A novel recombinant ethyl ferulate esterase from Burkholderia multivorans.
OBJECTIVE
Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity.


RESULTS
A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column.


CONCLUSIONS
Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF).


CONCLUSIONS
A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.
Rashamuse
K J
KJ
CSIR Biosciences, Modderfontein, Johannesburg, South Africa.
Burton
S G
SG
Cowan
D A
DA
D000595:Amino Acid Sequence; D001483:Base Sequence; D019149:Bioreactors; D019117:Burkholderia / Q000235:genetics / Q000378:metabolism*; D002109:Caffeic Acids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004950:Esterases / Q000235:genetics / Q000302:isolation & purification*; D005524:Food Technology; D015723:Gene Library; D005818:Genetic Engineering; D016678:Genome; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021521:Ribotyping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012820:Silage; D013379:Substrate Specificity; D018505:Waste Management; D003313:Zea mays
D002109:Caffeic Acids; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C099085:ethyl ferulate; D004950:Esterases