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

Dan-Ping Zhang,An-Wen Fan,Huan Liu,Xiao-Ran Jing,Yao Nie,Yan Xu

doi:10.3390/catal10020215

Dan-Ping Zhang, An-Wen Fan + Show 4 more

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https://doi.org/10.3390/catal10020215

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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.

Highlights

L-amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acid from L-amino acid and release ammonium and H2 O as well [1,2,3,4]
The PCR products of PmLAAD were inserted into the vector pET-21b cell lysate (pET-21b)
The membrane-bound protein PmLAAD was successfully expressed as a soluble active protein and lost its membrane-binding status

Summary

Introduction

L-amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acid from L-amino acid and release ammonium and H2 O as well [1,2,3,4]. LAADs have been identified in several bacterial genera including Proteus, Providencia, and Morganella [2,5] Some bacteria, such as Proteus mirabilis, express two types of LAAD which have similar sequences but have distinct substrate preferences: type I prefers aliphatic and aromatic amino acids, while type II shows significant activity with basic amino acids such as histidine and arginine [2,3,6,7,8,9]. Both types of LAAD contain a single membrane-spanning helix, which is anchored to the cytomembrane surface through the N-terminal transmembrane helix [2,9]. A series of LAAD-based technologies has been developed to efficiently transform L-amino acids to α-keto acids such as ketoglutaric acid, Catalysts 2020, 10, 215; doi:10.3390/catal10020215 www.mdpi.com/journal/catalysts

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