Production Of An Improved Enzyme Construction of an Expression Vector

To express the DCase gene effectively, the mutant DCase gene was inserted into a newly constructed vehicle, pUCNT. This vehicle was constructed as follows (21).

To insert the improved DCase gene just after the lac promoter of pUC19, a Ndel cleavage site was generated in the initiation codon of the lacZ gene, through PCR amplification of the HindIII-Cfr10I 1.3-kb fragment. The amplified fragment was used to replace the corresponding fragment of pUC19, and the plasmid constructed was designated as pUCNde. After generating a NdeI site instead of the NcoI site of pTrc99A in the same manner, a 0.6-kb Ndel-Sspl fragment was ligated into the 2.0-kb NdeI-SspI fragment, pUCNT being constructed.

The 455M mutant DCase gene was inserted into pUCNT between the NdeI and PstI cleavage sites, the recombinant plasmid pNT4553 being constructed (Fig. 7).

Table 7. Comparison of the Properties of Native and Mutant DCases
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