Gene cloning of a highly active phytase from Lactobacillus plantarum and further improving its catalytic activity and thermostability through protein engineering

Abstract

Phytase belongs to orthophosphate monoester hydrolase, which can catalyze the gradual hydrolysis of phytic acid to inositol phosphate. It can be added to animal feed to reduce the anti-nutritional factor of phytic acid in feed. The thermostability and specific activity of phytases are two key factors determining their potential applications. In this study, a highly active 233-aa phytase gene (LpPHY233) from Lactobacillus plantarum was cloned and expressed in Escherichia coli (E. coli), achieving 800 times higher activity than that expressed in L. plantarum. Next, the temperature characteristic and catalytic performance of LpPHY233 was improved by disulfide bond engineering and C-terminal truncation, respectively. Surprisingly, the specific activity of the C-terminal truncated mutant LpPHY200 was about 5.6 times higher than that of LpPHY233, and the optimal temperature for the mutant LpPHY233S58C/K61C introduced disulfide bond was 15 °C higher than that of LpPHY233. Moreover, these phytase mutants displayed excellent pH property and kinetic parameters, and have great application prospect in feed additives field. The molecular basis for its catalytic performance was preliminarily explained by in silico design methods. Our results provided a solid theoretical foundation for further molecular modification and industrial application of phytases.

Keywords: Disulfide bond engineering; Homology modeling; Phytase; Protein engineering; Truncated expression.