Hirokawa Kozo, Gomi Keiko, Bakke Mikio, Kajiyama Naoki
Research and Development Division, Kikkoman Corporation, 399 Noda, Chiba Pref. 278-0037, Noda-city, Japan.
Arch Microbiol. 2003 Sep;180(3):227-31. doi: 10.1007/s00203-003-0584-x. Epub 2003 Jul 17.
Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N(alpha)-fructosyl valyl-histidine (alpha-keto-amine) and N(epsilon)-fructosyl lysine (epsilon-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both alpha-keto-amine and epsilon-keto-amine, and (2) enzymes that preferably oxidize alpha-keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric ( M(r)=50,000), was most active at 40 degrees C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K(m) values for N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine were 2.30 and 1.69 mM, respectively. N(alpha)-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A(1C), the N-terminal valine residue of the beta-subunit of which is glycated.
我们对真菌培养物保藏库进行了筛选，以寻找果糖基肽氧化酶，这种酶可用于测定患有高血糖症的糖尿病患者的糖化血红蛋白。在八个属的菌株中发现了果糖基肽氧化酶：拟枝孢霉属、枝孢霉属、毛壳菌属、附球菌属、真青霉属、胶孢壳属、小囊菌属和嗜热栖热菌属。根据果糖基肽氧化酶对N（α）-果糖基缬氨酰-组氨酸（α-酮胺）和N（ε）-果糖基赖氨酸（ε-酮胺）的底物特异性，可将其分为两类：（1）氧化α-酮胺和ε-酮胺的酶，以及（2）优先氧化α-酮胺的酶。从绿色拟枝孢霉ATCC 32393中分离出一种对N（α）-果糖基缬氨酰-组氨酸和N（ε）-果糖基赖氨酸均有活性的果糖基肽氧化酶，并将其纯化至同质状态并进行了表征。该酶为单体（M（r）=50,000），在40℃和pH 8.0时活性最高，并且具有共价结合的黄素作为辅基。N（α）-果糖基缬氨酰-组氨酸和N（ε）-果糖基赖氨酸的表观K（m）值分别为2.30和1.69 mM。果糖基肽氧化酶反应消耗了N（α）-果糖基缬氨酰-组氨酸，并产生了相同摩尔量的缬氨酰-组氨酸。这种酶可用于测量血红蛋白A（1C），其β亚基的N末端缬氨酸残基被糖化。
