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Penicilium verruculosum 의 Glucose oxidase 정제
황현주,김두식 ( Hyun Joo Hwang,Doo Sik Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4
Glucose oxidase (EC 1.1.3.4) was purified to homogeneity from Penicillium verruculosum. A combination of ammonium sulfate fractionation, ion exchange and adsorption chromatographic procedure yields the pure enzyme that gives a single band on polyacrylamide gel electrophoresis. The purified glucose oxidase is characterized to be a dimeric glycoprotein with M.W. 150,000 composed of two identical size of polypeptide chains. It appears that the subunits are strongly associated by noncovalent interactions. Apparent K_m and V_(max) for the enzyme were determined to be 1.9×10^(-2) M and 0.15×10^(-3) ㏖/l/min for glucose. Catalytic function of the glucose oxidase is rapidly and irreversibly inhibited in the presence of either Ag^+ or Hg^(2+). Chemical modification studies suggest that histidine and lysine residues maybe involved in its catalytic mechanism.
Penicilium verruculosum의 Glucose oxidase 정제
황현주,김두식,Hwang, Hyun-Joo,Kim, Doo-Sik 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4
Penicilium verruculosum 배양액으로부터 황산암모늄 분별침전, 이온교환 및 흡착 크로마토그라피 과정을 거쳐 순수하게 정제한 glucose oxidase (EC 1.1.3.4)의 비활성도는 115.4 units/mg이었다. 정제된 효소단백질분자는 동일한 크기를 갖는 두개의 subunit으로 구성된 분자량 150,000의 당단백질임을 확인하였으며, 두 subunit은 비공유결합력에 의해 dimer 구조를 이루는 것으로 판명되었다. 효소의 글루코스에 대한 $K_m$과 $V_{max}$는 각각 $1.9{\times}10^{-2}\;M$과 $0.15{\times}10^{-3}\;mol/l/min$로 측정되었고, 촉매활성에 대한 최적 pH는 6.0, 최적온도는 $30^{\circ}C$로 나타났다. 효소의 촉매기능은 $Ag^+$ 혹은 $Hg^{2+}$에 의해 급격하게 비가역적으로 억제됨을 관찰하였으며, 화학변형실험의 결과로부터 glucose oxidase의 활성에 histidine의 imidazole group과 lysine의 $\varepsilon$-amino group이 관여하는 것으로 추정하였다. Glucose oxidase (EC 1.1.3.4) was purified to homogeneity from Penicillium verruculosum. A combination of ammonium sulfate fractionation, ion exchange and adsorption chromatographic procedure yields the pure enzyme that gives a single band on polyacrylamide gel electrophoresis. The purified glucose oxidase is characterized to be a dimeric glycoprotein with M.W. 150,000 composed of two identical size of polypeptide chains. It appears that the subunits are strongly associated by noncovalent interactions. Apparent $K_m$ and $V_{max}$ for the enzyme were determined to be $1.9{\times}10^{-2}\;M$ and $0.15{\times}10^{-3}\;mol/l/min$ for glucose. Catalytic function of the glucose oxidase is rapidly and irreversibly inhibited in the presence of either $Ag^+$ or $Hg^{2+}$. Chemical modification studies suggest that histidine and lysine residues may be involved in its catalytic mechanism.