Active Expression of Membrane-Bound L-Amino Acid Deaminase from Proteus mirabilis in Recombinant Escherichia coli by Fusion with Maltose-Binding Protein for Enhanced Catalytic Performance

Abstract

L-amino acid deaminases (LAADs) are membrane flavoenzymes that catalyze the deamination of neutral and aromatic L-amino acids to α-keto acids and ammonia. LAADs can be used to develop many important biotechnological applications. However, the transmembrane α-helix of LAADs restricts its soluble active expression and purification from a heterologous host, such as Escherichia coli. Herein, through fusion with the maltose-binding protein (MBP) tag, the recombinant E. coli BL21 (DE3)/pET-21b-MBP-PmLAAD was constructed and the LAAD from Proteus mirabilis (PmLAAD) was actively expressed as a soluble protein. After purification, the purified MBP-PmLAAD was obtained. Then, the catalytic activity of the MBP-PmLAAD fusion protein was determined and compared with the non-fused PmLAAD. After fusion with the MBP-tag, the catalytic efficiency of the MBP-PmLAAD cell lysate was much higher than that of the membrane-bound PmLAAD whole cells. The soluble MBP-PmLAAD cell lysate catalyzed the conversion of 100 mM L-phenylalanine (L-Phe) to phenylpyruvic acid (PPA) with a 100% yield in 6 h. Therefore, the fusion of the MBP-tag not only improved the soluble expression of the PmLAAD membrane-bound protein, but also increased its catalytic performance.

Highly selective synthesis of d-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory

“…At the same time, under the optimal induction conditions, E. coli pET-21b-MBP- laad /pET-28a- dapdh - fdh had the highest catalytic activity (the optimal IPTG concentration was 0.75 mM and the optimal induction temperature was 30°C). According to our previous research, the fusion of the MBP-tag not only improves the soluble expression of the membrane-bound LAAD, but also increases its catalytic performance [ 29 ]. Therefore, in the E. coli pET-21b-MBP- laad /pET-28a- dapdh - fdh co-expression system, the improvement of the catalytic activity of LAAD increased the catalytic activity of the E. coli pET-21b-MBP- laad /pET-28a- dapdh - fdh whole-cell catalyst.…”

“…The mixture was centrifuged at 12000× g for 5 min. The amount of PPA generated in the reaction process was determined using ferric chloride solution [ 8 , 29 , 40 ] and the amounts of l -Phe and d -Phe were determined by HPLC after derivation with 1-fluor-2,4-dinitrophenyl-5- l -alanine amide (FDAA) [ 2 , 8 , 29 ].…”

“…BAD40410.1), and fdh from B. stabilis (GenBank accession no. ACF35003.1) were stored in our laboratory [8,29,[35][36][37][38]. Enzymes, vectors, oligonucleotides, and other reagents for DNA cloning and amplification were obtained from Takara-Bio Co. (Kusatsu, Japan) and Novagen Co. (Madison, WI, USA).…”

“…We previously constructed a biocatalytic cascade system for the asymmetric synthesis of d -amino acids by the stereoinversion of l -amino acids, using a combination of LAAD whole-cells ( l -amino acid deaminase from Proteus mirabilis ) (oxidative deamination module), DAPDH (H227V variant of meso -diaminopimelate dehydrogenases from Symbiobacterium thermophilum ) (reductive amination module), and FDH (formate dehydrogenase from Burkholderia stabilis ) (cofactor regeneration) [ 8 , 29 ]. However, in this biocatalytic cascade system, the reaction intermediate needs to be transferred through the cell membrane for the connection between the two necessary steps of the stereoinversion reaction, which would affect the conversion efficiency of the entire biocatalytic system.…”

“…In the in vivo cascade catalytic system, LAAD catalyzing oxidative deamination and DAPDH catalyzing reductive amination were mainly used to catalyze the stereoinversion from l -amino acids to d -amino acids. By using only LAAD and DAPDH in the cells, it would be feasible to perform the stereoinversion transformation from l -amino acids to d -amino acids [ 8 , 29 ]. Because DAPDH is NADPH dependent, however, FDH was used to construct the NADPH recycling system, improving the cofactor regeneration and the conversion efficiency of the entire system.…”