1,3-1,4-β-D-Glucanase has been widely used as a
feed additive to help non-ruminant animals digest plant fibers,
with potential in increasing nutrition turnover rate and
reducing sanitary problems. Engineering of enzymes for better
thermostability is of great importance because it not only can
broaden their industrial applications, but also facilitate
exploring the mechanism of enzyme stability from structural
point of view. To obtain enzyme with higher thermostability
and specific activity, structure-based rational design was
carried out in this study. Eleven mutants of Fibrobacter
succinogenes 1,3-1,4-β-D-glucanase were constructed in
attempt to improve the enzyme properties. In particular, the
crude proteins expressed in Pichia pastoris were examined
firstly to ensure that the protein productions meet the need for
industrial fermentation. The crude protein of V18Y mutant
showed a 2 °C increment of Tm and W203Y showed ∼30%
increment of the specific activity. To further investigate the
structure-function relationship, some mutants were expressed
and purified from P. pastoris and Escherichia coli. Notably,
the specific activity of purified W203Y which was expressed
in E. coli was 63% higher than the wild-type protein. The
double mutant V18Y/W203Y showed the same increments of
Tm and specific activity as the single mutants did. When
expressed and purified from E. coli, V18Y/W203Y showed
similar pattern of thermostability increment and 75% higher
specific activity. Furthermore, the apo-form and substrate
complex structures of V18Y/W203Y were solved by X-ray
crystallography. Analyzing protein structure of V18Y/W203Y
helps elucidate how the mutations could enhance the protein
stability and enzyme activity.