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

Author:
Wang, Xiaowen, Lu, Zhenghui, Xu, Ting, Selvaraj, Jonathan Nimal, Yi, Li, Zhang, Guimin
Source:
Biochemical engineering journal 2018 v.129 pp. 74-83
ISSN:
1369-703X
Subject:
Bacillus subtilis, additive effect, biocatalysts, bioscouring, calcium, fabrics, half life, hardness, mutagenesis, mutants, pH, pectate lyase, textile industry, thermal stability, wettability
Abstract:
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.
Agid:
5859475