Semi-rational engineering membrane binding domain of L-amino acid deaminase from Proteus vulgaris for enhanced α-ketoisocaproate

α-Keto acids are important raw materials for pharmaceuticals and functional foods, which could be produced from cheap feed stock by whole cell biocatalysts containing L -amino acid deaminases ( L -AADs). However, the production capacity is limited by the low activity of L -AADs. The L -AAD mediated redox reaction employs the electron transport chain to transfer electrons from the reduced FADH 2 to O 2 , implying that the interaction between L -AAD and the cell membrane affects its catalytic activity. To improve the catalytic activity of L -AAD from Proteus vulgaris , we redesigned the membrane-bound hydrophobic insertion sequences (INS, residues 325–375) by saturation mutagenesis and high-throughput screening. Mutants D340N and L363N exhibited higher affinity and catalytic efficiency for L -leucine, with half-life 1.62-fold and 1.28-fold longer than that of wild-type L -AAD. D340N catalyzed L -leucine to produce 81.21 g⋅ L –1 α-ketoisocaproate, with a bioconversion rate of 89.06%, which was 17.57% higher than that of the wild-type. It is predicted that the mutations enhanced the interaction between the protein and the cell membrane.