Construction and Application of Strains that Constitutively Express the Arginase I Gene

详细

Suicide vectors typically contain an ori that can replicate only under specific conditions. The suicide plasmid pRE112 has a conditional R6K ori, requiring the π protein. As the Escherichia coli DH5α cells cannot secrete the π protein and this plasmid can survive only by integrating into the genome. In our study, insertion mutants were constructed using a method based on the suicide plasmid pRE112. After constructing a recombinant suicide plasmid pRE112 that included the arginase I gene, the vector was transformed into E. coli DH5α cells, producing the strain that constitutively expressed the arginase I gene. The E. coli strains were screened to determine the highest enzyme activity levels. Comparison of arginase I-induced expressed strains BL21/pET21a-ARG and BL21/pET35b-ARG constructed by our laboratory with the constitutively expressed strain did not reveal any significant differences in enzyme activity levels. The conversion efficiency of L-Arg was 97.8% under the optimum conditions (60°C, pH 9.5, 1 mM of Mn²⁺, 100 mg/g of wet cell weight, 3% L-Arg and 1 h of reaction time). After purification with macroreticular cation exchange resin 001×7, the purity of obtained L-Orn was 98.7%. Compared with induced expression, constitutive expression has improved economic benefits, convenience, stability and simplicity in preparation, thus overcoming the processing defects that lose plasmids. This approach may improve benefits in preserving the cultures in industrial production processes.