TY - JOUR T1 - Immobilization and Characterization of D-Lactate Dehydrogenase onto 3-aminopropyl Silica Gel Support TT - D-Laktat Dehidrogenazın 3-Aminopropil Silika Jel Üzerine İmmobilizasyonu ve Karakterizasyonu AU - Varan, Nazlı Ece AU - Tukel, Sevde Seyhan PY - 2025 DA - November Y2 - 2025 DO - 10.21597/jist.1683104 JF - Journal of the Institute of Science and Technology JO - J. Inst. Sci. and Tech. PB - Iğdır Üniversitesi WT - DergiPark SN - 2536-4618 SP - 1411 EP - 1422 VL - 15 IS - 4 LA - en AB - In this study, D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides was covalently immobilized onto 3-aminopropyl-functionalized silica gel using glutaraldehyde as a bifunctional crosslinker, with the aim of developing a catalytically active and thermally stable biocatalyst for D-lactic acid production. The immobilization protocol achieved an efficiency of 86% using 1 mg/mL of enzyme and 0.250 g of support material. Comparative biochemical characterization of both free and immobilized D-LDH was performed, assessing optimal pH and temperature, thermal stability and kinetic parameters. The immobilized enzyme preparation exhibited an optimal pH of 6.5 and a temperature optimum of 45 °C. These values corresponded to 7.0 and 37 °C for the free enzyme. Kinetic analysis revealed a Michaelis constant (Km) of 0.37 mM and maximum velocity (Vmax) of 86.9 U/mg protein for the free enzyme, whereas the immobilized enzyme displayed a significantly reduced Km of 0.08 mM and a lower Vmax of 19.2 U/mg protein, indicating increased substrate affinity but reduced catalytic turnover. Thermal stability assays demonstrated enhanced resistance of the immobilized D-LDH to elevated temperatures. Furthermore, reuse studies in a batch reactor showed that the immobilized enzyme preserved 38% of its original activity after 10 successive uses, underscoring its potential for repeated use in biotechnological applications. KW - D-lactate dehydrogenase KW - 3-aminopropyl silica gel KW - Glutaraldehyde N2 - Bu çalışmada, Leuconostoc mesenteroides'ten elde edilen D-Laktat dehidrogenazın (D-LDH) glutaraldehit ara kolu kullanılarak kovalent olarak immobilizasyonu gerçekleştirilip optimize edildi. 1 mg/mL protein ve 0,250 g destek kullanılarak gerçekleştirilen immobilizasyonda verimlilik %86 olarak belirlendi. Hem serbest hem de immobilize edilmiş D-LDH'ler karakterize edildi ve optimum pH, sıcaklık ve kinetik parametreleri belirlendi. Ayrıca, serbest ve immobilize edilmiş D-LDH'lerin stabilitesi termal stabilite açısından değerlendirildi. İmmobilize edilmiş D-LDH'nin yeniden kullanım stabilitesi de bir kesikli reaktörde test edildi. Serbest ve immobilize edilmiş D-LDH'lar için optimum pH değerleri sırasıyla 7,0 ve 6,5 olarak bulundu. Serbest ve immobilize edilmiş D-LDH enzimleri için optimum sıcaklıklar sırasıyla 37 °C ve 45 °C idi. Serbest D-LDH için kinetik parametreler 0,37 mM'lik bir Km ve 86,9 U/mg protein Vmax gösterirken, immobilize edilmiş D-LDH'ın Km'si 0,08 mM ve Vmax'ı 19,2 U/mg protein olarak elde edildi. Ek olarak, immobilize edilmiş D-LDH, bir kesikli reaktörde 10 kullanım döngüsünden sonra aktivitesinin %38'ini korudu. CR - Alagöz, D., Toprak, A., Yildirim, D., Tükel, S. S., & Fernandez-Lafuente, R. (2021). Modified silicates and carbon nanotubes for immobilization of lipase from Rhizomucor miehei: Effect of support and immobilization technique on the catalytic performance of the immobilized biocatalysts. Enzyme and Microbial Technology, 144, 109739. https://doi.org/10.1016/j.enzmictec.2020.109739 CR - Ashkan, Z., Hemmati, R., Homaei, A., Dinari, A., Jamlidoost, M., & Tashakor, A. (2021). Immobilization of enzymes on nanoinorganic support materials: An update. International Journal of Biological Macromolecules, 168, 708-721. https://doi.org/10.1016/j.ijbiomac.2020.11.127 CR - Bié, J., Sepodes, B., Fernandes, P. C., & Ribeiro, M. H. (2022). Enzyme immobilization and co-immobilization: main framework, advances and some applications. Processes, 10, 494. https://doi.org/10.3390/pr10030494 CR - Carlsson, N., Gustafsson, H., Thörn, C., Olsson, L., Holmberg, K., Åkerman, B. (2014). Enzymes immobilized in mesoporous silica: a physical-chemical perspective. Advances in Colloid and Interface Science, 205, 339-360. https://doi.org/10.1016/j.cis.2013.08.010 CR - Cavalcante, F. T., Cavalcante, A. L., de Sousa, I. G., Neto, F. S., & dos Santos, J. C. (2021). Current status and future perspectives of supports and protocols for enzyme immobilization. Catalysts, 11, 1222. https://doi.org/10.3390/catal11101222 CR - Chapman, J., Ismail, A. E., & Dinu, C. Z. (2018). Industrial applications of enzymes: Recent advances, techniques, and outlooks. Catalysts, 8, 238. https://doi.org/10.3390/catal8060238 CR - Cheng, Y. Y., Park, T. H., Seong, H., Kim, T. J., & Han, N. S. (2022). Biological characterization of D‐lactate dehydrogenase responsible for high‐yield production of D‐phenyllactic acid in Sporolactobacillus inulinus. Microbial Biotechnology, 15, 2717-2729. https://doi.org/10.1111/1751-7915.14125 CR - Chikurova, N. Y., Shemiakina, A. O., Kryzhanovskaya, D. S., Shpigun, O. A., & Chernobrovkina, A. V. (2023). Comparison of the properties of 3-aminopropyl silica with different nitrogen content in HILIC mode. Moscow University Chemistry Bulletin, 78, 118-125. https://doi.org/10.3103/S0027131423030057 CR - Donga, C., Mishra, S. B., Abd-El-Aziz, A. S., Ndlovu, L. N., Mishra, A. K., & Kuvarega, A. T. (2022). (3-Aminopropyl) Triethoxysilane (APTES) functionalized magnetic nanosilica graphene oxide (MGO) nanocomposite for the comparative adsorption of the heavy metal [Pb (II), Cd (II) and Ni (II)] ions from aqueous solution. Journal of Inorganic and Organometallic Polymers and Materials, 32, 2235-2248. https://doi.org/10.21203/rs.3.rs-1184162/v1 CR - Inoue, Y., Yamada, R., Matsumoto, T., & Ogino, H. (2024). Enhancing D-lactic acid production by optimizing the expression of D-LDH gene in methylotrophic yeast Komagataella phaffii. Biotechnology for Biofuels and Bioproducts, 17, 149. https://doi.org/10.1186/s13068-024-02596-0 CR - Jin, S., Chen, X., Yang, J., & Ding, J. (2023). Lactate dehydrogenase D is a general dehydrogenase for D-2-hydroxyacids and is associated with D-lactic acidosis. Nature Communications, 14, 6638. https://doi.org/10.1038/s41467-023-42456-3 CR - Kim, S., Ga, S., Bae, H., Sluyter, R., Konstantinov, K., Shrestha, L. K., ... & Ariga, K. (2024). Multidisciplinary approaches for enzyme biocatalysis in pharmaceuticals: protein engineering, computational biology, and nanoarchitectonics. EES Catalysis, 2, 14-48. https://doi.org/10.1039/D3EY00239J CR - Liu, J., Jiang, X., Zheng, Y., Li, K., Zhang, R., Xu, J., Wang, Z., Zhang, Y., Yin, H., & Li, J. (2024). Expression, characterization, and immobilization of a novel D-lactate dehydrogenase from Salinispirillum sp. LH 10-3-1. Processes, 12, 1349. https://doi.org/10.3390/pr12071349 CR - Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. https://doi.org/10.1016/S0021-9258(19)52451-6 CR - Meghwanshi, G. K., Kaur, N., Verma, S., Dabi, N. K., Vashishtha, A., Charan, P. D., ... & Kumar, R. (2020). Enzymes for pharmaceutical and therapeutic applications. Biotechnology and Applied Biochemistry, 67, 586-601. https://doi.org/10.1002/bab.1919 CR - Putz, A. M., Ciopec, M., Negrea, A., Grad, O., Ianăşi, C., Ivankov, O. I., ... & Almásy, L. (2021). Comparison of structure and adsorption properties of mesoporous silica functionalized with aminopropyl groups by the co-condensation and the post grafting methods. Materials, 14, 628. https://doi.org/10.3390/ma14030628 CR - Satomura, T., Hayashi, J., Sakamoto, H., Nunoura, T., Takaki, Y., Takai, K., Takami, H., Ohshima, T., Sakuraba, H., &Suye, S. (2018). d-Lactate electrochemical biosensor prepared by immobilization of thermostable dye-linked d-lactate dehydrogenase from Candidatus Caldiarchaeum subterraneum. Journal of Bioscience and Bioengineering, 126, 425-430. https://doi.org/10.1016/j.jbiosc.2018.04.002 CR - Sosa, N., Chanlek, N., & Wittayakun, J. (2020). Facile ultrasound-assisted grafting of silica gel by aminopropyltriethoxysilane for aldol condensation of furfural and acetone. Ultrasonics Sonochemistry, 62, 104857. https://doi.org/10.1016/j.ultsonch.2019.104857 CR - Tan, Z., Bilal, M., Li, X., Ju, F., Teng, Y., & Iqbal, H. M. (2022). Nanomaterial-immobilized lipases for sustainable recovery of biodiesel–A review. Fuel, 316, 123429. http://dx.doi.org/10.1016/j.fuel.2022.123429 CR - Torkzadeh-Mahani, M., Zaboli, M., Barani, M., Torkzadeh-Mahani, M. (2020). A combined theoretical and experimental study to improve the thermal stability of recombinant D-lactate dehydrogenase immobilized on a novel superparamagnetic Fe3O4NPs@ metal–organic framework. Applied Organometallic Chemistry, 34. https://doi.org/10.1002/aoc.5581 CR - Tülek, A., Günay, E., Servili, B., Essiz, S., Binay, B., & Yildirim, D. (2023). Sustainable production of formic acid from CO2 by a novel immobilized mutant formate dehydrogenase. Separation and Purification Technology, 309, 123090. https://doi.org/10.1016/j.seppur.2022.123090 CR - Umaru, I. J., Adam, B. R., Habibu, B., Umaru, K. I., & Chizaram, B. C. (2021). Biochemical impact of microorganism and enzymatic activities in food and pharmaceutical industries. International Journal of Advanced Biochemistry Research, 5, 42-57. https://doi.org/10.33545/26174693.2021.v5.i1a.147 CR - Vidhya, K., Parveen, S., Rajkumar, P., Arulmari, R., Nisha, K., & Pandiselvam, R. (2024). A comprehensive review on minimizing acrylamide in foods: rethinking ingredients, process tweaks, culinary techniques, and advanced analysis. Journal of Food Measurement and Characterization, 1-21. http://dx.doi.org/10.1007/s11694-024-03020-9 CR - Wang, Y., Luo, X., Sun, X., Hu, J., Guo, Q., Shen, B., & Fu, Y. (2022). Lactate dehydrogenase encapsulated in a metal-organic framework: A novel stable and reusable biocatalyst for the synthesis of D-phenyllactic acid. Colloids and Surfaces B: Biointerfaces, 216, 112604. https://doi.org/10.1016/j.colsurfb.2022.112604 CR - Yan, Y., Li, X., Yang, Q., Zhang, H., Hettinga, K., Li, H., & Chen, W. (2022). Dietary D‐lactate intake facilitates inflammatory resolution by modulating M1 macrophage polarization. Molecular Nutrition & Food Research, 66, 2200196. https://doi.org/10.1002/mnfr.202200196 CR - Yuping, W. E. N., Jinxi, L. I. U., Qing, J. I. N., & Hushan, C. U. I. (2024). Characterization of D-lactate dehydrogenase in Leuconostoc citreum KM20. Food and Machinery, 40(2), 36-42. https://doi.org/10.13652/j.spjx.1003.5788.2023.80549 CR - Zaboli, M., Raissi, H., Zaboli, M., Farzad, F., Torkzadeh-Mahani, M. (2019). Stabilization of D-lactate dehydrogenase diagnostic enzyme via immobilization on pristine and carboxyl-functionalized carbon nanotubes, a combined experimental and molecular dynamics simulation study. Archives of Biochemistry and Biophysics, 661, 178-186. https://doi.org/10.1016/j.abb.2018.11.019 CR - Zaboli, M., Saeidnia, F., Zaboli, M., Torkzadeh-Mahani, M. (2021). Stabilization of recombinant D-lactate dehydrogenase enzyme with trehalose: Response surface methodology and molecular dynamics simulation study. Process Biochemistry, 101, 26-35. http://dx.doi.org/10.1016/j.procbio.2020.11.001 CR - Zhou, W., Zhang, W., & Cai, Y. (2021). Laccase immobilization for water purification: A comprehensive review. Chemical Engineering Journal, 403, 126272. http://dx.doi.org/10.1016/j.cej.2020.126272 UR - https://doi.org/10.21597/jist.1683104 L1 - https://dergipark.org.tr/tr/download/article-file/4803554 ER -