ENTEROCOCCUS DURANS İLE EKSTRASELÜLER LİPAZ ÜRETİMİ VE KAREKTERİZASYONU

Yıl 2021, , 474 - 487, 23.03.2021

Esra Acu , Volkan Kılıç , Merih Kıvanç

https://doi.org/10.15237/gida.GD21020

Cited By: 1

Öz

Süt ve ürünlerinden izole edilen 50 laktik asit bakterisinin lipaz aktivitesi taranmıştır. E114 ve E114.11 numaralı izolatlarda yüksek aktivite görülmesi nedeniyle enzim üretimi için seçilmiştir. 16S rRNA gen bölgesine göre test bakterilerinin dizi analizi sonuçları Enterococcus durans olarak belirlenmiştir. Lipaz üretimi için optimum üretim koşulları belirlenmiştir. Bu koşullar: Azot ve karbon kaynağı olarak %5 pepton ve %5 glikoz ile hazırlanan besi ortamının sağlanması, pH 6.5'te 48 saat 120 rpm'de çalkalama ve 40-60°C de inkübasyondur. E. durans izolatlarına ait enzimler en yüksek aktiviteyi pH’ı 9 olan ve %20 tuz içeren ortamda göstermiştir. Çeşitli katyonların ve yüzey aktif maddelerin etkisinin farklı olduğu saptanmıştır. Enzimler düşük ve yüksek sıcaklıklarda aktivite göstermektedir. Ayrıca 5°C de 48 saat boyunca enzim aktivitesinin stabil kalması da gıda endüstrisi için önem taşımaktadır. Özellikle et ve süt ürünleri gibi fermente ürünlerde lezzet kazandırılması amacıyla kullanılabilirliği umut vericidir.

Anahtar Kelimeler

Enterococcus durans, lipaz, enzim aktivitesi

PRODUCTION AND CHARACTERIZATION OF EXTRACELLULAR LIPASE FROM ENTEROCOCCUS DURANS

Öz

Lipase activity of 50 lactic acid bacteria isolated from milk and milk products were screened. E114 and E114.11 isolates were chosen for enzyme production due to their high activity. Sequence analysis results of test bacteria according to 16S rRNA gene region were determined as Enterococcus durans. Optimum production conditions for lipase production have been determined. These conditions are: Providing a medium prepared with 5% peptone and 5% glucose as nitrogen and carbon source, shaking at 120 rpm for 48 hours at pH 6.5 and incubating at 40-60°C. Enzymes belonging to E. durans isolates showed the highest activity in the environment with pH 9 and containing 20% salt. It has been found that the effects of various cations and surfactants are different. Enzymes show activity at low and high temperatures. Stability of enzyme activity for 48 hours at 5°C is also important for the food industry. Its usability in fermented products such as meat and dairy products is promising.

Anahtar Kelimeler

Enterococcus durans, lipase, enzyme activity

Kaynakça

• Adrio, J.L. ve Demain, A.L. (2014). Microbial enzymes: tools for biotechnological processes. Biomolecules, 4(1): 117-139.
• Amato, P. ve Christner, B.C. (2009). Energy metabolism response to low-temperature and frozen conditions in Psychrobacter cryohalolentis. Appl Environ Microbiol, 75: 711-718.
• Amid, M., Manap, M., Hussin, M., Mustafa, S. (2015). A novel aqueous two phase system composed of surfactant and xylitol for the purification of lipase from pumpkin (Cucurbita moschata) seeds and recycling of phase components. Molecules, 20(6): 11184-11201.
• Aravindan, R., Anbumathi, P. ve Viruthagiri, T. (2007). Lipase applications in food industry. Int J Biotechnol, 6: 141-158.
• Arora, P.K. (2013). Staphylococcus lipolyticus sp. nov., a new cold-adapted lipase producing marine species. Annals of Microbiol, 63(3): 913-922.
• Bautista‐Gallego, J., Rantsiou, K., Garrido‐Fernandez, A., Cocolin, L., Arroyo‐Lopez, F.N. (2013). Salt reduction in vegetable fermentation: reality or desire?. Food Sci, 78(8): R1095-R1100.
• Bharathi, D., Rajalakshmi, G.ve Komathi, S. (2018). Optimization and production of lipase enzyme from bacterial strains isolated from petrol spilled soil. King Saud Univ-Sc. https://doi.org/10.1016/j.jksus.2017.12.018.
• Bhargavi, P.L., Manjushri, R. ve Reddy, P.N. (2010). Lipase production by lactic acid bacteria in submerged and solid state fermentation. BTAIJ, 4(3): 126-129.
• 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 biochem, 72(1-2): 248-254.
• Brockerhoff, H. ve Jensen, R.G. (1974). Lipolytic Enzymes. Academic Press, NewYork, pp. 1–340.
• Choo, D.W., Kurihara, T., Suzuki, T., Soda, K., Esaki, N. (1998). A cold-adapted lipase of an alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterisation. Appl Environ Microb, 64 (2): 486– 491.
• Çitak, S., Yucel, N. ve Orhan, S. (2004). Antibiotic resistance and incidence of Enterococcus species in Turkish white cheese. Int Dairy Technol, 57(1): 27-31.
• de Almeida, A.F., Tauk-Tornisielo, S.M. ve Carmona, E.C. (2013). Acid lipase from Candida viswanathii: production, biochemical properties, and potential application. BioMed Res Int, ID 435818, 10.
• Dellali, A. Karam H.Z., and Karam, N-E. (2020). Lipase and esterase activities of lactic acid bacteria isolated from different biotopes African Biotech, 19(4): 156-164.
• Eaton, T.J. ve Gasson, M.J. (2001). Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol, 67(4): 1628-1635.
• Enger, E.D., Ross, F.C. ve Bailey, D.B. (2012). Concepts in Biology, 14th ed.; McGraw- Hill: New York, NY, USA.
• Esteban-Torres, M., Mancheno, J. M., de las Rivas, B., Munoz, R. (2015). Characterization of a halotolerant lipase from the lactic acid bacteria Lactobacillus plantarum useful in food fermentations. LWT-Food Sci and Technol, 60(1): 246-252.
• Franz, C.M., Stiles, M.E., Schleifer, K.H., Holzapfel, W.H. (2003). Enterococci in foods- a conundrum for food safety. Int Food Microbiol, 88(2-3): 105-122. Giraffa, G. (2003). Functionality of enterococci in dairy products. Int Food Microbiol, 88(2-3): 215-222.
• Handwerger, S., Pucci, M.J., Volk, K.J., Liu, J.I.N.P.I.N. G., Lee, M.S. (1994). Vancomycin-resistant Leuconostoc mesenteroides and Lactobacillus casei synthesize cytoplasmic peptidoglycan precursors that terminate in lactate. Bacteriol, 176(1): 260-264.
• Hugas, M., Garriga, M. ve Aymerich, M.T. (2003). Functionalty of enterococci in meat products. Int Food Microbiol, 88(2-3): 223-233.
• Javed, S., Azeem, F., Hussain, S., Rasul, I., Siddique, M.H., Riaz, M., Afzal, M., Kouser, A., Nadeem, H. (2018). Bacterial lipases: A review on purification and characterization. Prog in biophysics and molecular biol, 132, 23-34.
• Johnson, M.E., Kapoor, R., McMahon, D.J., McCoy, D.R., Narasimmon, R.G. (2009). Reduction of sodium and fat levels in natural and processed cheeses: Scientific and technological aspects. Comprehensive Reviews in Food Sci and Food Safety, 8(3): 252-268.
• Karigar, C.S. ve Rao, S.S. (2011). Role of microbial enzymes in the bioremediation of pollutants: a review. Enzyme esearch, 1-11.
• Ko, W.H., Wang, I.T., Ann, P.J. (2005). "A simple method for detection of lipolytic microorganisms in soils, " Soil Biology & Biochemistry. 37: 597, 599.
• Kumar, A., Kanwar, S.S. (2012). Lipase production in solid-state fermentation (SSF): recent developments and biotechnological applications. Dyn. Biochem. Process Biotech Mol Biol, 6(1): 13-27.
• Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-885.
• Lopes, M.D.F.S., Leitao, A.L., Regalla, M., Marques, J.F., Carrondo, M.J.T., Crespo, M.T.B. (2002). Characterization of a highly thermostable extracellular lipase from Lactobacillus plantarum. Int Food Microbiol, 76(1-2): 107- 115.
• Meyers, S.A., Cuppett, S.L. ve Hutkins, R.W. (1996). Lipase production by lactic acid bacteria and activity on butter oil. Food Microbiol, 13: 383–389.
• Petrovic, S.E., Skrinjar, M., Becarevic, A., Vujicic, I.F., Banka, L. (1990). Effect of various carbon sources on microbial lipases biosynthesis. Biotech Letters, 12(4): 299-304.
• Piatkiewicz, A. (1987). Lipase and esterase formation by mutants of lactic acid streptococci and lactobacilli. Milchwissenschaft, 42(9): 561–564.
• Pratuangdejkul, J. ve Dharmsthiti, S. (2000). Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009. Microbiol research, 155(2): 95-100.
• Rajendran, A., Palanisamy, A. ve Thangavelu, V. (2009). Lipase catalyzed ester synthesis for food processing industries. Brazilian Arc of Biol and Tech, 52(1): 207-219.
• Ramyasree, S. ve Dutta, J.R. (2013). The effect of process parameters in enhancement of lipase productionby co-culture of lactic acid bacteria and their mutagenesis study. Biocatalysis and Agricultural Biotech, 2: 393–398.
• Ramakrishnan, V., Balakrishnan, B., Rai, A. K., Narayan, B., Halami, P. M. (2012). Concomitant production of lipase, protease and enterocin by Enterococcus faecium NCIM5363 and Enterococcus durans NCIM5427 isolated from fish processing waste. Int Aquatic Res, 4(1): 14.
• Ramakrishnan, V., Goveas, L.C., Suralikerimath, N., Jampani, C., Halami, P.M., Narayan, B. (2016). Extraction and purification of lipase from Enterococcus faecium MTCC5695 by PEG/phosphate aqueous-two phase system (ATPS) and its biochemical characterization. Biocatalysis and Agricultural Biotech, 6: 19– 27.
• Samad, M.Y.A., Razak, C.N.A., Salleh, A.B., Yunus, W.M.Z., Ampton, K and Basri, M (1989). A plate assay for primary screening of lipase activity. J Microbiol Methods, 9: 51-56.
• Sangeetha, R., Geetha, A. ve Arulpandi, I. (2008). Optimization of protease and lipase production by Bacillus pumilus SG 2 isolated from an industrial effluent. Internet J Microbiol, 5(2).
• Sharma, D., Sharma, B., Shukla, A.K. (2011). Biotechnological approach of microbial lipase: a review. Biotechnology, 10 (1): 23-40.
• Sukohidayat N.H.E., Zarei M., Baharin, B.S., and Manap, M.Y. (2018). Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol. Molecules, 23: 180. https://doi.org/10.3390/molecules23071800.
• Tanasupawat, S., Phoottosavako, M., Keeratipibul, S. (2015). Characterization and lipolytic activity of lactic acid bacteria isolated from Thai fermented meat. Appl Pharmal Sci, 5 (03): 006-012.
• Toole, G. ve Toole, S. (2004). Essential AS Biology for OCR. Nelson Thornes ltd. Treichel, H., de Oliveira, D., Mazutti, M. A., Di Luccio, M. D., Oliveira, J. V. (2010). A review on microbial lipases production. Food and Rioprocess Technol, 3(2): 182- 196.
• Treichel, H., de Oliveira, D., Mazutti, M. A., Di Luccio, M. D., Oliveira, J. V. (2010). A review on microbial lipases production. Food and rioprocess technol, 3(2): 182- 196.
• Uppada, S.R., Gupta, A.K. ve Dutta, J.R. (2012). Statistical optimization of culture parameters for lipase production from Lactococcus lactis and its application in detergent industry. Int Chem Tech Res, 4(4): 1509-1517.