Molecular characterization of a novel family VIII esterase from Burkholderia multivorans UWC10.
An esterase producing Burkholderia multivorans UWC10 strain was isolated by culture enrichment. A shotgun library of B. multivorans UWC10 genomic DNA was screened for esterase activity and a recombinant clone conferring an esterolytic phenotype was identified. Full-length sequencing of the DNA insert showed that it consisted of a single open reading frame (ORF1) encoding a predicted protein of 398 amino acids. ORF1 (termed EstBL) had a high protein sequence identity to family VIII esterases. The EstBL primary structure showed two putative serine motifs, G-V-S(149)-D-G and S(74)-V-T-K. The estBL gene was successfully over-expressed in E. coli and the encoded protein purified by a combination of ammonium sulphate fractionation, hydrophobic interaction, ion exchange and size exclusion chromatographies. Biochemical assays confirmed EstBL esterase activity and revealed a preference for short-chain p-nitrophenyl and beta-naphthyl esters (C2-C4) with no activity against beta-lactam substrates. Secondary structure predictions indicated that EstBL adopts the alpha/beta fold, which is common to all esterases.
Rashamuse
Konanani J
KJ
CSIR Biosciences, Modderfontein, Johannesburg, South Africa.
Burton
Stephanie G
SG
Stafford
William H L
WH
Cowan
Don A
DA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D019117:Burkholderia / Q000201:enzymology* / Q000235:genetics; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015698:Genomic Library; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D043182:Carboxylesterase