Semi-rational engineering membrane binding domain of L-amino acid deaminase from Proteus vulgaris for enhanced α-ketoisocaproate.
Semi-rational engineering membrane binding domain of L-amino acid deaminase from Proteus vulgaris for enhanced α-ketoisocaproate.
复制标题
普通变形杆菌L-氨基酸脱氨酶的半理性工程膜结合域增强α-酮异己酸
DOI：
10.3389/fmicb.2022.1025845
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发表时间：
2022
影响因子：
5.2
通讯作者：
Yu, Xiaoping
中科院分区：
文献类型：
--
作者：
Song, Yang;Wang, Rui;Zhang, Zixuan;Liu, Xinran;Qi, Lulu;Shentu, Xuping;Yu, Xiaoping
关键词：
α-酮酸是重要的药物和功能食品原料，利用含L-氨基酸脱氨酶（L-AAD s）的全细胞生物催化剂，可以从廉价的原料中生产α-酮酸。然而，L-AAD的低活性限制了生产能力。L-AAD介导的氧化还原反应利用电子传递链将电子从还原的FADH 2转移到O2，这意味着L-AAD与细胞膜之间的相互作用影响其催化活性。为了提高普通变形杆菌L-AAD的催化活性，我们通过饱和诱变和高通量筛选，重新设计了膜结合疏水插入序列（INS，残基325-375）。突变体D340 N和L363 N对L-亮氨酸表现出更高的亲和力和催化效率，半衰期分别是野生型L-AAD的1.62倍和1.28倍。D340 N菌株发酵L-亮氨酸的转化率为89.06%，比野生型菌株提高了17.57%。据预测，突变增强了蛋白质和细胞膜之间的相互作用。
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.
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影响因子：
3.7
作者：
Song Y;Li J;Shin HD;Liu L;Du G;Chen J
通讯作者：
Chen J
影响因子：
5.6
作者：
Huang, Harris K-H.;Taneva, Svetla G.;Cornell, Rosemary B.
通讯作者：
Cornell, Rosemary B.
影响因子：
3.9
作者：
Li, Ruoxi;Sakir, Hossain Gazi;Liu, Long
通讯作者：
Liu, Long
影响因子：
4.4
作者：
JORGENSEN, WL;CHANDRASEKHAR, J;KLEIN, ML
通讯作者：
KLEIN, ML
影响因子：
5.8
作者：
ORELAND, L;EKSTEDT, B
通讯作者：
EKSTEDT, B