AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND 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. CONCLUSION: 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). SIGNIFICANCE AND IMPACT OF THE STUDY: 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.
AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND 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. CONCLUSION: 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). SIGNIFICANCE AND IMPACT OF THE STUDY: 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.