Takahashi Shouji, Furukawara Makoto, Omae Keishi, Tadokoro Namiho, Saito Yayoi, Abe Katsumasa, Kera Yoshio
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
Appl Environ Microbiol. 2014 Dec;80(23):7219-29. doi: 10.1128/AEM.02193-14. Epub 2014 Sep 12.
d-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic d-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested d-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain d-amino acids, with the highest specific activity toward d-valine and catalytic efficiency (kcat/Km) toward d-leucine. Substrate inhibition was observed in the case of d-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain d-amino acids, and as a scaffold to generate a novel DAO via protein engineering.
D-氨基酸氧化酶（DAO）是一种在生物技术领域具有吸引力的酶，可用于多种应用，但其效用受到相对较差稳定性的限制。对细菌基因组数据库的搜索揭示了嗜热细菌嗜热栖热放线菌（RxDAO）中一个编码与DAO同源蛋白的基因。在大肠杆菌中表达的重组蛋白是一种单体蛋白，含有非共价结合的黄素腺嘌呤二核苷酸作为辅因子。该蛋白对中性和碱性D-氨基酸表现出氧化酶活性，并被DAO抑制剂苯甲酸显著抑制，但不受任何测试的D-天冬氨酸氧化酶（DDO）抑制剂抑制，因此表明该蛋白是DAO。RxDAO对支链D-氨基酸表现出更高的活性和亲和力，对D-缬氨酸的比活性最高，对D-亮氨酸的催化效率（kcat/Km）最高。在D-酪氨酸的情况下观察到底物抑制。该酶的最佳pH范围和温度分别为pH 7.5至10和65°C，在pH 5.0至pH 8.0之间稳定，T50（初始酶活性丧失50%时的温度）为64°C。在30°C孵育1周后未观察到酶活性丧失。该酶被苯甲基磺酰氟显著灭活，但不被已知抑制某些DAO的硫醇修饰试剂和焦碳酸二乙酯灭活。这些结果表明RxDAO是一种高度稳定的DAO，并表明该酶可能对实际应用有价值，例如支链D-氨基酸的测定和定量，以及作为通过蛋白质工程产生新型DAO的支架。
