Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1.

Abstract:

:A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+). The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).

journal_name

Appl Environ Microbiol

authors

Baek DH,Kwon SJ,Hong SP,Kwak MS,Lee MH,Song JJ,Lee SG,Yoon KH,Sung MH

doi

10.1128/aem.69.2.980-986.2003

keywords:

subject

Has Abstract

pub_date

2003-02-01 00:00:00

pages

980-6

issue

2

eissn

0099-2240

issn

1098-5336

journal_volume

69

pub_type

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