효소 및 미생물의 고정화에 관한 연구 제1보. 방사선조사에 의한 Glucose Oxidase의 고정화법

Studies on the Immobilization of Enzymes and Microoganism Part 1. Immobilizing Method of Glucose Oxidase by Gamma Radiation

혈액 및 생체반응물질등에 함유된 미량의 glucose를 효소적으로 간편하게 측량하기 위하여 먼저 고가의 glucose oxidase를 고정화 시켰다. 전리 방사선조사에 의하여 이 효소를 쉽게 고정화 시킬수 있는 방법과 그 알맞는 조건 및 높은 잔여 활성에 관한 결과는 다음과 같다. 1) 여러가지 monomer 및 polymer의 combination 중에서 AA-Bis, NK ester 23G, 물 (1:1:2)에 가용성 효소 1ml를 고정화 시켰을 경우를 비롯하여 GOD의 잔여활성이 50%이상인 여러 monomer Combination 찾았다 2) Carrier의 방사선중합에 필요한 선량은 100 krad 이상 이였으나, 400~500krad가 적당하였고, solvent는 toluene, n-haxane, petoleum ether chloroform 등을 이용할 때 고정화된 GOD의 잔여 활성 및 이화학적 성장이 좋았다. 3) GOD 고정화에 이용될 수 있는 완충 용액은 tris-glycerol buffer (pH 7.0)가 phosphate (pH 7.0) 보다 높은 활성을 보여 주었다. 4) 고정화된 GOD의 최적 pH는 6.0~6.5 또 온도는 30~4$0^{\circ}C$로서 가용성 효소보다 작용범위가 넓었고, pH 및 온도의 변화도 완만하였다. The author acknowledges with gratitude financial assistance from International Atomic Energy Agency, Vienna, Austria and also sincerely thanks Dr. K. Kawashima and his colleagues, National Food Research Institute, Tokyo, Iapan for encouragement and assistance.

A new method for immobilization of glucose oxidate by the aerobic gamma radiation of synthetic monomers was developed. The radiocopolymerization was conducted aerobically at -70 to -8$0^{\circ}C$ with the mixture of several polyfunctional esters, acrylates and native enzyme. The retained activity of immobilized glucose oxidase was about 50 to 55％ when a NK 23G ester, acrylamide-bis and water mixture (1:1:2) in cold toluene treated with 450 krad of gam-ma radiation. The radiation dose did not influence significantly to the enzyme activity. The solvents used to prepare the beads of glucose oxidase and monomers were toluene, n-hexane, petoleum ether and chloroform. 0.05M tris-gycerol (pH 7.0) was a more suitable bugger solution for immobilizing the enzyme than was 0.02M phosphate. Immobilization of glucose oxidase shifted the optimum pH for its reaction from 6.0 to 6.5. The pH profile for the immobilized enzyme showed a broad range of optimum activity while the native enzyme gave a sharp pick for its optimum pH value. The immobilized enzyme reaction temperature was at the range of 30~4$0^{\circ}C$.