(191co) Building Disulfide Bonds Between Subunits to Improve the Stability of Nitrile Hydratase

Stability is an indispensable property of enzymes for industrial applications. Nitrile hydratase (NHase) with two subunits is a vital biocatalyst for commercial bioproduction of acrylamide. The bioconversion process shows many advantages such as mild reaction conditions, low energy consumption, high conversion rate and product purity. However, the NHase was not stable enough in the late catalytic process because of the high concentration acrylamide immersion. Enhancing stability of NHase is thus urgently required in industry. Generally, the tertiary structure of protein is maintained by noncovalent interactions such as hydrophobic interactions, hydrogen bonds and salt bridges, which are all relatively weak interactions. To significantly improve the stability of NHase, covalent disulfide bond is specifically highlighted in this study. By using the Disulfide by Design software, 9 disulfide bridges between the two subunits were designed. And corresponding mutant enzymes were constructed with genetic engineering tools. In the host of Rhodococcus ruber TH3, one NHase mutant, 23-307, displayed significant enhanced activity (3915 U/mL), 8.2% higher than the original one, although the other mutants completely lost their activities due to the conformation changes. The in vivo stabilizing effect of the R. ruber TH3/23-307 cells harboring the mutant NHase was assessed using both heat shock and acrylamide immersion experiments. While immersing the cells of R. ruber TH3/23-307 and the original R. ruber TH3/NHase at 60°C for 10 min, 52.5% of NHase activity remained in the mutant R. ruber TH3/23-307, but this value dropped to 29.4% in the original control. The deactivation behavior of R. ruber TH3/23-307 against acrylamide immersion was conducted in a solution with dynamically increased acrylamide concentrations. When the acrylamide concentration was increased to 150 g/L, the remaining NHase activity in R. ruber TH3/23-307 was as high as 92%, compared with 37% of that in the original R. ruber TH3/NHase. The hydration synthesis was further conducted to produce 500 g/L acrylamide with continuous acrylonitrile feeding. The bacteria were recovered through centrifugation for multiple batches of hydration after the acrylamide concentration reached 500 g/L. Results showed that the cells of R. ruber TH3/23-307 could complete three batches production of 500 g/L acrylamide, but the cells of original R. ruber TH3/NHase could only complete one batch.