Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library.
OBJECTIVE
The aim of this study was to demonstrate the application of environmental sample pre-enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s).


RESULTS
A positive recombinant clone (UVCL29), conferring an esterase phenotype was identified from a shotgun gene library. The complete sequence of the 3.0 kb DNA insert from the pUVCL29 recombinant plasmid was obtained using primer-walking strategies. Nucleotide sequence analysis revealed a complete 945 bp open reading frame (ORF1). Translational analysis of the ORF1 showed a protein of 314 amino acids (named EstAM) with a predicted molecular weight of 34 kDa. EstAM's primary structure showed a classical (-G-D-S-A-G-) motif, corresponding with the generally conserved (G-x-S-x-G) esterase signature motif. Identity searches indicated that EstAM has high sequence similarity with esterases from family IV. EstAM was successfully expressed in Escherichia coli in a biologically active form. Partial purification was achieved using a one-step Pro-PurTM IMAC column. Biochemical characterization revealed that EstAM has a temperature optimum of 40 degrees C.


CONCLUSIONS
Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p-nitrophenyl ester substrates.


CONCLUSIONS
This study is a demonstration of the successful application of environmental sample pre-enrichment technology in accessing novel esterases from a mining environment.
Rashamuse
K
K
CSIR Biosciences, Modderfontein, Johannesburg, South Africa.
Ronneburg
T
T
Hennessy
F
F
Visser
D
D
van Heerden
E
E
Piater
L
L
Litthauer
D
D
Möller
C
C
Brady
D
D
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004783:Environmental Microbiology; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015698:Genomic Library; D008969:Molecular Sequence Data; D010802:Phylogeny
D043182:Carboxylesterase