Aacagttatcaagggtggaacgatcgtcgccgccgatcgcagctatgaagccgat Atc Ct Gat Cg A Aggcg A A A Ag At Cgc C C Ag At Cggc Aggg Atc T G 1800 Cagggcgacaagattgtcgac

Figure 2. Continued.

However, these enzymes showed that the N-carbamoyl compounds involved in the metabolism of pyrimidine and purine are not hydrolyzed at all. On characterization of these enzymes, the reactive molecular weights of the native enzymes were found to be about 120,000 and those of the subunits to be about 40,000. Enzymochemical analysis was also performed. Recently, Louwrier and Knowles purified a DCase from Agrobacterium sp. which was composed of genetically engineered self-cloning cells, and characterized it (8). This enzyme was able to cleave a variety of N-carbamoyl substrates but was strictly D-form specific. The active enzyme was suggested to be present as a dimer with a subunit molecular weight of 38,000 Da, differing from the trimer enzymes of Ogawa et al. (6,7).

Concerning the enzyme genes, some DCase genes have been cloned and analyzed. The DCase gene from Agrobac-terium radiobacter NRRL B11291 was cloned, analyzed, and expressed in Escherichia coli, and the recombinant enzyme was then characterized (9,10). By means of a site-directed mutagenesis experiment, the relationship between activity and amino acid substitutions was examined, and some mutations concerning enzyme stability were found. Neal et al. also isolated the DCase gene of Agrobacterium sp. and expressed the gene in E. coli and Agrobacterium sp. (11).

We also screened some strains producing the enzyme from mesophile (12) and thermotolerant strains (13) and cloned two enzyme genes. We tried to improve the native DCase to obtain a practical DCase that exhibits both high reactivity and sufficient stability for repeated use in a bio-reactor system by means of amino acid substitutions using recombinant DNA technology (14,15). We succeeded in creating a practical DCase by the substitution of three amino acids (16), and applied it to an industrial production process as an immobilized enzyme (17,18).

In this section, we report our attempts to establish a new D-amino acid production process, such as screening of DCase-producing bacteria from soil, purification of the enzymes, cloning of the enzyme genes, mutagenesis to obtain thermostabilized enzymes, and immobilization of the enzymes for a bioreactor system to produce D-amino acids.

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