ABSTRACT
A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M
r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k
cat
/Km
) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k
cat
/Km
)
l,d
/(k
cat
/Km
)
d,d
], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k
cat
/Km
)
l,d
/(k
cat
/Km
)
l,l
] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.
Nucleotide sequence of a 1.1 kb fragment of the pea chloroplast genome containing three tRNA genes, one of which is located within an open reading frame of 91 codons