A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

Abstract

BackgroundMicrobial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities.Methodology/Principal FindingsBacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220.ConclusionEstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.

INFLUENCIA DE MICROORGANISMOS EN LA DISPONIBILIDAD DE FÓSFORO EN PLÁNTULAS DE CAFÉ (Coffea arabica)

“…En el agar tributirina se observó el halo de degradación alrededor de los pozos positivos, pero como las lipasas verdaderas catalizan la hidrólisis en las cadenas relativamente largas de acilgliceroles mayores de 10 átomos de carbono y el sustrato aportado por la tributirina consta de menos de 10 átomos de carbono (sustrato para esterasas), no permitió seleccionar los aislamientos con lipasas verdaderas, no obstante estas enzimas son capaces de hidrolizar la tributirina [11,14]. Por lo tanto, su halo de hidrólisis se tomó como una referencia para la observación de la fluorescencia en el medio de cultivo mínimo FAM + aceite de oliva suplementado con Rodamina B, que fue el ensayo determinante.…”

Molecular characterization, overexpression and comparison of esterases-encoding LipRT, Lip4 and Lip20 from moderately thermophilic and mesophilic bacteria

“…The 14 families of lipolytic enzymes identified by Rao et al [7] were used for constructing a phylogenetic tree. Figure 1a shows that Lip20, Lip4 and LipRT were classified into the Family IV, III and VIII clades.…”

Expressão e caracterização estrutural e funcional de sequências genômicas codificadoras de enzimas lipolíticas.

“…A atividade da enzima aumentou na presença de quase todos os íons (RAO et al, 2013). Cinco esterases metagenômicas de ambiente marinho responderam diferentemente à presença de 2 a 50% de glicerol (TCHIGVINTSEV et al, 2015).…”

“…O mesmo perfil foi observado para a esterase EstOF4, que se manteve estável em 10% de DMSO, foi inibida por 2-propanol e 2-butanol e teve sua atividade fracamente estimulada em 10% de glicerol (RAO et al, 2013). O modelo estrutural da EST5 apresenta enovelamento alfa-beta, típico da família das α/β-hidrolases (NARDINI; DIJKSTRA, 1999), composto por oito folhas beta (apenas a β2 é antiparalela) e 11 alfa hélices (Met38-Asn40, Ser43-Arg52, Leu95-Leu109, Met121-Arg136, Leu152-Ala161, Arg172-Gln187, Asp195-Met209, Gln218-Ala220, Gly226-Lys234, Gly255-Ser261, Val279-Glu297 …”