Yano Shigekazu, Hori Yukari, Kijima Tatsuro, Konno Hiroyuki, Suyotha Wasana, Takagi Kazuyoshi, Wakayama Mamoru
1 Department of Biochemical Engineering, Graduate School of Sciences and Engineering, Yamagata University.
2 Department of Industrial Biotechnology, Faculty of Agro-industry.
J Appl Glycosci (1999). 2019 May 21;66(2):65-72. doi: 10.5458/jag.jag.JAG-2018_0011. eCollection 2019.
The cellulose binding domain (CBD) of cellulosome-integrating protein A from NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16-2.5 mM glucose, with a correlation coefficient, , of 0.998.
将来自日本微生物菌种保藏中心（NBRC）103400的纤维小体整合蛋白A的纤维素结合结构域（CBD）与来源于[具体来源未提及]的黄素单核苷酸（FMN）依赖性NADH偶氮还原酶（AZR）和葡萄糖1-脱氢酶（GDH）进行基因融合。融合酶AZR-CBD和CBD-GDH在Rosetta-gami B（DE3）中表达。这些酶从无细胞提取物中纯化得到，AZR-CBD的比活性为15.1 U/mg，CBD-GDH的比活性为22.6 U/mg。AZR-CBD和CBD-GDH分别以初始蛋白量的约95%和98%强烈结合到0.5%的膨胀纤维素上。固定到膨胀纤维素上后，AZR-CBD和CBD-GDH保留了它们的催化活性。两种酶与0.5%的微晶纤维素结合较弱，但添加高浓度的微晶纤维素（10%）提高了两种酶的结合率。一个用于流动注射分析的反应器填充了固定有微晶纤维素的AZR-CBD和CBD-GDH。该流动注射分析系统成功应用于葡萄糖的测定，在约0.16 - 2.5 mM葡萄糖范围内观察到线性校准曲线，相关系数为0.998。
