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Ionic and covalent immobilization of glucose isomerase of thermophilic Anoxybacillus gonensis on DEAE-sepharose

Year 2022, , 793 - 802, 15.07.2022
https://doi.org/10.17714/gumusfenbil.1028883

Abstract

High fructose corn syrup (HFCS), which is produced by the conversion of one sugar into another (glucose to fructose), has a marketing value. Hence, different glucose isomerases [(GI) (D-xylose ketol isomerase, EC 5.3.1.5)] isolated from different sources (macro-and microorganisms) were researched until today. In addition, the cost reduction of GI production for industrial applications has been investigated and applied with different techniques. Enzyme immobilization approaches have prominent features because they allow enzymes to be used repeatedly. In the current study, Anoxybacillus gonensis G2T glucose isomerase (AgoGI) (wild type) were immobilized with ionic and covalent binding on DEAE-sepharose matrix. Afterward, kinetic and biochemical parameters of the immobilized enzymes were evaluated. The pH and temperature parameters, in which the ionic and covalent immobilized enzymes showed the best activity, were determined as 6.50 and 85 °C, respectively. The kinetic data (Vmax and Km) of ionic bound AgoGI on DEAE-sepharose were 4.85±2.09 μmol/min/mg protein and 130,57±5,42 mM, as covalent immobilized AgoGI on the same matrix were 40.51± 0.81 μmol/min/mg protein µmol/min and 127,28±2,96 mM, respectively. Consequently, the usage of DEAE-sepharose for both covalent and ionic immobilization as immobilization matrix did not exhibit any negative effects on biochemical and kinetic parameters of glucose isomerase. Therefore, immobilized AgoGI on DEAE-sepharose was an excellent and promising tool for HFCS production.

Supporting Institution

RTEU-BAP Unit

Project Number

(Project No: 2014.103.01.03).

References

  • Bandlish, R.K., Hess, J.M., Epting, K.L., & Vieille, C. (2002). Glucose to fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and to hyperthermophilic Thermotoga species. Biotechnology and Bioengineering, 80(2), 185-194. http://dx.doi.org/ 10.1002/bit.10362.
  • Bashir, N., Sood, M., & Bandral, J.D. (2020). Enzyme immobilization and its applications in food processing: a review. International Journal of Chemical Studies, 8(2), 254-261. http://dx.doi.org/10.22271/chemi.2020.v8.i2d.8779.
  • Bhosale, S.H., Rao, M.B., & Deshpande, V.V. (1996). Molecular and industrial aspects of glucose isomerase. Microbiological Reviews, 60(2), 280-300. https://doi.org/10.1128/mr.60.2.280-300.1996.
  • Bor, Y.C., Moraes, C., Lee, S.P., Crosby, W.L., Sinskey, A.J., & Batt, C.A. (1992). Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase. Gene, 114(1), 127-132. https://doi.org/10.1016/0378-1119(92)90718-5.
  • Bradford, M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analitical Biochemistry, 72, 248-254. http://dx.doi.org/10.1006/abio.1976.9999.
  • Chang, M.Y., & Juang, R.S. (2007) Use of chitosan-clay composite as immobilization support for improved activity and stability of bglucosidase. Biochemical Engineering Journal, 35(1), 93-98. https://doi.org/ 10.1016/j.bej.2007.01.003.
  • Chen, L.F., Gong, C.S., & Tsao, T. (2010). Immobilized glucose isomerase on DEAE cellulose beads. Starch Stärke, 33(2), 58-63. https://doi.org/10.1002/star.19810330207.
  • Datta, S., Christena, L.R., Rani, Y., & Rajaram, S. (1976). Enzyme immobilization: an overview on techniques and support materials. 3 Biotech, 3, 1-9. http://dx.doi.org/10.1007/s13205-012-0071-7.
  • Demirel, G., Ozcetin, G., Sahin, F., Tumturk, H., Aksoy, S., & Hasırcı, N. (2006). Semi-interpenetrating polymer networks (IPNs) for entrapment of glucose isomerase. Reactive and Functional Polymers, 66(4), 389-394. https://doi.org/10.1016/j.reactfunctpolym.2005.08.015.
  • Elnashar, M.M.M., Wahba, M.I., Amin, M.A., & Eldiwany, A.I. (2014) Application of Plackett-Burman screening design to the modeling of grafted alginate-carrageenan beads for the immobilization of penicillinG-acylase. Journal of Applied Polymers Science, 131 (11): 40295. https://doi.org/10.1002/app.40295.
  • Flores-Maltos, A., Rodrı´guez-Dura´n, L.V., Renovato, J., Contreras, J.C., Rodrı´guez, R., & Aguilar, C.N. (2011) Catalytical properties of free and immobilized Aspergillus niger tannase. Enzyme Research. 2011, 1-6. https://doi.org/ 10.4061/2011/768183.
  • Ge, Y.B., Zhou, H., Kong, W., Tong, Y., Wang, S.Y.i & L.W. (1998). Immobilization of glucose isomerase and its application in continuous production of high fructose syrup. Applied Biochemistry and Biotechnology, 69, 17-29. https://doi.org/10.1007/BF02786018.
  • Han, S.L., & Juan, H. (2000). Kinetics of glucose isomerization to fructose by immobilized glucose isomerase: anomeric reactivity of D-glucose in kinetic model. Journal of Biotechnology, 84(2), 145-153. https://doi.org/ 10.1016/S0168 1656(00)00354-0.
  • Hartley, B.S., Hanlon, N., Jackson, R.J., & Rangarajan, M. (2000). Glucose isomerase: insights into protein engineering for increased thermostability. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1543(2), 294-335. https://doi.org/10.1016/s0167-4838(00)00246-6.
  • Hassan, M., Tamer, T., & Omer, A. (2016). Methods of enzyme immobilization. International Journal Current Pharmaceutical Research, 7(6), 385-392. https://doi.org/10.1016/j.procbio.2016.08.002.
  • Illeova, V., & Polakovic, M. (2018). Design of operational temperature for immobilized glucose isomerase using an accelerated inactivation method. Acta Chimica Slovaca, 11(2), 157-162. https://doi.org/10.2478/acs-2018-0022.
  • Jin, L.Q., Xu, Q., Liu, Z.Q., Jia, D.X., Liao, C.J., Chen, D.S., & Zheng, Y.G. (2017). Immobilization of recombinant glucose isomerase for efficient production of high fructose corn syrup. Applied Biochemistry and Biotechnology, 183, 293-306. https://doi.org/10.1007/s12010-017-2445-0.
  • 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 Moecular Catalysis B: Enzymatic, 97, 215–224. https://doi.org/10.1016/j.molcatb.2013.08.019.
  • Klein, M.P., Scheeren, C.W., Lorenzoni, A.S.G., Dupont, J., & Frazzon, Hertz, P.F. (2011). Ionic liquid-cellulose film for enzyme immobilization.. Process Biochemistry, 46(6), 1375–1379. https://doi.org/10.1016/j.procbio.2011.02.021.
  • Marwa, I.W., Mohamed, E.H., & Korany, A.A. (2021). Chitosan-glutaraldehyde activated carrageenan-alginate beads for β-D-galactosidase covalent immobilization. Biocatalisis and Biotransformation, 39(2), 138-151. https://doi.org/10.1080/10242422.2020.1832476.
  • Mbagwu, M.C., Egong, E.J., & Akan, O.D. (2018). Production, characterization and optimal performance studies of glucose isomerase by Achromobacter xylosoxidans mck-4 isolated from starch milling wastes. International Journal of Microbiology Research, 25(1), 1-17. https://doi.org/10.9734/MRJI/2018/43139.
  • Neifar, S., Hlima, H.B., Mhiri, S., Mezghani, M., Bouacem, K., Ibrahim, A.H., Jaouadi, B., Bouanane-Darenfed, A., & Bejar, S. (2019). A novel thermostable and efficient Class II glucose isomerase from the thermophilic Caldicoprobacter algeriensis: biochemical characterization, molecular investigation, and application in high fructose syrup production. International Journal of Biological Macromolecules, 15 (129), 31-40, https://doi.org/10.1016/j.ijbiomac.2019.01.150.
  • Nguyen, H.Y.T., & Tran, G.B. (2018). Optimization of fermentation conditions and media for production of glucose isomerase from Bacillus megaterium using response surface methodology. Hindawi Scientifica, 2011, 1-11, https://doi.org/10.1155/2018/6842843.
  • Parker, K., Salas, M., & Nwosu, V.C. (2010). High fructose corn syrup: production, uses and public health concerns. Biotechnology and Molecular Biology Reviews, 5(5), 71-78, https://doi.org/10.5897/BMBR2010.0009.
  • Raafat, A.I., Araby, E., & Lotfy, S. (2011). Enhancement of fibrinolytic enzyme production from Bacillus subtilis via immobilization process onto radiation synthesized starch/dimethylaminoethyl methacrylate hydrogel. Carbohydrate Polymers, 87(2), 1369-1374. https://doi.org/10.1016/j.carbpol.2011.09.029.
  • Rhimi, M., Messaoud, E.B., Borgi, M.A., Khadra, K.B., & Bejar, S. (2007). Co-expression of L-arabinose isomerase and D-glucose isomerase in E. coli and development of an efficient process producing simultaneously D-tagatose and D-fructose. Enzyme Microbial Technology, 40(6), 1531-1537. https://doi.org/10.1016/j.enzmictec.2006.10.032.
  • Shen, Q., Yang, R., Hua, X., Ye, F., Zhang, W., & Zhao, W. (2011). Gelatin templated biomimetic calcification for b-galactosidase immobilization. Process Biochemistry, 46(8), 1565-1571. https://doi.org/10.1016/j.procbio.2011.04.010.
  • Singh, K., & Kayastha, A.M. (2014). Optimal immobilization of α-amylase from wheat (Triticum aestivum) onto DEAE-cellulose using response surface methodology and its characterization Journal of Molecular Catalysis B: Enzymatic, 104, 75-81. https://doi.org/10.1016/j.molcatb.2014.03.014.
  • Tükel, S.S., & Alagöz, D. (2008). Catalytic efficiency of immobilized glucose isomerase in isomerization of glucose to fructose. Food Chemistry, 111(3), 658-662. https://doi.org/10.1016/j.foodchem.2008.04.035.
  • Tumturk, H., Demirel, G., Altınok, H., Aksoy, S., & Hasırcı, N. (2007). Immobılızatıon of Glucose Isomerase ın Surface-Modıfıed Algınate gel beads. Journal of Food Biochemistry, 32(2), 234-246. https://doi.org/10.1111/j.1745-4514.2008.00171.x.
  • Vaz, R.P., & Filho, E.X.F. (2019). Ion exchange chromatography for enzyme immobilization. In book: Applications of Ion Exchange Materials in Biomedical Industries. https://doi.org/10.1007/978-3-030-06082-4-2.
  • Yanmis, D., Karaoglu, H., Colak, D.N., Sal, F.A., Canakci, S., & Belduz, A.O. (2014). Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2T. Turkish Journal of Biology, 38, 586-592. https://doi.org/10.3906/biy-1403-76.
  • Yu, H., Guo, Y., Wu, D., Zhan, W., & Lu, G. (2011). Immobilization of glucose isomerase onto GAMM support for isomerization of glucose to fructose. Journal of Moecular Catalysis B: Enzymatic, 72(2011), 73-76. https://doi.org/10.1016/j.molcatb.2011.05.006.

Termofilik Anoxybacillus gonensis glukoz izomerazının DEAE-Sefaroz üzerine iyonik ve kovalent immobilizasyonu

Year 2022, , 793 - 802, 15.07.2022
https://doi.org/10.17714/gumusfenbil.1028883

Abstract

Bir şekerin diğerine (glukozun fruktoza) dönüştürülmesiyle üretilen yüksek fruktozlu mısır şurubu (HFCS), pazarlama değerine sahiptir. Bu nedenle günümüze kadar farklı kaynaklardan (makro ve mikroorganizmalar) izole edilen farklı glukoz izomerazlar [(GI) (D-ksiloz ketol izomeraz, EC 5.3.1.5)] araştırılmıştır. Ayrıca endüstriyel uygulamalar için GI üretiminin maliyetinin düşürülmesi araştırılmış ve bunun için farklı teknikler uygulanmıştır. Enzim immobilizasyon yaklaşımları, enzimlerin tekrar tekrar kullanılmasına izin verdiği için öne çıkan özelliklere sahiptir. Bu çalışmada Anoxybacillus gönensis G2T (AgoGI) yabani tip enzimlerinin DEAE-sefaroz matriksi üzerinde immobilizasyonu (iyonik ve kovalent) gerçekleştirildi. Çalışmanın bir sonraki aşamasında elde edilen enzimlerin kinetik ve biyokimyasal özellikleri belirlendi. İmmobilize enzimler için optimum sıcaklık ve pH değerleri sırasıyla 85 °C ve 6.50 olarak belirlendi. DEAE-sefaroz üzerinde iyonik bağlı AgoGI'nin kinetik verileri (Vmax ve Km) 4.85±2.09 μmol/dk/mg protein ve 130,57±5,42 mM, aynı matris üzerinde kovalent immobilize AgoGI 40.51± 0.81 μmol/dk/mg protein ve 127,28±2,96 mM’dır. Sonuç olarak, DEAE-sefarozun hem kovalent hem iyonik immobilizasyon için immobilizasyon matriksi olarak kullanılması, glukoz izomerazın biyokimyasal ve kinetik parametreleri üzerinde herhangi bir olumsuz etki göstermedi. Bu nedenle, DEAE-sefaroz üzerinde immobilize edilmiş AgoGI HFCS üretimi için mükemmel ve umut verici bir araçtır.

Project Number

(Project No: 2014.103.01.03).

References

  • Bandlish, R.K., Hess, J.M., Epting, K.L., & Vieille, C. (2002). Glucose to fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and to hyperthermophilic Thermotoga species. Biotechnology and Bioengineering, 80(2), 185-194. http://dx.doi.org/ 10.1002/bit.10362.
  • Bashir, N., Sood, M., & Bandral, J.D. (2020). Enzyme immobilization and its applications in food processing: a review. International Journal of Chemical Studies, 8(2), 254-261. http://dx.doi.org/10.22271/chemi.2020.v8.i2d.8779.
  • Bhosale, S.H., Rao, M.B., & Deshpande, V.V. (1996). Molecular and industrial aspects of glucose isomerase. Microbiological Reviews, 60(2), 280-300. https://doi.org/10.1128/mr.60.2.280-300.1996.
  • Bor, Y.C., Moraes, C., Lee, S.P., Crosby, W.L., Sinskey, A.J., & Batt, C.A. (1992). Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase. Gene, 114(1), 127-132. https://doi.org/10.1016/0378-1119(92)90718-5.
  • Bradford, M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analitical Biochemistry, 72, 248-254. http://dx.doi.org/10.1006/abio.1976.9999.
  • Chang, M.Y., & Juang, R.S. (2007) Use of chitosan-clay composite as immobilization support for improved activity and stability of bglucosidase. Biochemical Engineering Journal, 35(1), 93-98. https://doi.org/ 10.1016/j.bej.2007.01.003.
  • Chen, L.F., Gong, C.S., & Tsao, T. (2010). Immobilized glucose isomerase on DEAE cellulose beads. Starch Stärke, 33(2), 58-63. https://doi.org/10.1002/star.19810330207.
  • Datta, S., Christena, L.R., Rani, Y., & Rajaram, S. (1976). Enzyme immobilization: an overview on techniques and support materials. 3 Biotech, 3, 1-9. http://dx.doi.org/10.1007/s13205-012-0071-7.
  • Demirel, G., Ozcetin, G., Sahin, F., Tumturk, H., Aksoy, S., & Hasırcı, N. (2006). Semi-interpenetrating polymer networks (IPNs) for entrapment of glucose isomerase. Reactive and Functional Polymers, 66(4), 389-394. https://doi.org/10.1016/j.reactfunctpolym.2005.08.015.
  • Elnashar, M.M.M., Wahba, M.I., Amin, M.A., & Eldiwany, A.I. (2014) Application of Plackett-Burman screening design to the modeling of grafted alginate-carrageenan beads for the immobilization of penicillinG-acylase. Journal of Applied Polymers Science, 131 (11): 40295. https://doi.org/10.1002/app.40295.
  • Flores-Maltos, A., Rodrı´guez-Dura´n, L.V., Renovato, J., Contreras, J.C., Rodrı´guez, R., & Aguilar, C.N. (2011) Catalytical properties of free and immobilized Aspergillus niger tannase. Enzyme Research. 2011, 1-6. https://doi.org/ 10.4061/2011/768183.
  • Ge, Y.B., Zhou, H., Kong, W., Tong, Y., Wang, S.Y.i & L.W. (1998). Immobilization of glucose isomerase and its application in continuous production of high fructose syrup. Applied Biochemistry and Biotechnology, 69, 17-29. https://doi.org/10.1007/BF02786018.
  • Han, S.L., & Juan, H. (2000). Kinetics of glucose isomerization to fructose by immobilized glucose isomerase: anomeric reactivity of D-glucose in kinetic model. Journal of Biotechnology, 84(2), 145-153. https://doi.org/ 10.1016/S0168 1656(00)00354-0.
  • Hartley, B.S., Hanlon, N., Jackson, R.J., & Rangarajan, M. (2000). Glucose isomerase: insights into protein engineering for increased thermostability. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1543(2), 294-335. https://doi.org/10.1016/s0167-4838(00)00246-6.
  • Hassan, M., Tamer, T., & Omer, A. (2016). Methods of enzyme immobilization. International Journal Current Pharmaceutical Research, 7(6), 385-392. https://doi.org/10.1016/j.procbio.2016.08.002.
  • Illeova, V., & Polakovic, M. (2018). Design of operational temperature for immobilized glucose isomerase using an accelerated inactivation method. Acta Chimica Slovaca, 11(2), 157-162. https://doi.org/10.2478/acs-2018-0022.
  • Jin, L.Q., Xu, Q., Liu, Z.Q., Jia, D.X., Liao, C.J., Chen, D.S., & Zheng, Y.G. (2017). Immobilization of recombinant glucose isomerase for efficient production of high fructose corn syrup. Applied Biochemistry and Biotechnology, 183, 293-306. https://doi.org/10.1007/s12010-017-2445-0.
  • 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 Moecular Catalysis B: Enzymatic, 97, 215–224. https://doi.org/10.1016/j.molcatb.2013.08.019.
  • Klein, M.P., Scheeren, C.W., Lorenzoni, A.S.G., Dupont, J., & Frazzon, Hertz, P.F. (2011). Ionic liquid-cellulose film for enzyme immobilization.. Process Biochemistry, 46(6), 1375–1379. https://doi.org/10.1016/j.procbio.2011.02.021.
  • Marwa, I.W., Mohamed, E.H., & Korany, A.A. (2021). Chitosan-glutaraldehyde activated carrageenan-alginate beads for β-D-galactosidase covalent immobilization. Biocatalisis and Biotransformation, 39(2), 138-151. https://doi.org/10.1080/10242422.2020.1832476.
  • Mbagwu, M.C., Egong, E.J., & Akan, O.D. (2018). Production, characterization and optimal performance studies of glucose isomerase by Achromobacter xylosoxidans mck-4 isolated from starch milling wastes. International Journal of Microbiology Research, 25(1), 1-17. https://doi.org/10.9734/MRJI/2018/43139.
  • Neifar, S., Hlima, H.B., Mhiri, S., Mezghani, M., Bouacem, K., Ibrahim, A.H., Jaouadi, B., Bouanane-Darenfed, A., & Bejar, S. (2019). A novel thermostable and efficient Class II glucose isomerase from the thermophilic Caldicoprobacter algeriensis: biochemical characterization, molecular investigation, and application in high fructose syrup production. International Journal of Biological Macromolecules, 15 (129), 31-40, https://doi.org/10.1016/j.ijbiomac.2019.01.150.
  • Nguyen, H.Y.T., & Tran, G.B. (2018). Optimization of fermentation conditions and media for production of glucose isomerase from Bacillus megaterium using response surface methodology. Hindawi Scientifica, 2011, 1-11, https://doi.org/10.1155/2018/6842843.
  • Parker, K., Salas, M., & Nwosu, V.C. (2010). High fructose corn syrup: production, uses and public health concerns. Biotechnology and Molecular Biology Reviews, 5(5), 71-78, https://doi.org/10.5897/BMBR2010.0009.
  • Raafat, A.I., Araby, E., & Lotfy, S. (2011). Enhancement of fibrinolytic enzyme production from Bacillus subtilis via immobilization process onto radiation synthesized starch/dimethylaminoethyl methacrylate hydrogel. Carbohydrate Polymers, 87(2), 1369-1374. https://doi.org/10.1016/j.carbpol.2011.09.029.
  • Rhimi, M., Messaoud, E.B., Borgi, M.A., Khadra, K.B., & Bejar, S. (2007). Co-expression of L-arabinose isomerase and D-glucose isomerase in E. coli and development of an efficient process producing simultaneously D-tagatose and D-fructose. Enzyme Microbial Technology, 40(6), 1531-1537. https://doi.org/10.1016/j.enzmictec.2006.10.032.
  • Shen, Q., Yang, R., Hua, X., Ye, F., Zhang, W., & Zhao, W. (2011). Gelatin templated biomimetic calcification for b-galactosidase immobilization. Process Biochemistry, 46(8), 1565-1571. https://doi.org/10.1016/j.procbio.2011.04.010.
  • Singh, K., & Kayastha, A.M. (2014). Optimal immobilization of α-amylase from wheat (Triticum aestivum) onto DEAE-cellulose using response surface methodology and its characterization Journal of Molecular Catalysis B: Enzymatic, 104, 75-81. https://doi.org/10.1016/j.molcatb.2014.03.014.
  • Tükel, S.S., & Alagöz, D. (2008). Catalytic efficiency of immobilized glucose isomerase in isomerization of glucose to fructose. Food Chemistry, 111(3), 658-662. https://doi.org/10.1016/j.foodchem.2008.04.035.
  • Tumturk, H., Demirel, G., Altınok, H., Aksoy, S., & Hasırcı, N. (2007). Immobılızatıon of Glucose Isomerase ın Surface-Modıfıed Algınate gel beads. Journal of Food Biochemistry, 32(2), 234-246. https://doi.org/10.1111/j.1745-4514.2008.00171.x.
  • Vaz, R.P., & Filho, E.X.F. (2019). Ion exchange chromatography for enzyme immobilization. In book: Applications of Ion Exchange Materials in Biomedical Industries. https://doi.org/10.1007/978-3-030-06082-4-2.
  • Yanmis, D., Karaoglu, H., Colak, D.N., Sal, F.A., Canakci, S., & Belduz, A.O. (2014). Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2T. Turkish Journal of Biology, 38, 586-592. https://doi.org/10.3906/biy-1403-76.
  • Yu, H., Guo, Y., Wu, D., Zhan, W., & Lu, G. (2011). Immobilization of glucose isomerase onto GAMM support for isomerization of glucose to fructose. Journal of Moecular Catalysis B: Enzymatic, 72(2011), 73-76. https://doi.org/10.1016/j.molcatb.2011.05.006.
There are 33 citations in total.

Details

Primary Language Turkish
Subjects Engineering
Journal Section Articles
Authors

Züleyha Akpınar 0000-0003-0102-6651

Merve Kızaklı Yıldırım 0000-0002-2040-3881

Hakan Karaoğlu 0000-0003-4615-1157

Project Number (Project No: 2014.103.01.03).
Publication Date July 15, 2022
Submission Date November 26, 2021
Acceptance Date April 19, 2022
Published in Issue Year 2022

Cite

APA Akpınar, Z., Kızaklı Yıldırım, M., & Karaoğlu, H. (2022). Termofilik Anoxybacillus gonensis glukoz izomerazının DEAE-Sefaroz üzerine iyonik ve kovalent immobilizasyonu. Gümüşhane Üniversitesi Fen Bilimleri Dergisi, 12(3), 793-802. https://doi.org/10.17714/gumusfenbil.1028883