Identification and characterization of an amidase from Leclercia adecarboxylata for efficient biosynthesis of L-phosphinothricin

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

“…All tested hydrolases were not very strictly specific to 2'-hydroxylation of ribose; however, they preferred the nucleosides as substrates and most of the enzymes did not hydrolyse cytosine (15)(16) or isocytosine (17) derivatives. Only the D8_RL amidohydrolase accepted heterocyclic base derivatives (15)(16)(17) as substrates. The bulkiness of the acyl group played a significant role, since the selected hydrolases were specific towards longer acyls in the case of aliphatic radicals (1, 2, 3, 11, 13).…”

“…In the case of the clone P4FUMM07, it could be concluded that the growth complementation of E. coli DH10B ∆pyrFEC::Km cells depended on the activity of the amidohydrolase P4FUMM07_AmH, but not the P4FUMM07_AcAm. All tested hydrolases were not very strictly specific to 2'-hydroxylation of ribose; however, they preferred the nucleosides as substrates and most of the enzymes did not hydrolyse cytosine (15)(16) or isocytosine (17) derivatives. Only the D8_RL amidohydrolase accepted heterocyclic base derivatives (15)(16)(17) as substrates.…”

“…The hydrolytic activity of the recombinant enzymes was assayed qualitatively by testing 29 different substrates ( Figure 4A): N 4 -acylated (2'-deoxy)cytidines and cytosines (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16); N 2 -acetylisocytosines (17); chromogenic substrates such as p-nitroacetanilide (18) and p-nitrobenzanilide (19); various p-nitrophenyl (pNP) esters (20)(21)(22)(23)(24); terephthalate derivatives bis(2-hydroxyethyl) terephthalate (26), dimethyl terephthalate (27), and monomethyl terephthalate (28); and the beta-lactam nitrocefin (29). Hydrolysis of chromogenic substrates (18)(19)(20)(21)(22)(23)(24)(25) was analysed spectrophotometrically at 405 nm, and that of other substrates was analysed spectrophotometrically at 240-320 nm and using TLC or HPLC-MS ( Figures S4-S10).…”

“…Amidohydrolases have potential applications in chemical synthesis, pharmaceuticals, and effluent treatment due to their remarkable chemo-, regio-, and enantio-selectivity. At present, amidases are used for the biosynthesis 2 of 12 of β-lactam antibiotics [13], and the production of (S)-4-fluorophenylglycine (a chiral intermediate for the synthesis of antiemetic drugs) [14], nicotinic [15] and 2-chloronicotinic acids [16], and the herbicide L-phosphinothricin [17]. L-asparaginase and L-glutaminase are used as oncolytic enzymes [18], and in the food industry for the production of acrylamide-free food (to reduce the formation of acrylamide during the frying of starchy foods at high temperatures) [18].…”

A Single‐Transaminase‐Catalyzed Biocatalytic Cascade for Efficient Asymmetric Synthesis of l‐Phosphinothricin

“…amidase a PPT derivative [d] 150 0.03 [a] -6.8 49.7 [18] α-transaminase (single enzyme reaction) 1 a 20 34 [b] > 4 equiv. glutamate 14 91.2 [19] AspTA and GOT (coupled enzyme cascade) 1 a 89 0.08 for TA [a] , 0.02 for GOT [a] 0.2 equiv.…”