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Title 

Characteristics of a new enantioselective thermostable dipeptidase from Brevibacillus borstelensis BCS-1 and its application to synthesis of a D-amino-acid-containing dipeptide

Authors 

Dae Heoun BaekJae Jun SongSeok Joon KwonC ParkC M JungM H Sung

Publisher 

American Society for Microbiology

Issue Date 

2004

Citation 

Applied and Environmental Microbiology, vol. 70, no. 3, pp. 1570-1575

Keywords 

amino acidshydrolysissynthesis (chemical)thermodynamic stabilityamino acid sequencesenantioselectivitybacteriaalanineaspartic acidbeta lactamase

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 Mr 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 P 1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (kcat/Km) for L-enantioselectivity to the P1 site of Ala-Ala was 23.4 ± 2.2 [E = (kcat/Km)L,D/(kcat/K m)D,D], while the D-enantioselectivity to the P 1′ site of Ala-Ala was 16.4 ± 0.5 [E = (k cat/Km)L,D/(kcat/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 >-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.

ISSN 

0099-2240

Link 

http://dx.doi.org/10.1128/AEM.70.3.1570-1575.2004

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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