Ancestral enzymes are promising for industrial biotechnology due to high stability and catalytic promiscuity. An effective protocol was developed for the directed resurrection of ancestral enzymes. Employing genome mining with diaryl alcohol dehydrogenase KpADH as the probe, descendant enzymes D10 and D11 were firstly identified. Then through ancestral sequence reconstruction, A64 was resurrected with a specific activity of 4.3 U·mg⁻¹. The optimum pH of A64 was 7.5, distinct from 5.5 of D10. The T15 50 and T_m values of A64 were 57.5 °C and 61.7 °C, significantly higher than those of the descendant counterpart. Substrate spectrum of A64 was quantitively characterized with a Shannon-Wiener index of 2.38, more expanded than D10, especially, towards bulky ketones in Group A and B. A64 also exhibited higher enantioselectivity. This study provides an effective protocol for constructing of ancestral enzymes and an efficient ancestral enzyme of industrial relevance for asymmetric synthesis of chiral alcohols.

由于高稳定性和催化混杂性，祖先酶有望用于工业生物技术。为祖先酶的定向复活制定了有效的协议。采用以二芳基醇脱氢酶Kp ADH为探针的基因组挖掘，首次鉴定出后代酶D10和D11。然后通过祖先序列重建，A64以4.3 U·mg ^-1的比活性复活。A64 的最适 pH 值为 7.5，与 D10 的 5.5 不同。T15 50 和 T _mA64的值分别为57.5°C和61.7°C，显着高于后代对应物。A64 的底物光谱定量表征为香农-维纳指数为 2.38，比 D10 扩展更多，尤其是对 A 组和 B 组中的大酮体。A64 也表现出更高的对映选择性。本研究为构建祖先酶提供了一种有效的方案，并为手性醇的不对称合成提供了一种工业相关的高效祖先酶。