Journal
FRONTIERS IN MICROBIOLOGY
Volume 13, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2022.1025845
Keywords
membrane-binding domain; site-saturation mutagenesis; bioconversion; alpha-ketoisocaproate; L-amino acid deaminase
Categories
Funding
- National Natural Science Foundation of China
- Zhejiang Provincial Natural Science Foundation of China
- [31901930]
- [LGN22C140006]
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Alpha-keto acids are important raw materials for pharmaceuticals and functional foods. Improving the catalytic activity of L-AADs by modifying the membrane-bound region can enhance their efficiency.
alpha-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 alpha-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.
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