BACKGROUND:The metal-organic framework (MOF) ZIF-8 has the advantages of a large specific surface area and high stability. A new strategy for the assembly of amino acid dehydrogenase with peptide linker and for improving enzyme immobilization is proposed.RESULTS: Peptide linker enhanced enzyme activity after immobilization by 138.7%, and maintained 81.2% of its initial activity after seven recycles. The stability of PheDH_1D02/ZIF-8 was greatly enhanced at 70-80 °C and pH 10-11. The catalytic efficiency of PheDH_1D02/ZIF-8 was also enhanced compared with PheDH_1C1D/ZIF-8 and two free enzymes.CONCLUSIONS: Metal ions coordinated with the peptide linker and reoriented immobilization of enzyme onto the MOF with high enzyme loading capacity were achieved. The mesoporous structure of PheDH_1D02/ZIF-8 facilitates substrate diffusion. Combining the assembly of peptide linker and MOF immobilization is a simple, economical and efficient method for improving dehydrogenase stability.

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To design D-amino acid dehydrogenase (DAADH) for enhanced stability, the interactions of the subunit interfaces of DAADH were analyzed. Interaction network analysis of DAADH indicated that there are only weak interactions between the A and B subunits. Several co-evolved residue pairs were selected for mutation to enhance interfacial interactions of subunits, and 11 designed MDHs were obtained. DA06 and DA11 were selected for experimental verification for their salt bridges are 1.4 and 1.2-fold of that of DAwild, respectively. DA11 can maintain 93% activity in 80℃, while it was only 40% for DAwild. Thermostabiliy study indicated the half-life of DA11 was 2-fold of DAwild. Molecular dynamics simulations revealed that the extraordinary stability of the DA11 was due to the formation of extra interfacial salt bridges. The paper provided a strategy of mutations outside the active site of enzyme by co-evolutionary analysis which can reduce the effect of the activity-thermostability trade-off.