การตรึงเอนไซม์แซนทีนออกซิเดสเพื่อใช้วัดความสดของปลา / บริพันธ์ คงเผ่า = Immobilization of xanthine oxidase for determination of fish freshness / Boripan Kongpow
The objective of this study is the preparation of immobilized xanthine oxidase for the determination of fish freshness, by measuring the amount of hypoxanthine in fish muscle. Xanthine oxidase was immobilized on glass bead by covalent method. Two solutions were employed. Aminopropyltriethoxisilane (APTS) was used to activate glass bead, and then glutaraldyhyde was used to link between glass bead and the enzyme. It was found that the optimum condition was to use 10% v/v of APTS and 5% v/v of glutaraldyhyde for enzyme immobilization. The amount of glass bead (50-100 mesh) had to be 2 grams and the concentration of enzyme was 0.20 U/ml. The unbound enzyme was then rinsed twice with distilled water. An immobilized enzyme had optimum pH and temperature at 8.0 and 35 ํC, respectively. Enzyme was capable to react repeatedly at least 4 times at 25 ํC, and could last for 20 days at 4 ํC. An immobilized enzyme was then used to determine hypoxanthine in two species of Threadfin bream, Nemipterus hexodon and Nemipterus furcosus, and Bigeye fish (Priacanthus tayenus) which were kept on ice for less than 14 days. Results obtained from the use of free enzyme and immobilized enzyme were significantly related (r2 = 0.96 for Nemipterus hexodon muscle, r2 = 0.90 for Nemipterus furcosus muscle and r2 = 0.95 for Bigeye fish muscle). The determination of fish freshness from the amount of hypoxanthine for Nemipterus hexodon, Nemipterus furcosus and Bigeye fish were found to be less than 0.12, 0.20 and 0.20 mu mole/g sample respectively for very fresh fish and more than 0.80, 1.00 and 0.50 mu mole/g sample respectively for the fish which started to deteriorate.
