(9), 7838-7848.
• [30] El-Yazeed Abd El-Salam, B.A., El-Hamid Ibrahim, O.A., El-Sayed Amer, A. (2020). Efficient enzymatic conversion of lactose in milk using fungal β-galactosidase. Biocatalysis and Agricultural Biotechnology, 29, 101813.
• [31] Aziz, R.K., Bartels, D., Best, A., Dejongh, M. (2008). The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics, 9(75), 1-15.
• [32] Overbeek, R., Olson, R., Pusch, G.D., Olsen, G.J. (2014). The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Research, 42, 206-214.
• [33] Shirling, E.B., Gottlieb, D. (1966). Methods for Characterization of Streptomyces Species. International Journal of Systematic Bacteriology, 16(3), 313-340.
• [34] Bourneow, C., Benjakul, S., Sumpavapol P., H-Kıttıkun, A. (2012). Isolation and cultivation of transglutaminase-producing bacteria from seafood processing factories. Innovative Romanian Food Biotechnology, 10, 28-39.
• [35] Leblanc, A., Gravel, C., Labelle, J., Keillor, J.W. (2001). Kinetic studies of guinea pig liver transglutaminase reveal a general-base-catalyzed deacylation mechanism. Biochemistry, 40, 8335-8342.
• [36] Desai, M., Patel, K. (2019). Isolation, optimization, and purification of extracellular levansucrase from nonpathogenic Klebsiella strain L1 isolated from waste sugarcane bagasse. Biocatalysis and Agricultural Biotechnology, 19, 101-107.
• [37] Belghith, K.S., Dahech, I., Belghith, H., Mejdoub, H. (2012). Microbial production of levansucrase for synthesis of fructooligosaccharides and levan. International Journal of Biological Macromolecules, 50(2), 451-458.
• [38] Erdal, Ö., Kaplan-Türköz, B., Taştan, Ö., Göksungur, Y. (2017). Levansucrase production by Zyomonas mobilis: Optimization of process parameters and fructooligosaccharide production. Journal of Food Biochemistry, 41(3), 3-9.
• [39] Sorde, K.L., Ananthanarayan, L. (2019). Isolation, screening, and optimization of bacterial strains for novel transglutaminase production. Preparative Biochemistry and Biotechnology, 49(1), 64-73.
• [40] Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426-428.
• [41] Tille, P.M., Forbes, B.A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition). Elsevier, St. Louis, Missouri.
• [42] Chandler, V., Donovan, S., Goodwin, W., Sprague, S., Stiefbold, F. (1998). Enzyme kinetics. In Tested Studies for Laboratory Teaching. Edited by Karcher. S.J., Volume 19, 81-97.
• [43] Giordano, D., Facchiano, A. (2018). Classification of microbial transglutaminases by evaluation of evolution trees, sequence motifs, secondary structure topology and conservation of potential catalytic residues. Biochemical and Biophysical Research Communications, 509(2), 506-513.
• [44] Guan, T.W., Tang, S.K., Wu, Y.Z., Zhi, X.Y., Zhang, L.L., Li, W.J. (2009). Haloglycomyces albus gen. nov., sp. nov., a halophilic filamentous actinomycete of the family Glycomycetaceae. International Journal of Systematic and Evolutionary Microbiology, 59(6), 1297-1301.
• [45] Ho, M.L., Leu, S.Z., Hsieh, J.F., Jiang, S.T. (2000). Technical approach to simplify the purification method and characterization of microbial transglutaminase produced from Streptoverticillium ladakanum. Journal of Food Science, 65(1), 76-80.
• [46] Kim, H.S., Jung, S.H., Lee, I., Yu, T.S. (2000). Production and characterization of a novel microbial transglutaminase from Actinomadura sp. T-2. Journal of Microbiology and Biotechnology, 10(2), 187-194.
• [47] Yeo, S.H., Yoon, J.H., Lee, D.G., Kim, H.S. (2009). Screening and identification of a Streptomyces platensis YK-2, a new transglutaminase producer. Journal of Microbiology and Biotechnology, 19(6), 588-595.
• [48] Jin, M., Huang, J., Pei, Z., Gao, H., Chang, Z. (2016). Purification and characterization of a high-salt-resistant microbial transglutaminase from Streptomyces mobaraensis. Journal of Molecular Catalysis B: Enzymatic, 133, 6-11.
• [49] Zhang, N., Zhang, S., He, Y., Chen, X., Zhang, Y., Dong, Z. (2020). Intein-mediated intracellular production of active microbial transglutaminase in Corynebacterium glutamicum. Enzyme and Microbial Technology, 142, 109680.
• [50] Fiss, E., Brooks, G.F. (1991). Use of a siderophore detection medium, ethylene glycol degradation, and beta-galactosidase activity in the early presumptive differentiation of Nocardia, Rhodococcus, Streptomyces, and rapidly growing Mycobacterium species. Journal of Clinical Microbiology, 29(7), 1533-1535.
• [51] Sanchez, J., Hardisson, C. (1979). Induction of β-galactosidase in Streptomyces violaceus. Canadian Journal of Microbiology, 25(7), 833-840.
• [52] Hildebrandt, P., Wanarska, M., Kur, J. (2009). A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c-gene cloning, overexpression, purification and properties. BMC Microbiology, 9(1), 151.
• [53] Maity, M., Sanyal, S., Bhowal, J., Bhattacharyya, D.K. (2013). Studies on isolation and characterization of lactase produced from soil bacteria. International Journal of Recent Scientific Research, 2(8), 92-94.
• [54] Collinge, A.E., Neubeck, C., Udinsky, J. (1974). “Thermostable Lactase”. https://patentimages.storage.googleapis.com/56/d9/9c/7a577f198ab4fe/US3816259.pdf. (05.07.2020)
• [55] Nakao, M., Harada, M., Kodama, Y., Nakayama, T., Shibano, Y., Amachi, T. (1994). Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula. Applied Microbiology and Biotechnology, 40(5), 657-663.