Affiliation:
1. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
2. National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
3. University of Chinese Academy of Sciences, Beijing, China
Abstract
ABSTRACT
Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from
Bacillus
sp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca
2+
. However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in the
t
50
(time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in the
t
50
value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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