Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome SCIE SCOPUS

Cited 68 time in WEB OF SCIENCE Cited 73 time in Scopus
Title
Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome
Author(s)
Park, Hyo-Jung; Jeon, Jeong Ho; Kang, Sung Gyun; Lee, Jung-Hyun; Lee, Sung-A.; Kim, Hyung-Kwoun
KIOST Author(s)
Kang, Sung Gyun(강성균)Lee, Jung Hyun(이정현)
Publication Year
2007-04
Abstract
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 8.34 kcal/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. (c) 2006 Elsevier Inc. All rights reserved.
ISSN
1046-5928
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/4713
DOI
10.1016/j.pep.2006.10.010
Bibliographic Citation
PROTEIN EXPRESSION AND PURIFICATION, v.52, no.2, pp.340 - 347, 2007
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Subject
ESCHERICHIA-COLI; CODON USAGE; BIOCHEMICAL-PROPERTIES; INCLUSION-BODIES; LIPASES; SEQUENCE; GENOME; PURIFICATION; DNA; SUBSTRATE
Keywords
esterase; metagenome; refolding; alkaline
Type
Article
Language
English
Document Type
Article
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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