MEDIUM OPTIMIZATION FOR PROTEASE PRODUCTION VIA MODERATELY HALOTOLERANT VIRGIBACILLUS PANTOTHENTICUS USING RESPONSE SURFACE METHODOLOGY

Yıl 2020, Cilt: 9 Sayı: 1, 69 - 79, 31.01.2020

Gizem Banger , Emrah Nikerel

https://doi.org/10.18036/estubtdc.680602

Öz

Virgibacillus pantothenticus is an industrially promising, yet scarcely studied, moderately halotolerant microorganism (up to 10% NaCl) with high activity protease production potential. Following Response Surface Methodology, we employed a Central Composite Design for the experiments and constructed a second order polynomial model to represent the resulting data. For medium optimization for protease production we optimized the resulting model. 32 experiments (following the central composite design scheme) where, carbon (glucose), nitrogen (ammonium sulfate), potassium (potassium phosphate monobasic) and magnesium (magnesium sulfate) sources were studied in the media. Bacterial growth, residual glucose and protease activities were determined at the 48th hour of each experiment. The model was optimized and the concentrations were found for each parameter. Under the optimum conditions, the predicted protease activity is also experimentally verified and the model prediction was in very good agreement with experimental results. The interactions between medium components and their effect on cell growth and protease production are also sought. This work reports the improvements on protease production of a potentially interesting industrial host, Virgibacillus pantothenticus.

Anahtar Kelimeler

Virgibacillus pantothenticus, Response Surface Methodology, Alkali Protease, Medium optimization

ILIMLI HALOFILIK VIRGIBACILLUS PANTOTHENTICUS’TA PROTEAZ ÜRETIMI IÇIN, YANIT YÜZEY YÖNTEMI ILE BESIYERI OPTIMIZASYONU

Öz

Virgibacillus pantothenticus endüstriyel olarak umut vadeden, halotolerant (%10’a kadar tuzu tolere edebilen), yüksek aktiviteli proteaz üreticisi olma potansiyeli olan bir organizmadır. Bu çalışmada, Yanıt Yüzey Yöntemi metodu takip edilerek, deney tasarımı için Merkezi Kompozit Tasarımı, model olarak da ikinci derece polinom kullanılarak, biyokütle ve proteaz üretimi amaç fonskiyonu ile besiyeri optimizasyonu gerçekleştirilmiştir. Yanıt yüzeyi modelinin parametrelerinin belirlenmesi için, 32 deneyde faklı karbon (glukoz), azot (amonyum sülfat), potasyum (potasyum fosfat monobazik) ve magnezyum (magnezyum sülfat) miktarları, besiyerlerinin hazırlanması için kullanılmıştır. Her bir deneyde, bakterilerin büyümesi, 48 saat sonunda kalan glukoz miktarı ve proteaz aktivitesi belirlenmiştir. Kurulan model optimize edilmiş ve her bir besiyeri bileşeninin optimum seviyesi belirlenmiştir. Bu koşullarda, model tahmini deneysel olarak çok yakın bir sonuçla teyit edilmiştir. Aynı zamanda, besiyeri bileşenlerinin karşılıklı etkileşimleri ve hücre büyümesi ile proteaz üretimi üzerindeki etkileri araştırılmıştır. Bu çalışma, proteaz üreticisi olarak Virgibacillus pantothenticus’un potansiyelini geliştirmiştir.

Anahtar Kelimeler

: Virgibacillus pantothenticus, besiyeri optimizasyonu, Yanıt Yüzey Yöntemi, Alkalen Proteaz

Kaynakça

• Proom, H., Knight, B. C. J. G. Bacillus pantothenticus (n. sp.). Microbiology 1950; 4(3): 539-541.
• Knight, B. C. J. G., Proom, H. A comparative survey of the nutrition and physiology of mesophilic species in the genus Bacillus. Microbiology 1950; 4(3): 508-538.
• Heyndrickx, M., Lebbe, L., Kersters, K., De Vos, P., Forsyth, G., Logan, N. A. Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int J Syst Evol Micr 1998; 48(1): 99-106.
• Heyrman, J., Logan, N. A., Busse, H. J., Balcaen, A., Lebbe, L., Rodriguez-Diaz, M., Swings, J., De Vos, P. Virgibacillus carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus picturae sp. nov., three novel species isolated from deteriorated mural paintings, transfer of the species of the genus Salibacillus to Virgibacillus, as Virgibacillus marismortui comb. nov. and Virgibacillus salexigens comb. nov., and emended description of the genus Virgibacillus. Int J Syst Evol Micr 2003; 53(2): 501-511.
• Lee, J. S., Lim, J. M., Lee, K. C., Lee, J. C., Park, Y. H., Kim, C. J. Virgibacillus koreensis sp. nov., a novel bacterium from a salt field, and transfer of Virgibacillus picturae to the genus Oceanobacillus as Oceanobacillus picturae comb. nov. with emended descriptions. Int J Syst Evol Micr 2006; 56(1): 251-257.
• An, S. Y., Asahara, M., Goto, K., Kasai, H., Yokota, A. Virgibacillus halophilus sp. nov., spore-forming bacteria isolated from soil in Japan. Int J Syst Evol Micr 2007; 57(7): 1607-1611.
• Gupta, A., Joseph, B., Mani, A., Thomas, G. Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World J Microb Biot 2008; 24(2): 237-243.
• Wang, C. Y., Chang, C. C., Ng, C. C., Chen, T. W., Shyu, Y. T. Virgibacillus chiguensis sp. nov., a novel halophilic bacterium isolated from Chigu, a previously commercial saltern located in southern Taiwan. Int J Syst Evol Micr 2008; 58(2): 341-345.
• Cosa, S., Mabinya, L. V., Olaniran, A. O., Okoh, O. O., Bernard, K., Deyzel, S., Okoh, A. I.. Bioflocculant production by Virgibacillus sp. Rob isolated from the bottom sediment of Algoa Bay in the Eastern Cape, South Africa. Molecules 2011; 16(3): 2431-2442.
• Thillaimaharani, K. A., Logesh, A. R., Sharmila, K., Magdoom, B. K., Kalaiselvam, M. Studies on the intestinal bacterial flora of tilapia Oreochromis mossambicus (Peters, 1852) and optimization of alkaline protease by Virgibacillus pantothenticus. J. Microbiol. Antimicrob 2012; 4(5): 79-87.
• Sarkar, S., Roy, D., Mukherjee, J. Enhanced protease production in a polymethylmethacrylate conico-cylindrical flask by two biofilm-forming bacteria. Bioresource technol 2011; 102(2): 1849-1855.
• Abd-Elnaby, H., Beltagy, E. A., Abo-Elela, G. M., El-Sersy, N. A. Achromobacter sp. and Virgibacillus pantothenticus as models of thermo-tolerant lipase-producing marine bacteria from North Delta sediments (Egypt). Afr J Microbiol Res 2015; 9(14): 1001-1011.
• Kuhlmann, A. U., Hoffmann, T., Bursy, J., Jebbar, M., Bremer, E. Ectoine and hydroxyectoine as protectants against osmotic and cold stress: uptake through the SigB-controlled betaine-choline-carnitine transporter-type carrier EctT from Virgibacillus pantothenticus. J Bacteriol 2011; 193(18): 4699-4708.
• Torbaghan, M. E., Lakzian, A., Astaraei, A. R., Fotovat, A., Besharati, H. Salt and alkali stresses reduction in wheat by plant growth promoting haloalkaliphilic bacteria. J Soil Sci Plant Nut 2017; 17(4), 1058-1087.
• Kuhlmann, A. U., Bursy, J., Gimpel, S., Hoffmann, T., Bremer, E. Synthesis of the compatible solute ectoine in Virgibacillus pantothenticus is triggered by high salinity and low growth temperature. Appl Environ Microb 2008; 74(14): 4560-4563.
• Sinsuwan, S., Rodtong, S., Yongsawatdigul, J. NaCl Activated Extracellular Proteinase from Virgibacillus sp. SK37 Isolated from Fish Sauce Fermentation. J Food Sci 2007; 72(5): C264-C269
• Rao, M. B., Tanksale, A. M., Ghatge, M. S., Deshpande, V. V. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol R 1998; 62(3): 597-635.
• Kumar, C. G., Takagi, H. (1999). Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 1999; 17(7): 561-594.
• Sharma, K. M., Kumar, R., Panwar, S., Kumar, A. Microbial alkaline proteases: Optimization of production parameters and their properties. J Genet Eng Biotechnol 2017; 15(1): 115-126.
• Sawant, R., Nagendran, S. Protease: an enzyme with multiple industrial applications. World J Pharm Sci 2014; 3: 568-579.
• Soares, I., Barcelos, R. P., Baroni, S., Távora, Z. Microorganism-produced enzymes in the food industry. In: Benjamin Valdez, editor. Scientific, Health and Social Aspects of the Food Industry. IntechOpen, 2012.
• Korhonen, H. Milk-derived bioactive peptides: From science to applications. J Funct Foods 2009; 1(2): 177-187.
• Corrêa, A. P. F., Daroit, D. J., Coelho, J., Meira, S. M., Lopes, F. C., Segalin, J., Risso P. H., Brandelli, A. Antioxidant, antihypertensive and antimicrobial properties of ovine milk caseinate hydrolyzed with a microbial protease. J Sci Food Agr 2011; 91(12): 2247-2254.
• Nikerel, I. E., Verheijen, P. J., van Gulik, W. M., Heijnen, J. J. Model-based design of superior cell factory: an illustrative example of Penicillium chrysogenum. Syst Metab Eng 2012: 221-270.
• Puri, S., Beg, Q. K., Gupta, R. Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Curr Microbiol 2002; 44(4): 286-290.
• Myers, R. H., Montgomery, D. C., Anderson-Cook, C. M. Response surface methodology: process and product optimization using designed experiments. In: John Wiley & Sons, 2016.
• Maddox, I. S., Richert, S. H. Use of response surface methodology for the rapid optimization of microbiological media. J Appl Bacteriol 1977; 43(2): 197-204.
• Rao, K. J., Kim, C. H., Rhee, S. K. Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiae using response surface methodology. Process Biochem 2000; 35(7): 639-647.
• Yadav, K. K., Garg, N., Kumar, D., Kumar, S., Singh, A., Muthukumar, M. Application of response surface methodology for optimization of polygalacturonase production by Aspergillus niger. J Environ Biol 2015; 36(1): 255.
• Li, P. J., Xia, J. L., Shan, Y., Nie, Z. Y., Su, D. L., Gao, Q. R., Zhang, C., Ma, Y. L. Optimizing production of pectinase from orange peel by Penicillium oxalicum PJ02 using response surface methodology. Waste Biomass Valori 2015; 6(1): 13-22.
• Nagraj, A., Singhvi, M. S., Ravikumar, V., Gokhale, D. V. Optimization studies for enhancing cellulase production by Penicillium janthinellum mutant EU2D-21 using response surface methodology. BioResources 2014; 9(2): 1914-1923.
• Sathishkumar, R., Ananthan, G., Raghunathan, C. Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes. Ann Microbiol 2015; 65(3): 1481-1493.
• Kumar, R. S., Ananthan, G., Prabhu, A. S. Optimization of medium composition for alkaline protease production by Marinobacter sp. GA CAS9 using response surface methodology–A statistical approach. Biocatal Agric Biotechnol 2014; 3(2): 191-197.
• Sinsuwan, S., Jangchud, A., Rodtong, S., Roytrakul, S., Yongsawatdigul, J. Statistical optimization of the production of NaCl-tolerant proteases by a moderate halophile, Virgibacillus sp. SK37. Food Technol Biotech 2015; 53(2): 136-145.
• Saxena, R., Singh, R. Contemporaneous production of amylase and protease through CCD response surface methodology by newly isolated Bacillus megaterium strain B69. Enzyme Res 2014.
• Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959; 31(3): 426-428.
• Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
• Cupp-Enyard, Carrie. Sigma's non-specific protease activity assay-casein as a substrate. JoVE 2008: e899.
• Reese, E. T., and Maguire, A. Surfactants as stimulants of enzyme production by microorganisms. Appl Environ Microbiol 1969; 17(2): 242-245.
• Babu, K. R., and Satyanarayana, T. α-Amylase production by thermophilic Bacillus coagulans in solid state fermentation. Process Biochem 1995; 30(4): 305-309.
• Suberu, Y., Akande, I., Samuel, T., Lawal, A., Olaniran, A. Optimization of protease production in indigenous Bacillus species isolated from soil samples in Lagos, Nigeria using response surface methodology. Biocatal Agric Biotechnol 2019; 18: 101011.
• Zhang, J., Zhao, Y., Li, M., Liu, T. Optimization of defined medium for recombinant Komagataella phaffii expressing cyclodextrin glycosyltransferase. Biotechnol Progr 2019.
• Fitriani, S., Güven, K. Isolation, screening, partial purification and characterization of protease from halophilic bacteria isolated from Indonesian fermented food. AUBT-C 2018; 7(2)
• Sinha R., Khare S.K. Isolation of a halophilic Virgibacillus sp. EMB13: Characterization of its protease for detergent application, IJBT, 2012; 11: 416-426