Rational Engineering of Arginine Deiminase To Enhance the Acid Tolerance of Lactic Acid Bacteria

In this work, the arginine deiminase system in lactic acid bacteria was activated in response to acid stress. Subsequently, a protein engineering strategy was rationally proposed to enhance the robustness of arginine deiminase, thereby improving the acid tolerance of lactic acid bacteria. Through molecular docking and molecular dynamics simulations, arginine deiminase mutants possessing a lower binding energy with the substrate arginine and higher stability (lower RMSD, RMSF, and Rg values compared with the wild type) were identified. Furthermore, the mutants were constructed in <i>Lactococcus lactis</i> NZ9000, and the resulting variants D71L, D120M, and D71L-D120M exhibited a higher enzymatic activity than the wild-type strain. The survival rate of <i>L. lactis</i> variants D71L, D120M, and D71L-D120M increased 15.07, 31.98, and 113.47%, respectively, compared with the wild-type strain under acid stress, suggesting the feasibility of rationally engineered arginine deiminase for improving the acid tolerance of lactic acid bacteria. Findings showed in this work may be beneficial for rationally improving the industrial functionalities of lactic acid bacteria during food biomanufacture.