2003年8月
Cloning, heterologous expression, renaturation, and characterization of a cold-adapted esterase with unique primary structure from a psychrotroph Pseudomonas sp strain B11-1
PROTEIN EXPRESSION AND PURIFICATION
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- 巻
- 30
- 号
- 2
- 開始ページ
- 171
- 終了ページ
- 178
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/S1046-5928(03)00128-1
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
A Gene coding for an esterase (PsEst1, 1911 bp in length) of the psychrotrophic bacterium Pseudomonas sp. B11-1 isolated from Alaskan soil was cloned and sequenced. The deduced amino acid sequence revealed a protein of 637 amino acid residues with a molecular mass of 69 kDa. Although the expression product, PsEst1, showed no appreciable sequence similarity (less than 15% identity) to any known proteins with the established biochemical functions, it is expected to be related to the alpha/beta hydrolase superfamily because it shared sequence motifs that have been identified with this superfamily. For example, a unique "nucleophilic 6 40 38 elbow" motif. -Gly(36)-Asp-Ser-Leu-Asn(40)-, was identified, and Ser(38) was predicted to constitute a catalytic triad with Asp(162) and His(303). PsEst1 was overexpressed using a T7 RNA polymerase transcription (pET21a) system in the Escherichia coli BL21(DE3) cells as an inclusion body. A Soluble denatured form of the enzyme was purified to homogeneity in the presence of 8 M urea, and the catalytically active form of the enzyme could be obtained by subsequent removal of urea by dialysis, where the addition of 0.1% Triton X-100 was essential for the efficient renaturation of the enzyme. To our knowledge, this was the first example of the successful renaturation of the recombinant cold-adapted enzyme. The enzyme efficiently hydrolyzed vinyl and aryl esters with the C-4-C-6 acyl chain. The activation energy of the enzymatic p-nitrophenyl butyrate hydrolysis (20.1 kcal/mol at 10 degreesC) was significantly lower than the value (79.9 kcal/mol) of the mesophilic lipase. It was observed that the K-m values for p-nitrophenyl butyrate in the growth temperature range of strain B11-1 (5-15 degreesC) were lower than those at higher temperatures. (C) 2003 Elsevier Science (USA). All rights reserved.
- リンク情報
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- DOI
- https://doi.org/10.1016/S1046-5928(03)00128-1
- J-GLOBAL
- https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200902237872859189
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/12880765
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000184506500004&DestApp=WOS_CPL
- ID情報
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- DOI : 10.1016/S1046-5928(03)00128-1
- ISSN : 1046-5928
- J-Global ID : 200902237872859189
- PubMed ID : 12880765
- Web of Science ID : WOS:000184506500004