Organic solvent tolerant oxidoreductases are significant for both scientific research and biomanufacturing. However, it is really challenging to obtain oxidoreductases due to the shortages of natural resources and the difficulty to obtained it via protein modification. This review summarizes the recent advances in gene mining and structure-functional study of oxidoreductases from extremophiles for non-aqueous reaction systems. First, new strategies combining genome mining with bioinformatics provide new insights to the discovery and identification of novel extreme oxidoreductases. Second, analysis from the perspectives of amino acid interaction networks explain the organic solvent tolerant mechanism, which regulate the discrete structure-functional properties of extreme oxidoreductases. Third, further study by conservation and co-evolution analysis of extreme oxidoreductases provides new perspectives and strategies for designing robust enzymes for an organic media reaction system. Furthermore, the challenges and opportunities in designing biocatalysis non-aqueous systems are highlighted.

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Immobilization of D-amino acid dehydrogenase (DAADH) by the assembly of peptide linker was studied for biosynthesis of D-phenylalanine which is an unnatural amino acid. Hybrid material of ZIF-8 and reduced graphene oxide (RGO) were applied for the immobilization of DAADH from Ureibacillus thermosphaericus. Activity of DAADH/ZIF-8/RGO was enhanced by 1.65 folds than free enzyme. DAADH/ZIF-8/RGO remained 53.4% of its initial activity at 50 °C for 10 h. At the same time the free enzyme was inactivated. The result indicated that the immobilization greatly improved the thermostability of DAADH and the stability in hyperalkaline solution. Kinetic parameters indicated that DAADH/ZIF-8/RGO had greater affinity of phenylpyruvate as Vm /Km of DAADH/ZIF-8/RGO was 1.27-fold than free enzyme. After seven recycles, the activity of DAADH/ZIF-8/RGO remained 64.3%. Furthermore, one step separation and immobilization by ZIF-8/RGO/Ni-DAADH had 1.5-fold activity enhancement. Combination of peptide linker and MOF immobilization, thermostability of the dehydrogenase was significantly improved.

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