[Cloning, expression and characterization of a novel esterase from marine sediment microbial metagenomic library].
OBJECTIVE
To clone, express and characterize a novel esterase from marine sediment microbial metagenomic library.


METHODS
Using esterase segregation agar containing tributyrin, we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library. This fosmid was partially digested with Sau3A I to construct the sublibrary, from which the esterase positive subclone pFLS10 was obtained. The full length of the esterase gene was amplified and cloned into the expressing vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 cells. We analyse the enzyme activity and study the characterization of the esterase after its expression and purification.


RESULTS
An ORF (Open Reading Frame) of 924 bp was identified from the subclone pFLS10. Sequence analysis indicated that it showed 71% amino acid identity to esterase (ADA70030) from a marine sediment metagenomic library. The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate (C4). The optimum temperature of the esterase was 20 degrees C, the optimum pH was 7.5. The esterase in this study had good thermostability at 20 degrees C and good pH stability at pH8 -10. Significant increase in lipolytic activity was observed with addition of K+ and Mg2+, while decrease with Mn2+ etc.


CONCLUSIONS
We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library. The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions, which laid a basis for industrial application.
Xu
Shiqing
S
Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. xu.1127@126.com
Hu
Yongfei
Y
Yuan
Aihua
A
Zhu
Baoli
B
D000595:Amino Acid Sequence; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D019015:Geologic Sediments / Q000382:microbiology*; D056186:Metagenomics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004950:Esterases