Xiaoxiang Hu,Yajun Bai,Tai-Ping Fan,Xiaohui Zheng,Yujie Cai
A novel alanine dehydrogenase (ADH; EC.1.4.1.1) with high pyruvate reduced activity was isolated from Helicobacter aurati and expressed in Escherichia coli BL21 (DE3). The optimum pH of the reduction and oxidation reaction were 8.0 and 9.0, respectively, and the optimum temperature was 55 °C. With pyruvate and alanine as substrates, the specific activity of HAADH1 were 268 U·mg −1 and 26 U·mg −1 , respectively. HAADH1 had a prominent substrate specificity for alanine ( K m = 2.23 mM, k cat / K m = 8.1 s −1 ·mM −1 ). In the reduction reaction, HAADH1 showed the highest substrate affinity for pyruvate ( K m = 0.56 mM, k cat / K m = 364 s −1 ·mM −1 ). Compared to pyruvate, oxaloacetic acid, 2-ketobutyric acid, 3-fluoropyruvate, α-ketoglutaric acids, glyoxylic acid showed a residual activity of 93.30%, 8.93%, 5.62%, 2.57%, 2.51%, respectively. Phylogenetic tree analysis showed that this is a new type of ADH which have a low sequence similarity to available ADH reported in references. 3-Fluoropyruvate was effectively reduced to 3-fluoro-L-alanine by whole-cell catalysis.
Qi Li,Ruiying Gao,Yucheng Li,Bo Fan,Cuiluan Ma,Yu-Cai He
Lignin is a critical biopolymer for creating a large number of highly valuable biobased compounds. Vanillin, one of lignin-derived aromatics, can be used to synthesize vanillylamine that is a key fine chemical and pharmaceutical intermediate. To produce vanillylamine, a productive whole-cell-catalyzed biotransformation of vanillin was developed in deep eutectic solvent − surfactant − H 2 O media. One newly created recombinant E. coli 30CA cells expressing ω-transaminase and L -alanine dehydrogenase was employed to transform 50 mM and 60 mM vanillin into vanillylamine in the yield of 82.2% and 8.5% under 40 °C, respectively. The biotransamination efficiency was enhanced by introducing surfactant PEG-2000 (40 mM) and deep eutectic solvent ChCl:LA (5.0 wt%, pH 8.0), and the highest vanillylamine yield reached 90.0% from 60 mM vanillin. Building an effective bioprocess was utilized for transamination of lignin-derived vanillin to vanillylamine with newly created bacteria in an eco-friendly medium, which had potential application for valorization of lignin to value-added compounds.
Boyu Yang,Chao Li,Yongshuo Ren,Weichen Wang,Xiangxiang Zhang,Xiaojun Han
The bottom-up construction of artificial cells is beneficial for understanding cell working mechanisms. The glycolysis metabolism mimicry inside artificial cells is challenging. Herein, the glycolytic pathway (Entner–Doudoroff pathway in archaea) is reconstituted inside artificial cells. The glycolytic pathway comprising glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxygluconate aldolase (KDGA) converts glucose molecules to pyruvate molecules. Inside artificial cells, pyruvate molecules are further converted into alanine with the help of alanine dehydrogenase (AlaDH) to build a metabolic pathway for synthesizing amino acid. On the other hand, the pyruvate molecules from glycolysis stimulate the living mitochondria to produce ATP inside artificial cells, which further trigger actin monomers to polymerize to form actin filaments. With the addition of methylcellulose inside the artificial cell, the actin filaments form adjacent to the inner lipid bilayer, deforming the artificial cell from a spherical shape to a spindle shape. The spindle-shaped artificial cell reverses to a spherical shape by depolymerizing the actin filament upon laser irradiation. The glycolytic pathway and its further extension to produce amino acids (or ATP) inside artificial cells pave the path to build functional artificial cells with more complicated metabolic pathways.
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