Characterization and directed evolution of BliGO, a novel glycine oxidase from Bacillus licheniformis

• A cold-adapted glycine oxidase (BliGO) with glyphosate-degrading activity was cloned from Bacillus licheniformis.
• The catalytic efficiency of BliGO toward glyphosate was increased by 8-fold by in-vitro directed evolution.
• Residue Ser51 might involve in the binding of enzyme with glyphosate in the evolved mutant.

Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (Km, kcat and kcat/Km) of BliGO were 11.22 mM, 0.08 s−1, and 0.01 mM−1 s−1, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM−1 s−1) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.