Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida
The Ef-Man gene was determined to consist of 1131 bp and encode a 377 amino acid protein. The amino acid sequence showed similarity with the endo-1,4-beta-mannanases of Daphnia pulex (62%), Cryptopygus antarcticus (64%), Crassostrea gigas (61%), Mytilus edulis (60%), and Aplisia kurodai (58%). The g...
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ftnirs:oai:repo.qst.go.jp:00049415 2023-05-15T13:46:39+02:00 Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida 上田 光宏 平野 優 福原 宏章 中 裕規 中澤 昌美 阪本 龍司 Ogata Yoshiyuki 玉田 太郎 2018-05 https://repo.qst.go.jp/?action=repository_uri&item_id=49415 http://id.nii.ac.jp/1657/00049402/ en eng 10.1016/j.enzmictec.2018.05.014 https://repo.qst.go.jp/?action=repository_uri&item_id=49415 http://id.nii.ac.jp/1657/00049402/ Enzyme and Microbial Technology, 117, 15-22(2018-05) 0141-0229 https://www.sciencedirect.com/science/article/pii/S0141022918301984?via%3Dihub Journal Article 2018 ftnirs 2021-12-30T07:42:42Z The Ef-Man gene was determined to consist of 1131 bp and encode a 377 amino acid protein. The amino acid sequence showed similarity with the endo-1,4-beta-mannanases of Daphnia pulex (62%), Cryptopygus antarcticus (64%), Crassostrea gigas (61%), Mytilus edulis (60%), and Aplisia kurodai (58%). The gene encoding mature Ef-Man was expressed in Pichia pastoris (GS115 strain). Based on SDS-PAGE analysis, the molecular mass of the purified recombinant Ef-Man (rEf-Man) was estimated to be 39.5 kDa. All catalytically important residues of endo-1,4-beta-mannanases in the glycoside hydrolase (GH) family 5 were conserved in Ef-Man. The optimal temperature for rEf-Man was identified as 60°C. HPLC and HPAEC analyses suggest that Ef-Man requires at least six subsites for efficient hydrolysis and is capable of performing transglycosylation reactions. The overall structure of rEf-Man is similar to those of GH5 family proteins, and tertiary structures around the active site are conserved among endo-1,4-beta-mannanase families. X-ray crystallographic analysis supports the hydrolysis and transglycosylation reaction mechanism determined by HPLC and HPAEC analyses. Article in Journal/Newspaper Antarc* antarcticus Crassostrea gigas Cryptopygus antarcticus National Institute of Radiological Science: NIRS-Repository |
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National Institute of Radiological Science: NIRS-Repository |
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ftnirs |
language |
English |
description |
The Ef-Man gene was determined to consist of 1131 bp and encode a 377 amino acid protein. The amino acid sequence showed similarity with the endo-1,4-beta-mannanases of Daphnia pulex (62%), Cryptopygus antarcticus (64%), Crassostrea gigas (61%), Mytilus edulis (60%), and Aplisia kurodai (58%). The gene encoding mature Ef-Man was expressed in Pichia pastoris (GS115 strain). Based on SDS-PAGE analysis, the molecular mass of the purified recombinant Ef-Man (rEf-Man) was estimated to be 39.5 kDa. All catalytically important residues of endo-1,4-beta-mannanases in the glycoside hydrolase (GH) family 5 were conserved in Ef-Man. The optimal temperature for rEf-Man was identified as 60°C. HPLC and HPAEC analyses suggest that Ef-Man requires at least six subsites for efficient hydrolysis and is capable of performing transglycosylation reactions. The overall structure of rEf-Man is similar to those of GH5 family proteins, and tertiary structures around the active site are conserved among endo-1,4-beta-mannanase families. X-ray crystallographic analysis supports the hydrolysis and transglycosylation reaction mechanism determined by HPLC and HPAEC analyses. |
format |
Article in Journal/Newspaper |
author |
上田 光宏 平野 優 福原 宏章 中 裕規 中澤 昌美 阪本 龍司 Ogata Yoshiyuki 玉田 太郎 |
spellingShingle |
上田 光宏 平野 優 福原 宏章 中 裕規 中澤 昌美 阪本 龍司 Ogata Yoshiyuki 玉田 太郎 Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida |
author_facet |
上田 光宏 平野 優 福原 宏章 中 裕規 中澤 昌美 阪本 龍司 Ogata Yoshiyuki 玉田 太郎 |
author_sort |
上田 光宏 |
title |
Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida |
title_short |
Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida |
title_full |
Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida |
title_fullStr |
Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida |
title_full_unstemmed |
Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida |
title_sort |
gene cloning, expression, and x-ray crystallographic analysis of a β-mannanase from eisenia fetida |
publishDate |
2018 |
url |
https://repo.qst.go.jp/?action=repository_uri&item_id=49415 http://id.nii.ac.jp/1657/00049402/ |
genre |
Antarc* antarcticus Crassostrea gigas Cryptopygus antarcticus |
genre_facet |
Antarc* antarcticus Crassostrea gigas Cryptopygus antarcticus |
op_source |
https://www.sciencedirect.com/science/article/pii/S0141022918301984?via%3Dihub |
op_relation |
10.1016/j.enzmictec.2018.05.014 https://repo.qst.go.jp/?action=repository_uri&item_id=49415 http://id.nii.ac.jp/1657/00049402/ Enzyme and Microbial Technology, 117, 15-22(2018-05) 0141-0229 |
_version_ |
1766245010394578944 |