Metagenomic mining and structure-function studies of a hyper-thermostable cellobiohydrolase from hot spring sediment

Bacteria from hot springs are known for highly thermostable enzymes, which may have industrial potential. Here, a unique thermostable cellobiohydrolase is reported that can breakdown cellulose at temperature up to 95 degrees Celsius. 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 degrees C, while HmCel6A did it at 75 degrees C. Crystal structure showed conserved features among GH6, a (beta/alpha)(8)-barrel core and catalytic residues, and resembles TfCel6B, a bacterial CBH II of Thermobifida fusca, that had optimum temperature at 60 degrees 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.

Automated summary

A thermostable cellobiohydrolase, HmCel6A and its variant HmCel6A-3SNP, isolated from hot spring sediments, showed high thermostability with optimum temperatures at 75℃ and 95℃ respectively. Structural features, including hydrophobic and charge-charge interactions, metal bindings, disulphide bonds, and an additional tryptophan at the tunnel entrance, contributed to the high thermostability of the enzyme. These findings may contribute to the development of efficient enzymes and saccharification methods for cellulose working at high temperatures.