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Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming.

Applied and environmental microbiology (2015-06-14)
Cheng Zhou, Jintong Ye, Yanfen Xue, Yanhe Ma
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 t50 (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 t50 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.

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