Characterization and improvement of substrate-binding affinity of d-aspartate oxidase of the thermophilic fungus Thermomyces dupontii
Characterization and improvement of substrate-binding affinity of d-aspartate oxidase of the thermophilic fungus Thermomyces dupontii
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DOI：
10.1007/s00253-019-09787-y
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发表时间：
2019-04
影响因子：
5
通讯作者：
Shouji Takahashi;Kohei Osugi;Y. Shimekake;A. Shinbo;K. Abe;Y. Kera
中科院分区：
文献类型：
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作者：
Shouji Takahashi;Kohei Osugi;Y. Shimekake;A. Shinbo;K. Abe;Y. Kera
天冬氨酸氧化酶（DDO）是一种重要的酶，可用于酸性氨基酸的测定和酸性氨基酸外消旋混合物的光学拆分，这些都要求其具有较高的稳定性、催化活性和底物亲和力。本研究对一株嗜热真菌杜邦嗜热菌的DDO基因（TdDDO）进行了鉴定，并对重组酶在大肠杆菌中的表达进行了鉴定。此外，我们产生了具有更高底物结合亲和力的变体。重组TdDDO在E.大肠杆菌对酸性氨基酸和中性氨基酸d-Gln有氧化酶活性，对dd-Glu的酶活性最高。d-Glu的Kmandkcat值分别为2.16 mM和217 s-1。该酶的最适pH为8.0，最适温度为60 °C，在pH5.0 ~ 10.0范围内稳定，aT 50约为10.0。51 °C，远高于其他来源的DDO。随着蛋白质浓度的降低，酶的稳定性下降，并且外部添加FAD不能抑制酶的不稳定性。将TdDDO活性位点的Phe 248突变为DDO和D-氨基酸氧化酶中保守的Tyr残基，可显著提高底物结合亲和力。结果表明，TdDDO及其变体在实际应用中具有很大的潜力.
d-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidicd-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus,Thermomyces dupontii, and characterized the recombinant enzyme expressed inEscherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed inE. coliexhibited oxidase activity toward acidicd-amino acids and a neutrald-amino acid,d-Gln, with the highest activity towardd-Glu. TheKmandkcatvalues ford-Glu were 2.16 mM and 217 s−1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with aT50of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs andd-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.