Abstract
Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate (Pedotti et al., 2009). Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution.
Materials and Reagents
Equipment
Procedure
Representative data
Table 1. The apparent kinetic parameters on glycine and glyphosate measured for wild-type BceGO and variants obtained by random mutagenesis, site saturation mutagenesis and DNA shuffling
Especially, B3S1 demonstrated a 160-fold increase in substrate affinity for glyphosate, a 326-fold increase in catalytic efficiency towards glyphosate and a significant enhancement in the specificity constant over the wild-type BceGO, achieving the goal of efficient oxidation of glyphosate by evolution of glycine oxidase.
Notes
Recipes
Acknowledgments
We thank Drs. Ziduo Liu and Dexin Kong for valuable discussions about this article.
References
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