Gradually accumulating beneficial mutations to improve the thermostability of N -carbamoyl- d -amino acid amidohydrolase by step-wise evolution

To further enhance repeated batch reactions with immobilized N -carbamoyl- d -amino acid amidohydrolase (DCase), which can be used for the industrial production of d -amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T 50 (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met 31 , Asn 93 , Gln 207 , Asn 242 , Glu 266 , Thr 271 , Ala 273 ) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T 50 by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k cat and K m values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of d - p -hydroxyphenylglycine.