Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome.
A metagenomics approach is an efficient method of isolating novel and useful genes from uncultured microorganisms in diverse environments. In this research, a gene encoding a new esterase (EM2L8) was cloned and characterized from the metagenomic DNA library of a deep-sea sediment. The gene consisted of 804bp encoding a polypeptide of 267 amino acids with a molecular mass of 28,952. The deduced amino acid sequence showed similarities with the BioH of Kurthia, the 3-oxoadipate enol-lactonase of Haloarcula and the acyltransferase of Thermoanaerobacter, which feature identities of 38%, 32%, and 33%, respectively. Residues essential for esterase activity, such as pentapeptide (GXSXG) and catalytic triad sequences, were uncovered. While the protein was overproduced mainly as inclusion body at 37 degrees C, it was mainly produced as a soluble active enzyme at 18 degrees C. A zymogram analysis revealed that purified EM2L8 taken from the soluble fraction could hydrolyze tributyrin substrate. Furthermore, the protein from the inclusion body fraction also showed strong activity on gel, thus indicating that the protein was refolded during SDS-gel electrophoresis and the ensuing incubation period. When the inclusion body was mixed with some anionic detergent solutions and diluted with a non-detergent buffer, the insoluble EM2L8 refolded rapidly and recovered its full esterase activity. Although EM2L8 had an optimum temperature of 50-55 degrees C, its activation energy in the range of 10-40 degrees C was 8.34kcal/mol, indicating that it is a cold-adapted enzyme. Moreover, it was found to have an optimum pH of 10-11, thus revealing that it is an alkaline enzyme. In this paper, the new esterase EM2L8 buried in a deep-sea sediment became known on the surface and was characterized biochemically.
Park
Hyo-Jung
HJ
Division of Biotechnology, The Catholic University of Korea, 43-1 Yokkok 2-dong, Wonmi-gu, Bucheon, Gyeonggido 420-743, South Korea.
Jeon
Jeong Ho
JH
Kang
Sung Gyun
SG
Lee
Jung-Hyun
JH
Lee
Sung-A
SA
Kim
Hyung-Kwoun
HK
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology*; D015698:Genomic Library; D019015:Geologic Sediments / Q000737:chemistry*; D008969:Molecular Sequence Data; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
D004950:Esterases