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Applied and Environmental Microbiology, March 2004, p. 1570-1575, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1570-1575.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a D-Amino-Acid-Containing Dipeptide

Dae Heoun Baek,1 Jae Jun Song,1 Seok-Joon Kwon,1 Chung Park,2 Chang-Min Jung,2 and Moon-Hee Sung2,3*

Laboratory of Microbial Function, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong-Gu,1 BioLeaders Corporation, Joong-Gu, Daejeon,2 Department of Bio- & Nanochemistry, Kookmin University, Songbuk-gu, Seoul, South Korea3

Received 18 August 2003/ Accepted 3 December 2003

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 P1 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/Km)D,D], while the D-enantioselectivity to the P1' site of Ala-Ala was 16.4 ± 0.5 [E = (kcat/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 ß-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.

* Corresponding author. Mailing address: Department of Bio- & Nanochemistry, Kookmin University, 861-1 Chongnung-dong, Songbuk-gu, Seoul 136-702, South Korea. Phone: 82 2 910 4808. Fax: 82 2 910 4415. E-mail: smoonhee{at}kookmin.ac.kr.

Applied and Environmental Microbiology, March 2004, p. 1570-1575, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1570-1575.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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