Araştırma Makalesi
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Cloning, purification, and characterization of intracellular Anoxybacillus amyloliticus’ xylanase

Yıl 2023, Cilt: 8 Sayı: 3, 367 - 373, 30.09.2023
https://doi.org/10.35229/jaes.1315724

Öz

Xylanases [endo-1,4-β-D-ksilanaz (E.C. 3.2.1.8)] are extremely important enzymes used in many industrial applications where xylan needs to be processed with an environmentally friendly method. In this study, the gene of the Anoxybacillus amyloliticus’ intracellular xylana was expressed by cloning into the pET28a+ vector. Then, the enzyme (AnaXYN329) was purified, and its biochemical and kinetic properties were revealed. According to the obtained results, AnaXYN329 exhibited the optimum activity at pH 6.5, and 60 and 65 °C. Thermal stability experiments of AnaXYN329 maintained approximately 120 minutes and the enzyme activity was halved at the end of the 40th minutes. In addition, values of Km, Vmax, kcat, kcat/Km were determined as 3.631 ± 0.162 µg/µL, 100.05 ± 0.00292 µmoL/min/mg protein, 128.978 1/sec, and 35.514, respectively. Consequently, AnaXYN329 has potential to be used in industrial applications.

Kaynakça

  • Aygan, A. (2008). Haloalkalofil Bacillus sp. İzolasyonu, Amilaz, Selülaz ve Ksilanaz Enzimlerinin Üretimi, Karakterizasyonu ve Biyoteknolojik Uygulamalarda Kullanabilirliği. Doktora Tezi, Çukurova Üniversitesi, Fen Bilimleri Enstitüsü, Biyoloji Anabilim Dalı. Adana, Türkiye.
  • Basit, A., Liu, J., Miao, T., Zheng, F., Rahim, K., Lou, H. & Jiang, W. (2018). Characterization of two endo-beta-1, 4-xylanases from Myceliophthora thermophila and their saccharification efficiencies, synergistic with commercial cellulose. Frontiers in Microbiology, 9, 233. DOI: 10.3389/fmicb.2018.00233.
  • 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, 7(72), 248-54. DOI: 10.1006/abio.1976.9999.
  • Chakdar, H., Kumar, M., Pandiyan, K., Singh, A., Nanjappan, K., Kashyap, P.L. & Srivastava, A.K. (2016). Bacterial xylanases: biology to biotechnology. 3 Biotech, 6, 150. DOI: 10.1007/s13205-016-0457-z.
  • Colak, D.N., Bektas, K.I., Tokgoz, M., Canakcı, S. & Belduz, A. (2018). Screening of Xylanase and Glucose Isomerase Producing Bacteria Isolated from Hot Springs in Turkey. Sakarya University Journal of Science, 22(6), 1804-1811.
  • El Enshasy, H.A., Kandiyil, S.K., Malek, R. & Othman, N.Z. (2016). Microbial xylanases: sources, types, and their applications. Microbial Enzymes in Bioconversions of Biomass. Springer, Picassoplatz 4, CH-4052 Basel, Switzerland, 151- 213.
  • Ellis, J.T. & Magnuson, T.S. (2012). Thermostable and alkalistable xylanases produced by the thermophilic bacterium Anoxybacillus flavithermus TWXYL3. ISRN microbiology, 517- 524. DOI: 10.5402/2012/517524.
  • Garg, N., Kumar, A. & Mahatman, K.K. (2010). Xylanase: applications and biotechnological aspects: biotechnological aspects of xylanase. LAP LAMBERT Academic Publ, Lambert.
  • Georis, J., Giannotta, F., De-Buyl, E., Granier, B. & Frere, J.M. (2000). Purification and properties of three endo-beta-1, 4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps. Enzyme and Microbial Technology, 26, 178-186. DOI: 10.1016/s0141-0229(99)00141-6.
  • Girio, F.M., Fonseca, C., Carvalheiro, F., Duarte, L.C., Marques, S. & Bogel-Łukasik, R. (2010). Hemicelluloses for fuel ethanol: a review. Bioresource Technology, 101, 4775-4800.
  • Hauli, I., Sarkar, B., Mukherjee, T, Chattopadhyay, A. & Mukhopadhyay, S. (2013). Alkaline extraction of xylan from agricultural waste, for the cost effective production of xylooligosaccharides, using thermoalkaline xylanase of thermophilic Anoxybacillus sp. Ip-C. International Journal of Pure & Applied Bioscience, 1, 126-131. DOI: 10.18782/2320-7051.
  • Inan, K., Canakcı, S. & Belduz, A.O. (2011). Isolation and characterization of xylanolytic new strains of Anoxybacillus from some hot springs in Turkey. Turkish Journal of Biology, 35(5),1. DOI: 10.3906/biy-1003-75.
  • Jaf, Y., Hassan, W., Erez, M.E. & Ertas, M. (2022). Determination of extracellular hydrolytic enzyme capabilities of some Anoxybacillus isolated from hot spring environments. Frontiers in Life Sciences and Related Technologies, 3 (2) , 56-61. DOI: 10.51753/flsrt.1094629.
  • Kacagan, M., Canakci, S. & Sandalli, C. (2008). Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8. Biologia, 63,599-606 DOI: 10.2478/s11756-008-0134-8.
  • Kalemci, M. (2020). Termofilik Anoxybacillus amylolyticus bakterisinin gchı geninin klonlanması ve enzimin biyokimyasal karakterizasyonu. Yüksek Lisans Tezi, Recep Tayyip Erdoğan Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı. Rize, Türkiye.
  • Karaoglu, H., Yanmis, D., Sal, F.A., Celik, A., Canakci, S. & Belduz, A.O. (2013). Biochemical characterization of a novel glucose isomerase from Anoxybacillus gonensis G2T that displays a high level of activity and thermal stability. Journal of Molecular Catalysis B Enzymatic. 97, 215-224. DOI: 10.1016/j.molcatb.2013.08.019.
  • Kulkarni, N., Shendye, A. & Mala, R. (1999). Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews, 23, 411-456.
  • Kumar, P., Barrett, D.M., Delwiche, M.J. & Stroeve, P. (2009). Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Industrial & Engineering Chemistry Reasearch, 48(8), 3713-3729.
  • Liu, M.Q. & Liu, G.F. (2008). Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Protein expression and purification, 57, 101-107. DOI: 10.1016/j.pep.2007.10.020.
  • Marques, S., Alves, L., Ribeiro, S., Girio, F.M. & Amaral-Collaco, M.T. (1998). Characterization of a thermotolerant and alkalotolerant xylanase from a Bacillus sp. Applied Biochemistry and Biotechnology, 73, 159-172.
  • Miao, Y., Li, J., Xiao, Z., Shen, Q. & Miao, R. (2015). Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5. BMC Microbiology 15, 126. DOI: 10.1186/s12866-015-0463-z.
  • Miller, G.L. (1951). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 426-428.
  • Motta, F., Andrade, C. & Santana, M. (2013). A review of xylanase production by the fermentation of xylan: classification, characterization and applications, Sustainable Degradation of Lignocellulosic Biomass-Techniques, Applications and Commercialization, InTechOpen, Headquarters, IntechOpen Limited, 7th floor, 10 Lower Thames Street, London, EC3R 6AF, UK.
  • Singh, A.D. & Singh, B. (2018). Utility of acidic xylanase of Bacillus subtilis subsp. subtilis JJBS250 in improving the nutritional value of poultry feed. 3 Biotech, 8(503), 1-7. DOI: 10.1007/s13205-018- 1526-2.
  • Swain, M., Natarajan, V. & Krishnan C. (2017). Marine enzymes and microorganisms for bioethanol production. Advances in Food and Nutrition Research. Elsevier, United States, 181-197.
  • Turner, P., Mamo, G. & Karlsson, E.N. (2007). Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microbial cell factories, 15, 6-9. DOI: 10.1186/1475-2859-6-9.
  • Verma, D. & Satyanarayana, T. (2012). Molecular approaches for ameliorating microbial xylanases. Bioresource Technology, 117, 360-367. DOI: 10.1016/j.biortech.2012.04.034.
  • Walia, A., Guleria, S., Mehta, P., Chauhan, A. & Parkash, J. (2017). Microbial xylanases and their industrial application in pulp and paper biobleaching: a review, 3 Biotech, 7, 11.
  • Wang, G.O., Lapidot, A., Alchanati, I., Regueros, C. & Shoham, Y. (1994). Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase. Applied and Environmental Microbiology, 60(6), 1889-96. DOI: 10.1128/aem.60.6.1889-1896.1994.
  • Wang, J., Bai,Y., Yang, P., Shi, P., Luo, H. & Meng, K. (2010). A new xylanase from thermoalkaline Anoxybacillus sp. E2 with high activity and stability over a broad pH range. World Journal of Microbiology and Biotechnology 26, 917-924. DOI: 10.1007/s11274-009-0254-5.
  • Yadav, P., Maharjan, J., Korpole, S., Prasad, G.S., Sahni, G., Bhattarai, T. & Sreerama, L. (2018) Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13. Frontiers in Bioengineering and Biotechnology, 6, 65. DOI: 10.3389/fbioe.2018.00065.
  • Yanmıs, D., Karaoglu, H., Colak, D.N., Sal, F.A., Canakcı, S. & Belduz, A.O. (2014). Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2^T. Turkish Journal of Biology, 38(5), 5. DOI: 10.3906/biy-1403-76.

İntraselüler Anoxybacillus amyloliticus ksilanaz’ının klonlanması, saflaştırılması ve karakterizasyonu

Yıl 2023, Cilt: 8 Sayı: 3, 367 - 373, 30.09.2023
https://doi.org/10.35229/jaes.1315724

Öz

Ksilanazlar [endo-1,4-β-D-ksilanaz (E.C. 3.2.1.8)] ksilanın çevre dostu bir yöntemle işlenmesine ihtiyaç duyulan endüstriyel birçok uygulamada kullanılan son derece önemli enzimlerdir. Bu çalışmada Anoxybacillus amyloliticus bakterisine ait intraselüler ksilanaz geni pET28a+ vektörüne klonlanarak üretilmiştir. Daha sonra enzim (AnaXYN329) saflaştırılarak enzimin sahip olduğu biyokimyasal ve kinetik özellikler açığa çıkarılmıştır. Elde edilen sonuçlara göre, AnaXYN329 en yüksek aktiviteyi pH 6.5’da; 60 ve 65 °C’de, göstermiştir. AnaXYN329 enziminin ısıl kararlılık deneyleri yaklaşık 120 dakika sürmüştür ve enzim yarılanma ömrünü 40. dakikada tamamlamıştır. Ayrıca enzimin Km değeri 3.631 ± 0.162 µg/µL, Vmax değeri 100.05 ± 0.00292 µmoL/dk/mg protein, kcat değeri 128,978 1/sn, kcat/Km değeri ise 35.514 olarak hesaplandı. Sonuç olarak AnaXYN329, endüstriyel uygulamalarda kullanılma potansiyeline sahiptir.

Kaynakça

  • Aygan, A. (2008). Haloalkalofil Bacillus sp. İzolasyonu, Amilaz, Selülaz ve Ksilanaz Enzimlerinin Üretimi, Karakterizasyonu ve Biyoteknolojik Uygulamalarda Kullanabilirliği. Doktora Tezi, Çukurova Üniversitesi, Fen Bilimleri Enstitüsü, Biyoloji Anabilim Dalı. Adana, Türkiye.
  • Basit, A., Liu, J., Miao, T., Zheng, F., Rahim, K., Lou, H. & Jiang, W. (2018). Characterization of two endo-beta-1, 4-xylanases from Myceliophthora thermophila and their saccharification efficiencies, synergistic with commercial cellulose. Frontiers in Microbiology, 9, 233. DOI: 10.3389/fmicb.2018.00233.
  • 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, 7(72), 248-54. DOI: 10.1006/abio.1976.9999.
  • Chakdar, H., Kumar, M., Pandiyan, K., Singh, A., Nanjappan, K., Kashyap, P.L. & Srivastava, A.K. (2016). Bacterial xylanases: biology to biotechnology. 3 Biotech, 6, 150. DOI: 10.1007/s13205-016-0457-z.
  • Colak, D.N., Bektas, K.I., Tokgoz, M., Canakcı, S. & Belduz, A. (2018). Screening of Xylanase and Glucose Isomerase Producing Bacteria Isolated from Hot Springs in Turkey. Sakarya University Journal of Science, 22(6), 1804-1811.
  • El Enshasy, H.A., Kandiyil, S.K., Malek, R. & Othman, N.Z. (2016). Microbial xylanases: sources, types, and their applications. Microbial Enzymes in Bioconversions of Biomass. Springer, Picassoplatz 4, CH-4052 Basel, Switzerland, 151- 213.
  • Ellis, J.T. & Magnuson, T.S. (2012). Thermostable and alkalistable xylanases produced by the thermophilic bacterium Anoxybacillus flavithermus TWXYL3. ISRN microbiology, 517- 524. DOI: 10.5402/2012/517524.
  • Garg, N., Kumar, A. & Mahatman, K.K. (2010). Xylanase: applications and biotechnological aspects: biotechnological aspects of xylanase. LAP LAMBERT Academic Publ, Lambert.
  • Georis, J., Giannotta, F., De-Buyl, E., Granier, B. & Frere, J.M. (2000). Purification and properties of three endo-beta-1, 4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps. Enzyme and Microbial Technology, 26, 178-186. DOI: 10.1016/s0141-0229(99)00141-6.
  • Girio, F.M., Fonseca, C., Carvalheiro, F., Duarte, L.C., Marques, S. & Bogel-Łukasik, R. (2010). Hemicelluloses for fuel ethanol: a review. Bioresource Technology, 101, 4775-4800.
  • Hauli, I., Sarkar, B., Mukherjee, T, Chattopadhyay, A. & Mukhopadhyay, S. (2013). Alkaline extraction of xylan from agricultural waste, for the cost effective production of xylooligosaccharides, using thermoalkaline xylanase of thermophilic Anoxybacillus sp. Ip-C. International Journal of Pure & Applied Bioscience, 1, 126-131. DOI: 10.18782/2320-7051.
  • Inan, K., Canakcı, S. & Belduz, A.O. (2011). Isolation and characterization of xylanolytic new strains of Anoxybacillus from some hot springs in Turkey. Turkish Journal of Biology, 35(5),1. DOI: 10.3906/biy-1003-75.
  • Jaf, Y., Hassan, W., Erez, M.E. & Ertas, M. (2022). Determination of extracellular hydrolytic enzyme capabilities of some Anoxybacillus isolated from hot spring environments. Frontiers in Life Sciences and Related Technologies, 3 (2) , 56-61. DOI: 10.51753/flsrt.1094629.
  • Kacagan, M., Canakci, S. & Sandalli, C. (2008). Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8. Biologia, 63,599-606 DOI: 10.2478/s11756-008-0134-8.
  • Kalemci, M. (2020). Termofilik Anoxybacillus amylolyticus bakterisinin gchı geninin klonlanması ve enzimin biyokimyasal karakterizasyonu. Yüksek Lisans Tezi, Recep Tayyip Erdoğan Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı. Rize, Türkiye.
  • Karaoglu, H., Yanmis, D., Sal, F.A., Celik, A., Canakci, S. & Belduz, A.O. (2013). Biochemical characterization of a novel glucose isomerase from Anoxybacillus gonensis G2T that displays a high level of activity and thermal stability. Journal of Molecular Catalysis B Enzymatic. 97, 215-224. DOI: 10.1016/j.molcatb.2013.08.019.
  • Kulkarni, N., Shendye, A. & Mala, R. (1999). Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews, 23, 411-456.
  • Kumar, P., Barrett, D.M., Delwiche, M.J. & Stroeve, P. (2009). Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Industrial & Engineering Chemistry Reasearch, 48(8), 3713-3729.
  • Liu, M.Q. & Liu, G.F. (2008). Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Protein expression and purification, 57, 101-107. DOI: 10.1016/j.pep.2007.10.020.
  • Marques, S., Alves, L., Ribeiro, S., Girio, F.M. & Amaral-Collaco, M.T. (1998). Characterization of a thermotolerant and alkalotolerant xylanase from a Bacillus sp. Applied Biochemistry and Biotechnology, 73, 159-172.
  • Miao, Y., Li, J., Xiao, Z., Shen, Q. & Miao, R. (2015). Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5. BMC Microbiology 15, 126. DOI: 10.1186/s12866-015-0463-z.
  • Miller, G.L. (1951). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 426-428.
  • Motta, F., Andrade, C. & Santana, M. (2013). A review of xylanase production by the fermentation of xylan: classification, characterization and applications, Sustainable Degradation of Lignocellulosic Biomass-Techniques, Applications and Commercialization, InTechOpen, Headquarters, IntechOpen Limited, 7th floor, 10 Lower Thames Street, London, EC3R 6AF, UK.
  • Singh, A.D. & Singh, B. (2018). Utility of acidic xylanase of Bacillus subtilis subsp. subtilis JJBS250 in improving the nutritional value of poultry feed. 3 Biotech, 8(503), 1-7. DOI: 10.1007/s13205-018- 1526-2.
  • Swain, M., Natarajan, V. & Krishnan C. (2017). Marine enzymes and microorganisms for bioethanol production. Advances in Food and Nutrition Research. Elsevier, United States, 181-197.
  • Turner, P., Mamo, G. & Karlsson, E.N. (2007). Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microbial cell factories, 15, 6-9. DOI: 10.1186/1475-2859-6-9.
  • Verma, D. & Satyanarayana, T. (2012). Molecular approaches for ameliorating microbial xylanases. Bioresource Technology, 117, 360-367. DOI: 10.1016/j.biortech.2012.04.034.
  • Walia, A., Guleria, S., Mehta, P., Chauhan, A. & Parkash, J. (2017). Microbial xylanases and their industrial application in pulp and paper biobleaching: a review, 3 Biotech, 7, 11.
  • Wang, G.O., Lapidot, A., Alchanati, I., Regueros, C. & Shoham, Y. (1994). Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase. Applied and Environmental Microbiology, 60(6), 1889-96. DOI: 10.1128/aem.60.6.1889-1896.1994.
  • Wang, J., Bai,Y., Yang, P., Shi, P., Luo, H. & Meng, K. (2010). A new xylanase from thermoalkaline Anoxybacillus sp. E2 with high activity and stability over a broad pH range. World Journal of Microbiology and Biotechnology 26, 917-924. DOI: 10.1007/s11274-009-0254-5.
  • Yadav, P., Maharjan, J., Korpole, S., Prasad, G.S., Sahni, G., Bhattarai, T. & Sreerama, L. (2018) Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13. Frontiers in Bioengineering and Biotechnology, 6, 65. DOI: 10.3389/fbioe.2018.00065.
  • Yanmıs, D., Karaoglu, H., Colak, D.N., Sal, F.A., Canakcı, S. & Belduz, A.O. (2014). Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2^T. Turkish Journal of Biology, 38(5), 5. DOI: 10.3906/biy-1403-76.
Toplam 32 adet kaynakça vardır.

Ayrıntılar

Birincil Dil Türkçe
Konular Gen İfadesi
Bölüm Makaleler
Yazarlar

Hakan Karaoğlu 0000-0003-4615-1157

Züleyha Akpınar 0000-0003-0102-6651

Erken Görünüm Tarihi 15 Eylül 2023
Yayımlanma Tarihi 30 Eylül 2023
Gönderilme Tarihi 16 Haziran 2023
Kabul Tarihi 26 Ağustos 2023
Yayımlandığı Sayı Yıl 2023 Cilt: 8 Sayı: 3

Kaynak Göster

APA Karaoğlu, H., & Akpınar, Z. (2023). İntraselüler Anoxybacillus amyloliticus ksilanaz’ının klonlanması, saflaştırılması ve karakterizasyonu. Journal of Anatolian Environmental and Animal Sciences, 8(3), 367-373. https://doi.org/10.35229/jaes.1315724


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