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Improving the specific activity and thermo-stability of alkaline pectate lyase from Bacillus subtilis 168 for bioscouring

Wang, Xiaowen, Lu, Zhenghui, Xu, Ting, Selvaraj, Jonathan Nimal, Yi, Li, Zhang, Guimin
Biochemical engineering journal 2018 v.129 pp. 74-83
Bacillus subtilis, additive effect, biocatalysts, bioscouring, calcium, fabrics, half life, hardness, mutagenesis, mutants, pH, pectate lyase, textile industry, thermal stability, wettability
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 improvement in specific activity than PEL168. Furthermore, R272W was introduced to K47D/V132F based on evolutionary trace analysis. The K47D/V132F/R272W specific activity reached to 5610U/mg at 1mM Ca²⁺, showing highest activity of reported alkaline pectate lyases, with T50 extended to 330min. 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.