Engineering of dual-functional hybrid glucanases.
1,3-1,4-β-D-Glucanase (lichenase) and 1,3-β-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-β-D-glucanase gene (TFs(W203F)) from Fibrobacter succinogenes, and a 1,3-β-D-glucanase gene (TmLam) from hyperthermophilic Thermotoga maritima were used as target enzymes. The substrate-binding domains (TmB₁ and TmB₂) and the catalytic domain (TmLam(CD)) of TmLam were ligated to the N- or C-terminus of TFsW203F to create four hybrid enzymes, TmB₁-TFs(W203F), TFs(W203F)-TmB₂, TmB₁-TFs(W203F)-TmB₂ and TFs(W203F)-TmLam(CD). The results obtained from kinetic studies show that increased specific activities and turnover rate for lichenan and laminarin were observed in TmB₁-TFs(W203F)-TmB₂ and TFs(W203F)-TmLam(CD), respectively. Furthermore, fluorescence and circular dichroism spectrometric analyses indicated that the hybrid TFs(W203F)-TmLam(CD) was structurally more stable than the parental TFs(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.
Agricultural Biotechnology Research Center,  Academia Sinica, Taipei 115, Taiwan.
Wei-Chun
WC
Liu
Yu-Shiun
YS
Lin
Wen-Yih
WY
Jeng
Je-Hsin
JH
Chen
Andrew H-J
AH
Wang
Lie-Fen
LF
Shyur
D001665:Binding Sites; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D045848:Fibrobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D044602:Cellulases; D006026:Glycoside Hydrolases; C028020:licheninase