Preparation of ascorbate oxidase enzyme electrode and its usage for L-ascorbic acid determination
Küçük Resim Yok
Tarih
1996
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tezde, salatalıktan (Cucumis sativus L.) izole edilip kısmi olarak saflaştınlan Askorbat oksidaz [(L-Askorbat: oksijen oksidoredüktaz) (EC 1.10.3.3)] glutaraldehid yardımıyla jelatin ile çapraz bağlanarak, çözünmüş oksijen probu membranı üzerinde bir biyoaktif tabaka oluşturuldu ve L-askorbik aside duyarlı bir askorbat oksidaz enzim eiektrodu geliştirildi. Ölçümler enzimatik reaksiyon uyarınca L-askorbik asit konsantrasyonu ile orantılı olarak tüketilen oksijen düzeyinin belirlenmesi yoluyla çizilen standard grafikler yardımıyla gerçekleştirildi. Hazırlanan biyosensörün optimizasyon çalışmalarında en uygun askorbat oksidaz, jelatin ve glutaraldehid oranlan sırasıyla 24,8 U/cm^, 5,9 mgjelatin/cm^ ve %2,5 olarak bulundu. Optimum çalışma ortamı olarak 50 mM'lik pH:7,5 olan fosfat tamponunun kullanılması ve ölçümlerin 35°C de yapılması gerektiği sonucuna varıldı. Geliştirilen biyosensörün karakterizasyonu amacıyla yapılan çalışmalar sonucunda 5,0xl0~5 - 1,2x10" 3 M L-askorbik asit konsantrasyon aralığında doğrusal sonuçların alınabildiği tespit edildi. Tekrarlanabilirlik denemelerinde (n=ll), 4,0xl0"4 MTlık L-askorbik asit konsantrasyonu için ortalama değer (x)=3,99xl0"4 M, standard sapma (S.S):±0,001 ve varyasyon katsayısı (V.K):%0,251 olarak bulundu. Ayrıca substrat spesifikliği, depo kararlılığı ve bazı maddelerin girişim etkileri araştırıldı. Son olarak bazı meyva ekstraktları ve vitamin tabletlerinde, geliştirilen biyosensörle L-askorbik asit tayini yapıldı ve elde edilen sonuçlar ve metodun duyarlılığı 2,6 diklorofenolindofenol metodu ile kıyaslandı. Anahtar ' Kelimeler : Askorbat Oksidaz, L-Askorbik Asit, Enzim Elektrodlan, Biosensörler
In this thesis, ascorbate oxidase [(L-Ascorbate:oxygen oxidoreductase) (EC1. 10.3.3)], isolated and partially purified from cucumber (Cucumis sativus L.) was crosslinked with gelatine by using glutaraldehyde and a bioactive layer was formed on the dissolved oxygen probe, therefore, ascorbate oxidase enzyme electrode which was sensitive for L-ascorbic acid determination was developed. Measurements were carried out by standard curves which were obtained by the determination of consumed oxygen level, related to L-ascorbic acid concentration in the enzymatic reaction. In the optimization studies of the biosensor prepared, the most suitable ascorbate oxidase, gelatine and glutaraldehyde ratios were determined as 24.8 U/cm-, 5.9 mggelatine/cm^ and, 2.5% respectively. The phosphate buffer (50 mM, pH:7.5) and 35° C were established as providing the optimum conditions. The characterization studies proved a linearity in the ascorbic acid concentration range 5.0xl0~5-1.2xl0~3 M. The reproducibility experiments (n=ll) revealed that for 4x10-4 M L-ascorbic acid, the average value (x) was 3.99x10-4 M,standard deviation (S.D) was ±0.001 and, variation coefficient (C.V) was 0.251%. Moreover, substrate specification, storage stability and the interference effects of some substances were investigated. Finally, L-ascorbic acid determination in some fruit extracts and vitamin tablets was carried out. Both the results obtained and the sensitivity of the method were compared with the 2,6 dichlorophenolindophenol method. Keywords: Ascorbate Oxidase, L- Ascorbic Acid, Enzyme Electrodes, Biosensors
In this thesis, ascorbate oxidase [(L-Ascorbate:oxygen oxidoreductase) (EC1. 10.3.3)], isolated and partially purified from cucumber (Cucumis sativus L.) was crosslinked with gelatine by using glutaraldehyde and a bioactive layer was formed on the dissolved oxygen probe, therefore, ascorbate oxidase enzyme electrode which was sensitive for L-ascorbic acid determination was developed. Measurements were carried out by standard curves which were obtained by the determination of consumed oxygen level, related to L-ascorbic acid concentration in the enzymatic reaction. In the optimization studies of the biosensor prepared, the most suitable ascorbate oxidase, gelatine and glutaraldehyde ratios were determined as 24.8 U/cm-, 5.9 mggelatine/cm^ and, 2.5% respectively. The phosphate buffer (50 mM, pH:7.5) and 35° C were established as providing the optimum conditions. The characterization studies proved a linearity in the ascorbic acid concentration range 5.0xl0~5-1.2xl0~3 M. The reproducibility experiments (n=ll) revealed that for 4x10-4 M L-ascorbic acid, the average value (x) was 3.99x10-4 M,standard deviation (S.D) was ±0.001 and, variation coefficient (C.V) was 0.251%. Moreover, substrate specification, storage stability and the interference effects of some substances were investigated. Finally, L-ascorbic acid determination in some fruit extracts and vitamin tablets was carried out. Both the results obtained and the sensitivity of the method were compared with the 2,6 dichlorophenolindophenol method. Keywords: Ascorbate Oxidase, L- Ascorbic Acid, Enzyme Electrodes, Biosensors
Açıklama
Anahtar Kelimeler
Biyomühendislik, Bioengineering, Askorbik asit, Ascorbic acid, Enzimler, Enzymes
