A Chromatographic Approach for Partial Purification of Polygalacturonase Produced by Aspergillus sojae

Year 2017, Volume: 15 Issue: 1, 8 - 16, 15.04.2017

İlknur Sen Marco A. Mata-gomez Marco Rito-palomares Canan Tari , Melike Dinç

https://doi.org/10.24323/akademik-gida.305767

Abstract

The aim of this study was to produce
polygalacturonase from A. sojae
mutant and partially purify the crude extract by chromatographic methods. As a preliminary
step for the confirmation of its peptides, matrix-assisted
laser-desorption-ionization-time-of-flight mass spectrometry (Maldi-TOF MS)
analysis was performed on in-gel digested sodium-dodecyl-sulphate-polyacrylamide-gel-electrophoresis
(SDS-PAGE) gels. Three different carbon sources were employed in submerged and
solid-state fermentations for the production of polygalacturonase. Crude
extract was first purified by ion-exchange chromatography (IEXC) and followed
further by size exclusion chromatography. Crude extracts obtained from
sub-merged [of bitter orange peel, sugar beet molasses and (NH₄)₂SO₄]
and solid-state [of wheat bran, sugar beet and HCl] fermentation exhibited high
levels of polygalacturonase enzyme activity (95.22 and 50.27 U/mL,
respectively). Size exclusion of IEXC pooled fraction (180, 200 and 220 mM salt
fractions) revealed the highest yield (36%) and purification fold (2.00). The
likely polygalacturonase bands from SDS-PAGE were in-gel digested and analyzed
by Maldi-TOF MS in route for peptides confirmation.

Keywords

Aspergillus sojae, Polygalacturonase, Maldi-TOF MS, Size exclusion chromatography, Ion exchange chromatography

Aspergillus sojae Tarafından Üretilen Poligalakturonazın Kısmi Saflaştırılması için Kromatografik Bir Yaklaşım

Abstract

Bu çalışmanın amacı, A. sojae mutantından poligalakturonaz
üretilmesi ve ham ekstraktın kromatografik yöntemlerle kısmi
saflaştırılmasıdır. Peptitlerin konfirmasyonu için ilk basamak olarak, jel
içinde sindirilmiş sodyum-dodesil-sülfat-poliakrilamid-jel-elektroforezi
(SDS-PAGE) jellerinde matriks-yardımlı lazer desorpsiyon/iyonlaştırmalı-uçuş
zamanlı-kütle spektrometresi (Maldi-TOF MS) analizi yapılmıştır. Poligalakturonaz
üretimi için, katı-faz ve derin fermentasyonlarda üç farklı karbon kaynağı
kullanılmıştır. Ham ekstrakt ilk olarak iyon değişim kromatografisi (IEXC) ile
saflaştırılmıştır ve ardından bunu boyut eleme kromatografisi izlemiştir. Derin
[acı portakal kabuğu, şeker pancarı melası ve (NH₄)₂SO₄]
ve katı-faz (buğday kepeği, şeker pancarı ve HCl) fermentasyonlarından elde
edilen ham ekstraktlar yüksek seviyede poligalakturonaz enzim aktivitesi
(sırasıyla 95.22 and 50.27 U/mL) göstermiştir. IEXC toplanmış fraksiyonunun
(180, 200 ve 220 mM tuz fraksiyonları) boyut elemesi, en yüksek verimi (%36) ve
saflaştırma katını (2.00) göstermiştir. SDS-PAGE’den elde edilen olası
poligalakturonaz bantları jel içinde sindirilmiş ve peptit konfirmasyonu için
Maldi-TOF-MS ile analiz edilmiştir. 

Keywords

Aspergillus sojae, Poligalakturonaz, Maldi-TOF MS, Boyut eleme kromatografisi, İyon değişim kromatografisi

References

• [1] Gummadi, S.N., Kumar, D.S., 2005. Microbial pectic transeliminases. Biotechnology Letters 27(7): 451-458.
• [2] Hoondal, G.S., Tiwari, R.P., Tewari, R., Dahiya, N., Beg, Q.K., 2002. Microbial alkaline pectinases and their ındustrial applications: A review. Applied Microbiology and Biotechnology 59(4-5): 409-18.
• [3] Jayani, R.S., Saxena, S., Gupta, R., 2005. Microbial pectinolytic enzymes: A review. Process Biochemistry 40(9): 2931-2944.
• [4] Naidu, G.S.N., Panda, T., 1998. Production of Peectolytic Enzymes-a Review. Bioprocess Engineering 19(5): 355-361.
• [5] Castilho, L.R., Medronho, R.A., Alves, T.L.M., 2000. Production and extraction of pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger. Bioresource Technology 71: 45-50.
• [6] Silva, D., Martins, E.S., Da Silva, R., Gomes, E., 2002. Pectinase prodcution by Penicillium viridicatum Rfc3 by Solid State Fermentation Using Agricultural Wastes and Agro-Industrial by-Products. Brazilian Journal of Microbiology 33: 318-324.
• [7] Patil, S.R., Dayanand, A., 2006. Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-State Conditions. Bioresource Technology 97(18): 2340-2344.
• [8] Anuradha, K., Padma, P.N., Venkateshwar, S., Reddy, G., 2010. Fungal ısolates from natural pectic substrates for polygalacturonase and multienzyme production. Indian Journal of Microbiology 50(3): 339-344.
• [9] Palaniyappan, M., Vijayagopal, V., Viswanathan, R., Viruthagiri, T., 2009. Screening of natural substrates and optimization of operating variables on the production of pectinase by submerged fermentation using Aspergillus niger Mtcc 281. African Journal of Biotechnology 8(4): 682-686.
• [10] El-Sheekh, M.M., Ismail, A.M.S., El-Abd, M.A., Hegazy, E.M., El-Diwany, A.I., 2009. Effective technological pectinases by Aspergillus carneus Nrc1 utilizing the Egyptian orange juice industry scraps. International Biodeterioration & Biodegradation 63(1): 12-18.
• [11] Blandino, T.I.S.P.A., 2002. Polygalacturonase Production by Aspergillus awamori on wheat in solid-state fermentation. Applied Microbiology and Biotechnology 58(2): 164-169.
• [12] Demir, H., Göğüş, N., Tarı, C., Heerd, D., Lahore, M.F., 2012. Optimization of the process parameters for the utilization of orange peel to produce polygalacturonase by solid-state fermentation from an Aspergillus sojae mutant strain. Turkish Journal of Biology 36: 394-404.
• [13] Gögus, N., Tari, C., Oncü, S., Unluturk, S., Tokatli, F., 2006. Relationship between morphology, rheology and polygalacturonase production by Aspergillus sojae Atcc 20235 in submerged cultures. Biochemical Engineering Journal 32(3): 171-178.
• [14] Mata-Gomez, M.A., Heerd, D., Oyanguren-Garcia, I., Barbero, F., Rito-Palomares, M., et al., 2015. A novel pectin-degrading enzyme complex from Aspergillus sojae ATCC 20235 mutants. Journal of the Science of Food and Agriculture 95(7): 1554-61.
• [15] Ustok, F.I., Tari, C., Gogus, N., 2007. Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235. Journal of Biotechnology 127(2): 322-34.
• [16] Dey, T.B., Adak, S., Bhattacharya, P., Banerjee, R., 2014. Purification of polygalacturonase from Aspergillus awamori Nakazawa Mtcc 6652 and Its Application in Apple Juice Clarification. LWT - Food Science and Technology 59(1): 591-595.
• [17] Gomes, E., Leite, R.S., Da Silva, R., Silva, D., 2009. Purification of an exopolygalacturonase from Penicillium viridicatum Rfc3 produced in submerged fermentation. International Journal of Microbiology 2009: 631942.
• [18] Contreras Esquivel, J.C., Voget, C.E., 2004. Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii. Journal of Biotechnology 110(1): 21-8.
• [19] Devi, N.A., Rao, A.G.A., 1996. Fractionation, purification and preliminary characterization of polygalacturonases produced by Aspergillus carbonarius. Enzyme and Microbial Technology 18: 59-65.
• [20] Hirose, N., Kishida, M., Kawasaki, H., Sakai, T., 1999. Purification and characterization of an endo-polygalacturonase from a mutant of Saccharomyces cerevisiae. Bioscience, Biotechnology, and Biochemistry 63(6): 1100-1103.
• [21] Jacob, N., Asha Poorna, C., Prema, P., 2008. Purification and partial characterization of polygalacturonase from streptomyces lydicus. Bioresource Technology 99(14): 6697-701.
• [22] Pathak, N., Mishra, S., Sanwal, G.G., 2000. Purification and characterization of polygalacturonase from banana fruit. Phytochemistry 54: 147-152.
• [23] Cohen, S.L., Chait, B.T., 1997. Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Analytical Biochemistry 247: 257-267.
• [24] Blackstock, W.P., Weir, M.P., 1999. Proteomics: Quantitative and physical mapping of cellular proteins. Trends in Biotechnology 17(3): 121-127.
• [25] Lei, Z., Anand, A., Mysore, K.S., Sumner, L.W., 2007. Electroelution of ıntact proteins from sds-page gels and their subsequent maldi-tof ms analysis. Methods in Molecular Biology 355: 353-363.
• [26] Heerd, D., Tari, C., Fernandez-Lahore, M., 2014. Microbial strain ımprovement for enhanced polygalacturonase production by Aspergillus sojae. Applied Microbiology and Biotechnology 98(17): 7471-81.
• [27] Buyukkileci, A.O., Tari, C., Fernandez-Lahore, M., 2011. Enhanced production of exo-polygalacturonase from agro-based products by Aspergillus sojae. BioResources 6(3): 3452-3468.
• [28] 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: 248-254.
• [29] Panda, T., Naidu, G.S.N., Sinha, J., 1999. Multiresponse analysis of microbiological parameters affecting the production of pectolytic enzymes by Aspergillus niger: A Statistical View. Process Biochemistry 35: 187-195.
• [30] Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
• [31] Kinter, M., Sherman, N.E., 2005. Protein Sequencing and Identification Using Tandem Mass Spectrometry. New York: John Wiley & Sons, Inc.
• [32] Niture, S., 2008. Comparative biochemical and structural characterizations of fungal polygalacturonases. Biologia 63(1): 1-19.
• [33] Demir, H., Tari, C., 2016. Effect of physicochemical parameters on the polygalacturonase of an Aspergillus sojae mutant using wheat bran, an agro-ındustrial waste, via solid-state fermentation. Jornal of the Science of Food and Agriculture 96(10): 3575-82.
• [34] Acuna-Arguelles, M.E., Gutierrez-Rojas, M., Viniegra-Gonzales, G., Favela-Torres, E., 1995. Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation. Applied Microbiology and Biotechnology 43: 808-814.
• [35] Dinu, D., Nechifor, M.T., Stoian, G., Costache, M., Dinischiotu, A., 2007. Enzymes with new biochemical properties in the pectinolytic complex produced by Aspergillus niger miug 16. Journal of Biotechnology 131(2): 128-37.
• [36] Freitas, P., Martin, N., Silva, D., Silva, R., Gomes, E., 2006. Production and partial characterization of polygalacturonases produced by thermophilic Monascus Sp. N8 and by thermotolerant Aspergillus Sp. N12 on solid-state fermentation. Brazilian Journal of Microbiology 37: 302-306.
• [37] Nagai, M., Katsuragi, T., Terashita, T., Yoshikawa, T., Sakai, T., 2000. Purification and characterization of an endo-polygalacturonase from Aspergillus awamori. Bioscience, Biotechnology and Biochemistry 64(8): 1729-1732.
• [38] Dogan, N., Tari, C., 2008. Characterization of three-phase partitioned exo-polygalacturonase from Aspergillus sojae with unique properties. Biochemical Engineering Journal 39(1): 43-50.
• [39] Siddiqui, M.A., Pande, V., Arif, M., 2012. Production, purification and characterization of polygalacturonase from Rhizomucor pusillus ısolated from decomposting orange peels. Enzyme Research 2012: 138634.
• [40] Kant, S., Vohra, A., Gupta, R., 2013. Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323. Protein Expression and Purification 87(1): 11-6.
• [41] Medina, M.L., Kiernan, U.A., Francisco, W.A., 2004. Proteomic analysis of rutin-ınduced secreted proteins from Aspergillus flavus. Fungal Genetics and Biology 41(3): 327-35.
• [42] Yuan, P., Meng, K., Huang, H., Shi, P., Luo, H., Yang, P., Yao, B., 2011. A novel acidic and low-temperature-active endo-polygalacturonase from Penicillium Sp. Cgmcc 1669 with potential for application in apple juice clarification. Food Chemistry 129(4): 1369-1375.
• [43] Zhang, J., Henriksson, H., Szabo, I.J., Henriksson, G., Johansson, G., 2005. The active component in the flax-retting system of the zygomycete Rhizopus oryzae Sb Is a family 28 polygalacturonase. Journal of Industrial Microbiology and Biotechnology 32(10): 431-8.
• [44] Rodrigo, D., Cortés, C., Clynen, E., Schoofs, L., Loey, A.V., Hendrickx, M., 2006. Thermal and high-pressure stability of purified polygalacturonase and pectinmethylesterase from four different tomato processing varieties. Food Research International 39(4): 440-448.