Analysis of the Enzyme Genes

The DNA sequences of the genes in the 1.8-kb fragments were analyzed (Fig. 2), there being one open reading frame in each case, consisting of 912 and 936 bases with a starting triplet, ATG, and predicted to encode polypeptides of 304 and 312 amino acids with calculated molecular weights of 34,285 and 35,438, respectively. Upstream of the open reading frame, sequences similar to the — 35 and — 10 consensus sequences for E. coli promoters and a putative ribosomal-binding site (Shine-Dalgarno sequence) were found. The G + C contents of the open reading frames were 60.3% and 62.9%, respectively (see Table 2).

The homologies of the DNA sequences and the deduced amino acid sequences between these two DCases were 62% and 60%, respectively. An 8-amino acid length of the C-terminal region was different in these two enzymes. The homologies among those of Agrobacterium sp. KNK712 and those of two other reported Agrobacterium strains (9,10) which had completely the same sequences, were 97.0% (amino acid sequences) and 93.0% (DNA sequences). The homologies of the N-terminal regions of 30 amino acids in five DCases (6,7,9,12,13) were about 50%, the homology between the enzyme of Pseudomonas sp. KNK003A and the Comamonas sp. E222c DCase being the highest (56.7%), besides the homology among Agrobacterium strains. Multiple alignment of the amino acid sequences of these enzymes is shown in Figure 3.

Similarity research using a database (PIR release, 45.0) showed that the aliphatic amidase of Pseudomonas aeruginosa (19) showed the highest similarity (30%) to DCase, but a closely related enzyme was not found. Multiple alignment with Clustal W (1.4) software of the DCase and 10 known related enzymes (PIR accession numbers [PIR Ac. No.] are given in the legend to Fig. 4) showed that 6 amino acids, that is the 119th amino acid of DCase, glycine (hereinafter abbreviated as 119th Gly), 126th Arg, 127th Lys, 146th Glu, 153rd Gly, and 172nd Cys, were all at corresponding positions.

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