In vivo immobilization of d-hydantoinase in Escherichia coli

d-p-Hydroxyphenylglycine (d-HPG) is a precursor required for the synthesis of semi-synthetic antibiotics. This unnatural amino acid can be produced by a transformation reaction mediated by d-hydantoinase (d-HDT) and d-amidohydrolase. In this study, a method was developed to integrate production and immobilization of recombinant d-HDT in vivo. This was approached by first fusion of the gene encoding d-HDT with phaP (encoding phasin) of Ralstonia eutropha H16. The fusion gene was then expressed in the Escherichia coli strain that carried a heterologous synthetic pathway for polyhydroxyalkanoate (PHA). As a result, d-HDT was found to associate with isolated PHA granules. Further characterization illustrated that d-HDT immobilized on PHA exhibited the maximum activity at pH 9 and 60°C and had a half-life of 95 h at 40°C. Moreover, PHA-bound d-HDT could be reused for 8 times with the conversion yield exceeding 90%. Overall, it illustrates the feasibility of this approach to facilitate in vivo immobilization of enzymes in heterologous E. coli strain, which may open a new avenue of enzyme application in industry.