热稳定性
水解
嗜热菌
糖苷水解酶
化学
纤维素
温泉
纤维素酶
酶
残留物(化学)
生物化学
生物
古生物学
作者
Migiwa Takeda,Seiki Baba,J. Okuma,Yoshitsugu Hirose,Akio Nishimura,Masaki Takata,Kōhei Oda,Daisuke Shibata,Takashi Kumasaka,Yasuhiro Kondo
标识
DOI:10.1038/s42003-022-03195-1
摘要
Enzymatic breakdown is an attractive cellulose utilisation method with a low environmental load. Its high temperature operation could promote saccharification and lower contamination risk. Here we report a hyper-thermostable cellobiohydrolase (CBH), named HmCel6A and its variant HmCel6A-3SNP that were isolated metagenomically from hot spring sediments and expressed in Escherichia coli. They are classified into glycoside hydrolases family 6 (GH6). HmCel6A-3SNP had three amino acid replacements to HmCel6A (P88S/L230F/F414S) and the optimum temperature at 95 °C, while HmCel6A did it at 75 °C. Crystal structure showed conserved features among GH6, a (β/α)8-barrel core and catalytic residues, and resembles TfCel6B, a bacterial CBH II of Thermobifida fusca, that had optimum temperature at 60 °C. From structure-function studies, we discuss unique structural features that allow the enzyme to reach its high thermostability level, such as abundance of hydrophobic and charge-charge interactions, characteristic metal bindings and disulphide bonds. Moreover, structure and surface plasmon resonance analysis with oligosaccharides suggested that the contribution of an additional tryptophan located at the tunnel entrance could aid in substrate recognition and thermostability. These results may help to design efficient enzymes and saccharification methods for cellulose working at high temperatures.
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