Substitutability of metal-binding sites in an alcohol dehydrogenase / 生物工程学报

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

Structure-function relationships of industrial enzymes / 生物工程学报

Industrial enzymes are the "chip" of modern bio-industries, supporting tens- and hundreds-fold of downstream industries development. Elucidating the relationships between enzyme structures and functions is fundamental for industrial applications. Recently, with the advanced developments of protein crystallization and computational simulation technologies, the structure-function relationships have been extensively studied, making the rational design and de novo design become possible. This paper reviews the progress of structure-function relationships of industrial enzymes and applications, and address future developments.

Protein engineering: from directed evolution to computational design / 生物工程学报

By constructing mutant libraries and utilizing high-throughput screening methods, directed evolution has emerged as the most popular strategy for protein design nowadays. In the past decade, taking advantages of computer performance and algorithms, computer-assisted protein design has rapidly developed and become a powerful method of protein engineering. Based on the simulation of protein structure and calculation of energy function, computational design can alter the substrate specificity and improve the thermostability of enzymes, as well as de novo design of artificial enzymes with expected functions. Recently, machine learning and other artificial intelligence technologies have also been applied to computational protein engineering, resulting in a series of remarkable applications. Along the lines of protein engineering, this paper reviews the progress and applications of computer-assisted protein design, and current trends and outlooks of the development.

Recent advances in directed evolution / 生物工程学报

Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e.g. stereo/regioselectivity and activity. This review summarized recent advances in directed evolution and its applications in biocatalysis field.