Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida

• Main Authors: 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
• Format: Article in Journal/Newspaper
• Language: English
• Published: 2018
• Subjects: Antarc*; antarcticus; Crassostrea gigas; Cryptopygus antarcticus
• Online Access: https://repo.qst.go.jp/?action=repository_uri&item_id=49415; http://id.nii.ac.jp/1657/00049402/

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.