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Immobilization of phenylalanine dehydrogenase : a comparative study of various supports, optimal conditions and characterization

หน่วยงาน จุฬาลงกรณ์มหาวิทยาลัย

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ชื่อเรื่อง : Immobilization of phenylalanine dehydrogenase : a comparative study of various supports, optimal conditions and characterization
นักวิจัย : Orada Chumphukam, 1980-
คำค้น : Phenylalanine , Amino acids , Immobilized enzymes
หน่วยงาน : จุฬาลงกรณ์มหาวิทยาลัย
ผู้ร่วมงาน : Manchumas Prousoontorn , Kanoktip Packdibamrung , Chulalongkorn University. Faculty of Science
ปีพิมพ์ : 2547
อ้างอิง : 9745313807 , http://cuir.car.chula.ac.th/handle/123456789/3995
ที่มา : -
ความเชี่ยวชาญ : -
ความสัมพันธ์ : -
ขอบเขตของเนื้อหา : -
บทคัดย่อ/คำอธิบาย :

Thesis (M.Sc.)--Chulalongkorn University, 2004

Phenylalanine dehydrogenase (PheDH) produced by Escherichia coli transformant was partially purified by ammonium sulfate precipitation, DEAE-Toyopearl and Butyl-Toyopearl column chromatography with a 23.9% yield and 10.1 purification fold. The relative molecular weight of subunit was estimated to be 45,000 Da. The enzyme preferably acted on L-phenylalanine in oxidative deamination and phenylpyruvate in reductive amination and required NAD[superscript +] as a natural coenzyme. The optimum pH for the oxidative deamination and reductive amination were 10.4 and 8.7, respectively whereas optimum temperatures were 45 ํC and 40ํC, respectively. This enzyme was stable over the pH range of 6.5-12.5 and retained full activity when subjected to temperature at 35 ํC for 10 minutes. PheDH was then covalently immobilized or adsorbed on various supports including alumina, silica, polyvinyl (alcohol) bead and chitosan. Different activating agents were used: 1, 4-butanediol diglycidyl ether, tresyl chloride and glutaraldehyde. Silica with glutaraldehyde as a cross-linking agent was found to be the most appropriate support and method. The optimum condition for enzyme immobilization was to activate silica with 6% (v/v) [gamma]-aminopropyltriethoxysilane and 0.1 % (v/v) glutaraldehyde. PheDH was then added and incubated at 4 ํC for 6 hours, using an enzyme to support ratio of 16 units per gram silica. The activity of the immobilized enzyme was 0.14 U/g silica (1.05% of its original activity). When compared to the free enzyme, using coupled reaction with diaphorase, there was no change in pH optima (9.0) whereas the optimum temperature of the immobilized enzyme showed a broad range of 30 ํC to 55 ํC. The pH stability and thermostability of immobilized PheDH were closely similar to that of free enzyme. The immobilized PheDH exhibited good storage stability. For the batch production of amino acids, immobilized PheDH produced amino acids from their keto substrates with various % conversions from 63.7 to 100%

บรรณานุกรม :
Orada Chumphukam, 1980- . (2547). Immobilization of phenylalanine dehydrogenase : a comparative study of various supports, optimal conditions and characterization.
    กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย.
Orada Chumphukam, 1980- . 2547. "Immobilization of phenylalanine dehydrogenase : a comparative study of various supports, optimal conditions and characterization".
    กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย.
Orada Chumphukam, 1980- . "Immobilization of phenylalanine dehydrogenase : a comparative study of various supports, optimal conditions and characterization."
    กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย, 2547. Print.
Orada Chumphukam, 1980- . Immobilization of phenylalanine dehydrogenase : a comparative study of various supports, optimal conditions and characterization. กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย; 2547.