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Bacillus spp. Suşlarından Ksilanaz Enziminin Kısmi Saflaştırılması ve Karakterizasyonu

Year 2019, Volume: 9 Issue: 2, 335 - 348, 15.12.2019
https://doi.org/10.31466/kfbd.623603

Abstract

β-1,4 bağlı ksilopiranozil
rezidülerinden oluşan ksilanın β-1,4 glikozidik bağlarını yıkan ksilanaz, başta
kağıt ve kağıt hamuru endüstrileri olmak üzere tekstil, yem, fırın ve biyoyakıt
endüstrilerinde de geniş potansiyel uygulamalara sahip endüstriyel bir
enzimdir. Bu çalışmada, Kilis 7 Aralık Üniversitesi merkez kampüs toprak
örneğinden ksilanaz üretici Bacillus spp suşlarının izolasyonu ve saflaştırılan
ksilanazın çeşitli endüstrilerdeki uygulanabilirliği araştırılmıştır. Ksilan
içeren sıvı fermantasyon ortamında geliştirilen Bacillus spp suşundan hücre
serbest elde edilen ham ksilanaz preparatına amonyum sülfat ve alkol çöktürme
uygulanarak saflaştırılmıştır. En yüksek total ve spesifik enzim aktivitesi 723
U/L ve 64.81 U/mg olarak ham enzim preparatında saptanmıştır. Ksilanazın
optimum aktivitesi 90°C ve pH 10.6’da belirlenmiştir. Enzimin 90°C sıcaklıkta
15 dakika ön inkübasyonu sonrası aktivitenin %167’e kadar arttığı saptanmıştır.
Karakterizasyon çalışmalarındaki en yüksek aktivitesi 2935 U/L olarak hesaplanmıştır.
SDS-PAGE ve zimogram analizlerinde, kısmi saflaştırılmış ekstraselüler
ksilanaza ait ortalama 38.75 kDa moleküler ağırlığında tek protein bandı
gözlenmiştir. Ksilanın ksilanaz enzimi ile hidrolizi sonucunda parçalama
ürünleri olarak ksiloz ve ksilo-oligosakkaritler belirlenmiştir.

Thanks

Bu yüksek lisans tezinin kimyasal desteği "Bacillus spp. suşlarından ksilanaz enziminin kısmi saflaştırması ve plasmid profillerinin belirlenmesi" konulu proje kapsamında TÜBİTAK BIDEB 2209/A Üniversite Öğrencileri Yurt İçi Araştırma Projeleri Destek Programı tarafından sağlanmıştır. Bu sebeple TÜBİTAK’a desteklerinden dolayı teşekkür ederim.

References

  • Referans1 Abo-State, M. A. M., Fadel, M., Abdellah, E. M., and Ghaly, M. F., (2013). Studying the stability of cellulases and xylanase produced by thermophilic and alkaliphilic bacterial strains ısolated from agricultural wastes. American-Eurasian Jouranl of Agriculture and Environmental Sciences, 13(11), 1568-1575.
  • Referans2 Aygan, A. (2008). Haloalkalofil Bacillus spp. izolasyonu, amilaz, selülaz ve ksilanaz enzimlerinin üretimi, karakterizasyonu ve biyoteknolojik uygulamalarda kullanılabilirliği. Doktora Tezi, Çukurova Üniversitesi, Fen Bililmleri Enstitüsü, Adana.
  • Referans3 Butt, M. S., Nadeem, M. T., Ahmad, Z., and Sultan, M. T. (2008). Xylanases and their applications in baking ındustry. National Institute of Food Science and Technology, 46(1) 22–31.
  • Referans4 Chakdar, H., Kumar, M., Pandiyan, K., Singh, A., Nanjappan, K., Kashyap1, P. L., and Srivastava, A.K, (2016). Bacterial xylanases: biology to biotechnology. 3 Biotech, 6(2), 150.
  • Referans5 Chaturvedi, S., and Spaul Khurana, S. M. (2016). Partial purification and characterization of cellulase-free xylanase productıon from Bacillus licheniformis. International Journal of Pharma and Bio Sciences, 7(4), 144-149.
  • Referans6 Cordeiro, C. A. M., Martins, M. L. L., Luciano, A. B., and Silva, R. F. (2002). Production and properties of xylanase from thermophilic Bacillus spp. Brazilian Archives of Biology and Technology an İnternational Journal, 45(4), 413-418.
  • Referans7 Duarte, M. C. T., Pellegrino, A. C. A., Portugal, E. P, Ponezi, A. N., and Franco, T. T. (2000). Characterization of alkaline xylanases from Bacillus pumilus. M.C.T. Brazilian Journal of Microbiology, 31, 90-94.
  • Referans8 Hiremath, K. S., and Patil, C. S. (2011). Isolation, production and characterization of alkalo thermostable xylanase from newly isolated Bacillus spp. International Journal of Biotechnology Applications, 3(1), 48-51.
  • Referans9 Irfan, M., Asghar, U., Nadeem, M., Nelofer, R., and Syed, Q. (2016). Optimization of process parameters for xylanase production by Bacillus spp. in submerged fermentation. Journal of Radiation Research and Applied Sciences, 9, 139-147.
  • Referans10 İnce, E. (2006). Ksilanaz üreten ekstrem termofil anaerobik bakterilerin izolasyonları ve enzimlerinin özelliklerinin belirlenmesi. Yüksek Lisans Tezi, Ankara Üniversitesi Fen Bilimleri Enstitüsü, Ankara.
  • Referans11 Kalim, B., and Nazish, M. A. (2016). Optimization of fermentation media and growth conditions for microbial xylanase production. 3 Biotech, 6(2), 122.Kamble, R. D., and Jadhav, A. R. (2012). Xylanase production under solid-state and submerged fermentation conditions by bacterial strains. African Journal of Microbiology Research, 6, 4292-4297.
  • Referans12 Kapilan, R. (2015). Purification of xylanase from Bacillus subtilis Bs166. Journal of Science, 5(7), 2277-3290.
  • Referans13 Kırkpınar, F., ve Açıkgöz, Z. (2003). Kanatlı hayvanlarda nişasta tabiatında olmayan polisakkaritlerin sindirim sistemi mikroflorası üzerine etkileri. Hayvansal Üretim, 44(2), 20-28.
  • Referans14 Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of bacteriophage T4. Nature, 227, 680-685.
  • Referans15 Lowry, O. H., Rosebrough, N.J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.
  • Referans16 Mahilrajan, S., Balakumar, S., and Arasaratnam, V. (2012). Effect of xylan on xylanase production by Bacillus pumilus under submerged fermentation. Ving. Journal of Science, 11(1), 12-18.
  • Referans17 Mittal, A., Nagar, S., Simran Jot Kaur, K., and Gupta, V. (2012). Isolation, purification and characterization of alkali and thermo stable xylanase from Bacillus spp. International Journal of Research and Development in Pharmacy and Life Sciences, 1(2), 63-68.
  • Referans18 Motta, F. L., Andrade, C. C. P., and Santana, M. H. A. (2013). A Review of xylanase production by the fermentation of xylan: classification, characterization and applications. In: Chandel A.K., da Silva S.S. (Eds) sustainable degradation of lignocellulosic biomass -techniques, applications and commercialization, InTech, Croatia. 251-266.
  • Referans19 Pithadiya, D., Nandha, D., and Thakkar, A. (2016). Partial purification and optimization of xylanase from Bacillus circulans. Scholars Research Library, 8(2), 1-10.
  • Referans20 Roy, N., and Rowshanul, M. (2009). Isolation and characterization of xylanase producing strain of Bacillus cereus from soil. Iranian Journal of Microbiologgy, 2, 49-53.
  • Referans21 Sargın, S., ve Öngen, G. (2003). Kanatlı yemi katkısı olarak kullanılan ksilanaz enziminin katı kültür fermantasyon yöntemi ile üretiminde ölçek büyütme çalışmaları, Ege Üniversitesi Ziraat Fakültesi Dergisi, 40(3), 145-152.
  • Referans22 Sharma, S. C. D., Shovon, M. S., Asaduzzaman, A. K. M., Sarowar Jahan, M. G.,Yeasmin, T., and Roy, N. (2011). Optimization of alkali-thermostable and cellulase-free xylanase production from Bacillus spp. Journal of Biosciences, 19, 7-14..
  • Referans23 Shrinivas, D., Savitha, G., and Raviranjan, K. (2010). A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus spp. JB 99 suitable for paper and pulp industry: Purification and characterization. Applied Biochemistry and Biotechnology, 162, 2049-2057.
  • Referans24 Sarı, B. (2016). Rize ayder kaplıcalarından alınan su örneklerinden termofilik bakterilerin izolasyonu, identifikasyonu, ksilanaz enziminin Geobacillus galactosidasius BS61 bakterisinden saflaştırılması ve karakterizasyonu. Yüksek Lisans Tezi, Atatürk Üniversitesi Fen Bilimleri Enstitüsü, Erzurum.
  • Referans25 Silva, L. G., Trugo, L. C., Terzi, S. C., and Couri, S. (2005). Low phytate lupin flour based biomass obtained by fermentation with a mutant of Aspergillus niger. Process Biochemistry, 40, 951-954.
  • Referans26 Tamariz-Angeles, C., Olivera-Gonzales, P., Villena, G. K., and Gutierrez-Correa, M. (2014). Isolation and identification of cellulolytic and xylanolytic bacteria from huancarhuaz hot spring, Peru. Annual Research & Review in Biology, 4(19), 2920-2930.
  • Referans27 Yeğin, S., ve Büyükkilleci, A.O. (2015). Mikrobiyal ksilanazlar ve gıda endüstrisinde kullanım alanları. Akademik Gıda, 13(4), 317-326.Zambare, V., Zambare, A., Muthukumarappan, K., and Christopher, L. P. (2011). Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing. International Journal of Energy and Environment, 2(1), 99-112.

Partial Purification and Characterization of Xylanase Enzyme from Bacillus spp. Strains

Year 2019, Volume: 9 Issue: 2, 335 - 348, 15.12.2019
https://doi.org/10.31466/kfbd.623603

Abstract

Xylanase that cleaves
β-1,4-glycosidic linkages of xylan composed of β-1,4-linked xylopyranosyl
residues, is a industrial enzyme, have widespread potential applications in the
textile, feed, beverage and biofuel industries, especially the paper and pulp industry.
In this study, we were examined to the isolation of Bacillus spp strains,
produced extracellular xylanase, from Kilis 7 Aralık University central campus
soil sample and the application in various industries of purificated xylanase.
The cell-free crude xylanase preparate obtained from Bacillus spp strain
developed in a liquid fermentation medium including xylan was purified by using
ammonium sulfate and alcohol precipitation. The highest total and specific
enzyme activities were detected as 723 U/L and 64.81 U/mg in cell-free crude
xylanase preparate. The optimum activity of xylanase was emerged at 90°C and pH
10.6. After the pre-incubation of enzyme at 90°C for 15 min, its activity was
detected to increase up to 167%. In characterization studies, the highest
enzyme activity was calculated as 2935 U/L. In SDS-PAGE and zymogram analysis,
single protein band in 38.75 kDa molecular weight belonging to the partially
purified extracellular xylanase was observed. Xylose and xylo-oligosaccharides
were detected as the products by hydrolysis of xylan with xylanase.

References

  • Referans1 Abo-State, M. A. M., Fadel, M., Abdellah, E. M., and Ghaly, M. F., (2013). Studying the stability of cellulases and xylanase produced by thermophilic and alkaliphilic bacterial strains ısolated from agricultural wastes. American-Eurasian Jouranl of Agriculture and Environmental Sciences, 13(11), 1568-1575.
  • Referans2 Aygan, A. (2008). Haloalkalofil Bacillus spp. izolasyonu, amilaz, selülaz ve ksilanaz enzimlerinin üretimi, karakterizasyonu ve biyoteknolojik uygulamalarda kullanılabilirliği. Doktora Tezi, Çukurova Üniversitesi, Fen Bililmleri Enstitüsü, Adana.
  • Referans3 Butt, M. S., Nadeem, M. T., Ahmad, Z., and Sultan, M. T. (2008). Xylanases and their applications in baking ındustry. National Institute of Food Science and Technology, 46(1) 22–31.
  • Referans4 Chakdar, H., Kumar, M., Pandiyan, K., Singh, A., Nanjappan, K., Kashyap1, P. L., and Srivastava, A.K, (2016). Bacterial xylanases: biology to biotechnology. 3 Biotech, 6(2), 150.
  • Referans5 Chaturvedi, S., and Spaul Khurana, S. M. (2016). Partial purification and characterization of cellulase-free xylanase productıon from Bacillus licheniformis. International Journal of Pharma and Bio Sciences, 7(4), 144-149.
  • Referans6 Cordeiro, C. A. M., Martins, M. L. L., Luciano, A. B., and Silva, R. F. (2002). Production and properties of xylanase from thermophilic Bacillus spp. Brazilian Archives of Biology and Technology an İnternational Journal, 45(4), 413-418.
  • Referans7 Duarte, M. C. T., Pellegrino, A. C. A., Portugal, E. P, Ponezi, A. N., and Franco, T. T. (2000). Characterization of alkaline xylanases from Bacillus pumilus. M.C.T. Brazilian Journal of Microbiology, 31, 90-94.
  • Referans8 Hiremath, K. S., and Patil, C. S. (2011). Isolation, production and characterization of alkalo thermostable xylanase from newly isolated Bacillus spp. International Journal of Biotechnology Applications, 3(1), 48-51.
  • Referans9 Irfan, M., Asghar, U., Nadeem, M., Nelofer, R., and Syed, Q. (2016). Optimization of process parameters for xylanase production by Bacillus spp. in submerged fermentation. Journal of Radiation Research and Applied Sciences, 9, 139-147.
  • Referans10 İnce, E. (2006). Ksilanaz üreten ekstrem termofil anaerobik bakterilerin izolasyonları ve enzimlerinin özelliklerinin belirlenmesi. Yüksek Lisans Tezi, Ankara Üniversitesi Fen Bilimleri Enstitüsü, Ankara.
  • Referans11 Kalim, B., and Nazish, M. A. (2016). Optimization of fermentation media and growth conditions for microbial xylanase production. 3 Biotech, 6(2), 122.Kamble, R. D., and Jadhav, A. R. (2012). Xylanase production under solid-state and submerged fermentation conditions by bacterial strains. African Journal of Microbiology Research, 6, 4292-4297.
  • Referans12 Kapilan, R. (2015). Purification of xylanase from Bacillus subtilis Bs166. Journal of Science, 5(7), 2277-3290.
  • Referans13 Kırkpınar, F., ve Açıkgöz, Z. (2003). Kanatlı hayvanlarda nişasta tabiatında olmayan polisakkaritlerin sindirim sistemi mikroflorası üzerine etkileri. Hayvansal Üretim, 44(2), 20-28.
  • Referans14 Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of bacteriophage T4. Nature, 227, 680-685.
  • Referans15 Lowry, O. H., Rosebrough, N.J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.
  • Referans16 Mahilrajan, S., Balakumar, S., and Arasaratnam, V. (2012). Effect of xylan on xylanase production by Bacillus pumilus under submerged fermentation. Ving. Journal of Science, 11(1), 12-18.
  • Referans17 Mittal, A., Nagar, S., Simran Jot Kaur, K., and Gupta, V. (2012). Isolation, purification and characterization of alkali and thermo stable xylanase from Bacillus spp. International Journal of Research and Development in Pharmacy and Life Sciences, 1(2), 63-68.
  • Referans18 Motta, F. L., Andrade, C. C. P., and Santana, M. H. A. (2013). A Review of xylanase production by the fermentation of xylan: classification, characterization and applications. In: Chandel A.K., da Silva S.S. (Eds) sustainable degradation of lignocellulosic biomass -techniques, applications and commercialization, InTech, Croatia. 251-266.
  • Referans19 Pithadiya, D., Nandha, D., and Thakkar, A. (2016). Partial purification and optimization of xylanase from Bacillus circulans. Scholars Research Library, 8(2), 1-10.
  • Referans20 Roy, N., and Rowshanul, M. (2009). Isolation and characterization of xylanase producing strain of Bacillus cereus from soil. Iranian Journal of Microbiologgy, 2, 49-53.
  • Referans21 Sargın, S., ve Öngen, G. (2003). Kanatlı yemi katkısı olarak kullanılan ksilanaz enziminin katı kültür fermantasyon yöntemi ile üretiminde ölçek büyütme çalışmaları, Ege Üniversitesi Ziraat Fakültesi Dergisi, 40(3), 145-152.
  • Referans22 Sharma, S. C. D., Shovon, M. S., Asaduzzaman, A. K. M., Sarowar Jahan, M. G.,Yeasmin, T., and Roy, N. (2011). Optimization of alkali-thermostable and cellulase-free xylanase production from Bacillus spp. Journal of Biosciences, 19, 7-14..
  • Referans23 Shrinivas, D., Savitha, G., and Raviranjan, K. (2010). A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus spp. JB 99 suitable for paper and pulp industry: Purification and characterization. Applied Biochemistry and Biotechnology, 162, 2049-2057.
  • Referans24 Sarı, B. (2016). Rize ayder kaplıcalarından alınan su örneklerinden termofilik bakterilerin izolasyonu, identifikasyonu, ksilanaz enziminin Geobacillus galactosidasius BS61 bakterisinden saflaştırılması ve karakterizasyonu. Yüksek Lisans Tezi, Atatürk Üniversitesi Fen Bilimleri Enstitüsü, Erzurum.
  • Referans25 Silva, L. G., Trugo, L. C., Terzi, S. C., and Couri, S. (2005). Low phytate lupin flour based biomass obtained by fermentation with a mutant of Aspergillus niger. Process Biochemistry, 40, 951-954.
  • Referans26 Tamariz-Angeles, C., Olivera-Gonzales, P., Villena, G. K., and Gutierrez-Correa, M. (2014). Isolation and identification of cellulolytic and xylanolytic bacteria from huancarhuaz hot spring, Peru. Annual Research & Review in Biology, 4(19), 2920-2930.
  • Referans27 Yeğin, S., ve Büyükkilleci, A.O. (2015). Mikrobiyal ksilanazlar ve gıda endüstrisinde kullanım alanları. Akademik Gıda, 13(4), 317-326.Zambare, V., Zambare, A., Muthukumarappan, K., and Christopher, L. P. (2011). Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing. International Journal of Energy and Environment, 2(1), 99-112.
There are 27 citations in total.

Details

Primary Language Turkish
Journal Section Articles
Authors

Hatice Aysun Mercimek Takcı 0000-0002-5394-4959

Gökhan Sertbaş This is me 0000-0003-1686-0921

Publication Date December 15, 2019
Published in Issue Year 2019 Volume: 9 Issue: 2

Cite

APA Mercimek Takcı, H. A., & Sertbaş, G. (2019). Bacillus spp. Suşlarından Ksilanaz Enziminin Kısmi Saflaştırılması ve Karakterizasyonu. Karadeniz Fen Bilimleri Dergisi, 9(2), 335-348. https://doi.org/10.31466/kfbd.623603