RESUMO
Molecular information about family VIII esterases, which have similarities with class C ß-lactamases and penicillin-binding proteins, remains largely unknown. In this study, a novel family VIII esterase with ß-lactamase activity (PsEstA) from Paenibacillus sp. was characterized using several biochemical and biophysical methods. PsEstA was effective on a broad range of substrates including tertiary butyl acetate, glyceryl tributyrate, glucose pentaacetate, olive oil, and p-nitrophenyl esters. Additionally, PsEstA hydrolyzed nitrocefin, cefotaxime, and 7-aminocephalosporanic acid. Interestingly, two forms of immobilized PsEstA (CLEAs-PsEstA and mCLEAs-PsEstA) showed high recycling property and enhanced stability, but hybrid nanoflowers (hNFs) of PsEstA require improvement. This study provides a molecular understanding of substrate specificities, catalytic regulation, and immobilization of PsEstA, which can be efficiently used in biotechnological applications.
Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Paenibacillus/enzimologia , beta-Lactamases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/ultraestrutura , Catálise , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Simulação de Acoplamento Molecular , Mutação , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Penicilinas/química , Filogenia , Especificidade por Substrato/genéticaRESUMO
A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 Å, showed that the enzyme has a canonical α/ß hydrolase fold with an α-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (≤C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80°C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications.
Assuntos
Bacillaceae/química , Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Esterases/química , Nitrofenóis/química , Ácido Peracético/química , Sequência de Aminoácidos , Bacillaceae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Esterases/genética , Esterases/metabolismo , Expressão Gênica , Temperatura Alta , Cinética , Modelos Moleculares , Mutação , Nitrofenóis/metabolismo , Ácido Peracético/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato , TermodinâmicaRESUMO
Lactic acid bacteria (LAB) are sources of a large variety of microbial ester hydrolases because they can produce a wide range of short-chain esters, phenolic alcohols, and fatty acids. Here, a novel SGNH-type esterase (LpSGNH1) from Lactobacillus plantarum WCFS1 was identified, functionally characterized, and immobilized for biotechnological applications. Homologs of LpSGNH1 are also found in many lactic acid bacteria (LAB) species. Biochemical features of LpSGNH1 were investigated using mass spectrometry, gel filtration chromatography, enzyme kinetics, fluorescence, and circular dichroism (CD) spectroscopy. LpSGNH1 were retained its activity under conditions that would be encountered during fermentations. Interestingly, LpSGNH1 exhibited the ability to act on a broad range of substrates including ketoprofen acetate, cefotaxime (CTX), and 7-aminocephalosporanic acid (7-ACA) as well as glucose pentaacetate, acetylxylan, and acetylalginate, which make LpSGNH1 a great candidate for extensive industrial applications. Furthermore, cross-linked enzyme aggregates of LpSGNH1 (CLEA-LpSGNH1) displayed recycling ability and thermal stability compared to free LpSGNH1, which could be useful for industrial applications. This work highlights the importance of LpSGNH1 in the preparation of commercial compounds, and LpSGNH1 can be used as a model system of SGNH esterases in lactic acid bacteria.
