Biocatalysts requires enzymes with high activity and stability under process conditions for efficient application.
Several protein improvement strategies were used to improve pectate lyase PEL168 from Bacillus
subtilis. Initially, a rationally designed mutant V132F obtained showed 1.7-fold increase in activity with
wider pH stability. Meanwhile, highly advantageous mutant K47E selected from a random mutagenesis
library displayed 1.8-fold increase in activity, and half-life increased by 2.0-fold at 50 ◦C (T50). The additive
effect of these two advantageous mutants K47E/V132F showed 2.2-fold increase in activity than PEL168.
To identify beneficial substitution at 47th position, a smarter library was constructed... More
Biocatalysts requires enzymes with high activity and stability under process conditions for efficient application.
Several protein improvement strategies were used to improve pectate lyase PEL168 from Bacillus
subtilis. Initially, a rationally designed mutant V132F obtained showed 1.7-fold increase in activity with
wider pH stability. Meanwhile, highly advantageous mutant K47E selected from a random mutagenesis
library displayed 1.8-fold increase in activity, and half-life increased by 2.0-fold at 50 ◦C (T50). The additive
effect of these two advantageous mutants K47E/V132F showed 2.2-fold increase in activity than PEL168.
To identify beneficial substitution at 47th position, a smarter library was constructed by site-saturated
mutagenesis of K47E/V132F, and generated K47D/V132F mutant having 3.9-fold improvementin specific
activity than PEL168. Furthermore, R272W was introduced to K47D/V132F based on evolutionary trace
analysis. The K47D/V132F/R272W specific activity reached to 5610 U/mg at 1 mM Ca2+, showing highest
activity of reported alkaline pectate lyases, with T50 extended to 330 min. Structure comparisons revealed
that a much open catalytic clef and increased structure compactness of K47D/V132F/R272W were the
main contributors for increased specific activity and stability, respectively. The bioscouring assay showed
that K47D/V132F/R272W can significantly improve the wettability and softness of fabrics, suggesting its
potential application in textile industry.
