L-Aspartate transaminase (L-AspAT) from E.coli is an efficient enzyme, and its advantages include stereoselectivity and one-step synthesis, amongst others. The study of L-AspAT was essential for chiral amine biosynthesis, and in the wild E.coli L-AspAT was expressed in an E.coli engineering stain BL21(DE3) with high efficiency. The enzyme was purified to allow the identification of its catalytic function. The results showed that the enzyme was bifunctional with two catalytic abilities: the transamination of amino acids and a non-oxidative decarboxylation of dicarboxylic acid substrates. Transamination was first carried out in the reaction system during the reaction process. Then the decarboxylation reaction was started, and when the concentration of decarboxylation substrate in the reaction solution was sufficient, the two reactions influenced and promoted each other. With regard to the docking of L-AspAT with small molecules of the substrates of ammonia transfer and decarboxylation respectively, the molecular docking results showed a difference between the binding sites of L-AspAT for glutamate and ketoglutarate, and the bifunctional enzyme had two active catalytic centers that catalyze different reactions. The catalytic mechanism was also studied and it was found that the enzyme-catalyzed the enzymatic properties of the two reactions. The study also found that the optimum temperature of the transamination was 35 ℃ and that of the decarboxylation was 37 ℃. The optimum pH of both reactions was 8. 5 mmol/L at which level Cu2+ and Mg2+ can promote transamination, and 5 mmol/L Ni2+ can promote decarboxylation.
DING Xiaojie
,
LIU Jiali
,
YANG Jingwen
,
HU Xueqin
,
ZHANG Hongbin
. Clone, expression, functional characterization, and mechanism of amino acid transamination/decarboxylation bifunctional enzymes[J]. Food and Fermentation Industries, 2023
, 49(19)
: 7
-14
.
DOI: 10.13995/j.cnki.11-1802/ts.034741
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