Engineering of dual-functional hybrid glucanases super()

• Published in: Protein engineering, design and selection Vol. 25; no. 11; pp. 771 - 780
• Main Authors: Liu, Wei-Chun, Lin, Yu-Shiun, Jeng, Wen-Yih, Chen, Je-Hsin, Wang, Andrew H-J, Shyur, Lie-Fen
• Format: Journal Article
• Language: English
• Published: 01.11.2012
• Subjects: C-Terminus; Fibrobacter succinogenes
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzs083

1,3-1,4- beta -d-Glucanase (lichenase) and 1,3- beta -d-glucanase (laminarinase) are fibrolytic enzymes which play an important role in the hydrolysis of polysaccharide components. Both of these glucanases have been employed in a number of industrial applications. This study aims to improve or combine the novel properties of both glucanases in an attempt to create desirable hybrid enzymes with economic benefits for industrial applications. A truncated and mutated 1,3-1,4- beta -d-glucanase gene (TFs sub(W203F)) from Fibrobacter succinogenes, and a 1,3- beta -d-glucanase gene (TmLam) from hyperthermophilic Thermotoga maritima were used as target enzymes. The substrate-binding domains (TmB sub(1) and TmB sub(2)) and the catalytic domain (TmLam sub(CD)) of TmLam were ligated to the N- or C-terminus of TFs sub(W203F) to create four hybrid enzymes, TmB sub(1)-TFs sub(W203F), TFs sub(W203F)-TmB sub(2), TmB sub(1)-TFs sub(W203F)-TmB sub(2) and TFs sub(W203F)-TmLam sub(CD). The results obtained from kinetic studies show that increased specific activities and turnover rate for lichenan and laminarin were observed in TmB sub(1)-TFs sub(W203F)-TmB sub(2) and TFs sub(W203F)-TmLam sub(CD), respectively. Furthermore, fluorescence and circular dichroism spectrometric analyses indicated that the hybrid TFs sub(W203F)-TmLam sub(CD) was structurally more stable than the parental TFs sub(W203F), which was attributed to an improved thermal tolerance of the hybrid enzyme. This study has been successful in creating bifunctional hybrid glucanases with dual substrate catalytic functions which warrant further evaluation of their possible use in industrial applications.