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

Abstract

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 FADH2 to O2, 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.

Amino Acid Chirality: Stereospecific Conversion and Physiological Implications

“…The α-keto acids are intermediates in both amino acids as well the carbon metabolism pathways and precursors for the production of pharmaceutically important drugs. As such, LAADs have been suggested to be important biocatalysts for industrial production of α-keto acids such as ketoglutaric acid, methylthiobutyric acid, phenylpyruvic acid, etc., from the corresponding LAA s and potentially replace the harsh chemical methods currently used for α-keto acid synthesis . While LAOs and DAOs are potentially being employed for some of these applications, the recombinant LAAD overcomes certain limitations posed by these two enzymes; first, no H 2 O 2 is generated during catalysis, and second, the broader substrate specificity in comparison to the very narrow substrate selectivity in LAOs.…”

“…As such, LAADs have been suggested to be important biocatalysts for industrial production of α-keto acids such as ketoglutaric acid, methylthiobutyric acid, phenylpyruvic acid, etc., from the corresponding LAA s and potentially replace the harsh chemical methods currently used for α-keto acid synthesis. 121 While LAOs and DAOs are potentially being employed for some of these applications, the recombinant LAAD overcomes certain limitations posed by these two enzymes; first, no H 2 O 2 is generated during catalysis, and second, the broader substrate specificity in comparison to the very narrow substrate selectivity in LAOs. Moreover, recombinant LAADs can complement LAOs/DAOs as biosensors for resolution of racemic solutions of chiral amino acids, a role crucial in areas such as selection of chiral selective pharmaceutical drugs, determining nutritive values of processed food etc.…”