Characterization and its potential application of two esterases derived from the arctic sediment metagenome.
Two esterase genes (designated as estAT1 and estAT11, respectively) were cloned by activity-based screening of a fosmid library constructed with seashore sediment sample of the Arctic. The sequence analysis of the genes revealed that these esterase genes encoded proteins of 303 and 312 amino acids, respectively, and showed 40-50% identities to members of the hormone-sensitive lipase (HSL) family retaining a catalytic triad with a conserved GDSAG sequence and an oxyanion hole (HGGG). The esterases genes were overexpressed in Escherichia coli by co-expressing GroEL-GroES chaperonine, and the recombinant proteins (rEstAT1 and rEstAT11) were purified to homogeneity. The purified EstAT1 and EstAT11 were active in a broad range of temperature from 20 to 40 degrees C with an optimum temperature at 30 degrees C. The activation energies of rEstAT1 and rEstAT11 to hydrolyze p-nitrophenyl esters of butyrate were determined to be 12.65 kcal/mol and 11.26 kcal/mol, respectively, indicating that they are cold-adapted esterases. The purified EstAT1 and EstAT11 could hydrolyze racemic ofloxacin esters, and further rEstAT11 hydrolyzed preferentially (S)-racemic ofloxacin butyl ester with an enantiomeric excess (ee(p)) value of 70.3%. This work represents an example that develops enzymes from the Arctic using metagenomic approach, potentially applicable to chiral resolution of heat-labile substrates.
Jeon
Jeong Ho
JH
Marine Biotechnology Research Centre, Korean Ocean Research & Development Institute, Ansan, South Korea.
Kim
Jun-Tae
JT
Kang
Sung Gyun
SG
Lee
Jung-Hyun
JH
Kim
Sang-Jin
SJ
D000595:Amino Acid Sequence; D001110:Arctic Regions; D001483:Base Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000235:genetics*; D015723:Gene Library; D023281:Genomics; D019015:Geologic Sediments / Q000032:analysis*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry
D017931:DNA Primers; D004950:Esterases