Directed reconstruction of a novel ancestral alcohol dehydrogenase featuring shifted pH-profile, enhanced thermostability and expanded substrate spectrum. (November 2022)

• Record Type: Journal Article
• Authors: Chen, Xiaoyu; Dou, Zhe; Luo, Tianwei; Sun, Zewen; Ma, Hongmin; Xu, Guochao; Ni, Ye
• Is Part Of: Bioresource technology. Volume 363(2022)
• Publication Date: 2022-11
• Subjects: Alcohol dehydrogenase -- Ancestral sequence reconstruction -- pH-profile -- Thermostability -- Substrate spectrum; Biomass -- Periodicals; Biomass energy -- Periodicals; Bioremediation -- Periodicals; Agricultural wastes -- Periodicals; Factory and trade waste -- Periodicals; Organic wastes -- Periodicals; Bioénergie -- Périodiques; Déchets agricoles -- Périodiques; Déchets industriels -- Périodiques; Déchets organiques -- Périodiques; Déchets (Combustible) -- Périodiques; 662.88
• DOI: 10.1016/j.biortech.2022.127886
• Languages: English
• ISSNs: 0960-8524

Abstract

Graphical abstract: Highlights: An effective protocol for directed resurrection of ancestral enzymes. Ancestral ADH A64 was constructed with activity toward CPMK. A64 displays shifted pH profile, enhanced stability and expanded substrate spectrum. Shannon-Wiener index was used to describe the breadth of substrate spectrum of ancestral enzymes. Abstract: 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 Kp ADH 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 Tm 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.