D-Laktat Dehidrogenazın 3-Aminopropil Silika Jel Üzerine İmmobilizasyonu ve Karakterizasyonu

Öz

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.

Anahtar Kelimeler

• D-Laktat dehidrogenaz
• immobilizasyon
• 3-aminopropil silika jel

Immobilization and Characterization of D-Lactate Dehydrogenase onto 3-aminopropyl Silica Gel Support

Öz

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.

Anahtar Kelimeler

• D-lactate dehydrogenase
• 3-aminopropyl silica gel
• Glutaraldehyde

Kaynakça

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