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Isolation and biochemical analysis of novel lipolytic enzymes from diverse environments using metagenomic approach
메타게노믹 방법에 의한 다양한 환경으로부터의 신규 지방 분해 효소 탐색 및 생화학적 특성 분석

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Authors
전정호
Advisor
최상호
Major
농생명공학부
Issue Date
2012-02
Publisher
서울대학교 대학원
Abstract
Metagenomic libraries consisting of 81,100 (Edison Seamount), 80,050 (intertidal flat), 60,132 (Arctic), and 6912 (Upo wetland) clones were constructed using high molecular weight DNA samples isolated from three marine sediments and soil sample. To screen esterase/lipase-producing clones, the fosmid or cosmid clones were plated onto LB agar plates containing 1% tributyrin (TBN). Consequently, 18 positive clones (designated as pFosEM3L1, pFosEM3L2, pFosEM3L3, pFosEM3L4, pFosEM3L6, pFosEM3L7, pFosKT1, pFosKT3, pFosKT4, pFosKT7, pFosKT9, pFosAT1, pFosAT3, pFosAT5, pFosAT6, pFosAT7, pFosAT11, and pCosU1) were obtained by the appearance of clear zones around the colonies. The sequence analysis revealed that those clones contained open reading frames, showing 30-68% amino acid identity with putative lipolytic enzymes in the public database. Based on the phylogenetic analysis with previously characterized lipase/esterases, the enzymes were assigned to various families such as Family I (EM3L7), Family IV (EstKT4, EstKT7, EstKT9, EstAT1, EstAT5, EstAT7, and EstAT11), and Family V (EM3L1, EM3L3, and EM3L6), EstD2 group (EM3L2), Feruloyl esterase group (FeKT1) or Patatin-like protein group (PlAT6). EstKT4, EstKT7, and EstKT9 belonging to Family IV seemed encode distinct members in the family, forming a new subfamily in the Family IV of bacterial lipolytic enzymes. Of particular, 3 enzymes (EM3L4, EstKT3, and EstAT3) were assigned to three new groups that have never been described, and as discussed in the later section of abstract, EstU1 isolated from Upo swamp sample was assigned to Family VIII.
In an attempt to express the esterase/lipase genes in E. coli, problems with solubility were encountered. By employing a combination of approaches such as removing the hydrophobic region in the N-terminus, co-expression of chaperone genes, and low temperature induction, 14 lipolytic genes were expressed as soluble form, and the recombinant proteins were purified to homogeneity by metal chelating affinity chromatography, followed by biochemical characterization.
The optimum activities of the purified enzymes were determined in the temperature range of 20-45 °C and the cold-active profile that more than half of maxium activity remained even at 4 °C could be observed for seven enzymes. While 14 enzymes showed esterase activity toward short-chain fatty acid esters (C2-C10), EM3L4 showed lipase activity by hydrolyzing long-chain fatty acid esters (C16-C18), implicating that the probability of getting a true lipase is very low by applying this approach. EstKT4, EstKT7, and EstKT9 belonging to new subfamily displayed significant salt tolerance that over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). Among 14 purified enzymes tested in this study, EstAT1 and EstAT11could hydrolyze racemic ofloxacin esters, and further EstAT11 hydrolyzed preferentially (S)-racemic ofloxacin butyl ester with an enantiomeric excess (eep) value of 70.3%.
EstU1 classified as a member of Family VIII was investigated further since the sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining a S-X-X-K motif that is conserved in class C β-lactamases and Family VIII carboxylesterases. The relationship between the two families was suspected, but never been proven. To address the issue, estU1 gene was overexpressed in E. coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinity for both p-nitrophenyl butyrate and first-generation cephalosporins, while the turnover efficiency for the latter was much lower. The structure of EstU1 adopts a two-domain structure composed of a small helical domain including two β-sheets and a mixed α/β domain such as the structure of class C β-lactamases and penicillin-binding proteins (PBSs). The overall structure of EstU1 was also homologous to those of EstB carboxylesterase, R61 DD-peptidase and AmpC β-lactamase. Through structural-based phylogenetic analysis of EstU1 with DD-peptidases and β-lactamases, this result showed that the serine β-lactamases and the carboxylesterases including EstU1 are sister taxa and that the divergence of the DD-peptidase predated the divergence of the serine β-lactamases and the carboxylesterases
Accordingly, the lipolytic proteins obtained by metagenomic libraries in this study revealed novel enzymes in terms of the primary sequences, the activity profiles and substrate spectra. These enzymes might be useful in biotechnological application such as organic synthesis and industrial processes.
Language
eng
URI
https://hdl.handle.net/10371/156294

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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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