Thermostability and Specific-Activity Enhancement of an Arginine Deiminase from Enterococcus faecalis SK23.001 via Semirational Design for L-Citrulline Production

L-Citrulline is a nonessential amino acid with a variety of physiological functions and can be enzymatically produced by arginine deiminase (ADI, EC 3.5.3.6). The enzymatic-production approach is of immense interest because of its mild conditions, high yield, low cost, and environmental benignity. However, the major hindrances of L-citrulline industrialization are the poor thermostability and enzyme activity of ADI. Hence, in this work, directed evolution and site directed mutagenesis aided with in silico screening, including the use of b-factor values and HoTMuSiC, were applied to a previously identified ADI from Enterococcus faecalis SK23.001 (EfADI), and a triple-site variant R15K-F269Y-G292P was obtained. The triple-site variant displays a 2.5-fold higher specific enzyme activity (333 U mg(-1)), a lower K-m value of 6.4 mM, and a 6.1-fold longer half-life (t(1/2),(45 degrees C) = 86.7 min) than wild-type EfADI. This work provides a protein-engineering strategy to improve enzyme activity and thermostability, which might be transferrable to other ADIs and enzymes.