TY - JOUR
T1 - Engineering of dual-functional hybrid glucanases
AU - Liu, Wei Chun
AU - Lin, Yu Shiun
AU - Jeng, Wen Yih
AU - Chen, Je Hsin
AU - Wang, Andrew H.J.
AU - Shyur, Lie Fen
N1 - Funding Information:
This work was supported by grants from National Science Council [NSC 97-3114-P-001-001, 100-3111-Y-001-006 and 100-2325-B-001-029], Taiwan, R.O.C.
PY - 2012/11
Y1 - 2012/11
N2 - 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 (TFsW203F) 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 (TmLamCD) of TmLam were ligated to the N- or C-terminus of TFsW203F to create four hybrid enzymes, TmB 1-TFsW203F, TFsW203F-TmB2, TmB 1-TFsW203F-TmB2 and TFsW203F- TmLamCD. The results obtained from kinetic studies show that increased specific activities and turnover rate for lichenan and laminarin were observed in TmB1-TFsW203F-TmB2 and TFs W203F-TmLamCD, respectively. Furthermore, fluorescence and circular dichroism spectrometric analyses indicated that the hybrid TFs W203F-TmLamCD was structurally more stable than the parental TFsW203F, 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.
AB - 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 (TFsW203F) 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 (TmLamCD) of TmLam were ligated to the N- or C-terminus of TFsW203F to create four hybrid enzymes, TmB 1-TFsW203F, TFsW203F-TmB2, TmB 1-TFsW203F-TmB2 and TFsW203F- TmLamCD. The results obtained from kinetic studies show that increased specific activities and turnover rate for lichenan and laminarin were observed in TmB1-TFsW203F-TmB2 and TFs W203F-TmLamCD, respectively. Furthermore, fluorescence and circular dichroism spectrometric analyses indicated that the hybrid TFs W203F-TmLamCD was structurally more stable than the parental TFsW203F, 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.
KW - bi-functional enzyme
KW - laminarinase
KW - lichenase
KW - thermal tolerance
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U2 - 10.1093/protein/gzs083
DO - 10.1093/protein/gzs083
M3 - Article
C2 - 23081838
AN - SCOPUS:84868567651
SN - 1741-0126
VL - 25
SP - 771
EP - 780
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 11
ER -