Reconstruction of Hyper-Thermostable Ancestral L-Amino Acid Oxidase to Perform Deracemization to D-Amino Acids
Reconstruction of Hyper-Thermostable Ancestral L-Amino Acid Oxidase to Perform Deracemization to D-Amino Acids
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DOI：
10.1002/cctc.202101296
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发表时间：
2021-11-02
期刊：
影响因子：
4.5
通讯作者：
Nakano, Shogo
中科院分区：
文献类型：
--
作者：
Ishida, Chiharu;Miyata, Ryo;Nakano, Shogo
L-氨基酸氧化酶(LAAO)具有广泛的底物专一性，可用于D，L-氨基酸(D，L-氨基酸)的D-对映体去消旋反应。超高温LAAO(HTAncLAAO)是通过人工序列数据挖掘和祖先序列重建相结合的方式设计的。利用大肠杆菌系统可以实现HTAncLAAO(>；50 mg/L)的可溶性表达。HTAncLAAO可识别7种L-氨基酸作为底物，具有极高的热稳定性和长期稳定性；t(1/2)值为95℃和99%ee(D-对映体)。这些结果表明HTAncLAAO是一种很好的生物催化剂来执行这种去消旋作用。
L-amino acid oxidases (LAAOs) with broad substrate specificity can be used in the deracemization of D,L-amino acids (D,L-AAs) to their D-enantiomers. Hyper-thermostable LAAO (HTAncLAAO) was designed through a combination of manual sequence data mining and ancestral sequence reconstruction. Soluble expression of HTAncLAAO (>50 mg/L) can be achieved using an E. coli system. HTAncLAAO, which recognizes seven L-AAs as substrates, exhibits extremely high thermal stability and long-term stability; the t(1/2) value was 95 degrees C and 99 % ee, D-enantiomer). These results suggest that HTAncLAAO is an excellent biocatalyst to perform this deracemization.