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
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
复制标题
通过与麦芽糖结合蛋白融合，在重组大肠杆菌中主动表达来自奇异变形杆菌的膜结合 L-氨基酸脱氨酶，以增强催化性能
DOI：
10.3390/catal10020215
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发表时间：
2020-02-01
期刊：
影响因子：
3.9
通讯作者：
Xu, Yan
中科院分区：
文献类型：
--
作者：
Zhang, Dan-Ping;Jing, Xiao-Ran;Xu, Yan
L氨基酸脱氨酶(LAADs)是一种膜黄素酶，催化中性和芳香的L氨基酸脱氨为α-酮酸和氨。LAAD可用于开发许多重要的生物技术应用。然而，LAADs的跨膜α螺旋限制了其在异源宿主(如大肠杆菌)中的可溶性活性表达和纯化。因此，通过与麦芽糖结合蛋白(MBP)标签的融合，构建了重组大肠杆菌BL21(DE3)/pET-21b-MBP-PmLAAD，并以可溶性蛋白的形式高效表达了奇异变形杆菌LAAD。纯化后得到了纯化的MBP-PmLAAD。然后，测定了MBP-PmLAAD融合蛋白的催化活性，并与未融合的PmLAAD进行了比较。与MBP-Tag融合后，MBP-PmLAAD细胞裂解物的催化效率明显高于膜结合型PmLAAD全细胞。可溶性MBP-PmLAAD细胞裂解产物在6h内催化100mM L-苯丙氨酸(L-Phe)转化为苯丙酮酸(PPA)，产率为100%，因此，MBP-Tag的融合不仅提高了PmLAAD膜结合蛋白的可溶性表达，而且提高了其催化性能。
L-amino acid deaminases (LAADs) are membrane flavoenzymes that catalyze the deamination of neutral and aromatic L-amino acids to alpha-keto acids and ammonia. LAADs can be used to develop many important biotechnological applications. However, the transmembrane alpha-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.