Characterization and phylogenetic analysis of a thermostable N-carbamoyl-L-amino acid amidohydrolase from Bacillus kaustophilus CCRC11223

被引:18
作者
Hu, HY
Hsu, WH
Chien, HCR [1 ]
机构
[1] Chung Shan Med Univ, Dept Microbiol, Taichung 402, Taiwan
[2] Natl Chung Hsing Univ, Inst Mol Biol, Taichung 402, Taiwan
[3] Hung Kuang Inst Technol, Dept Food Sci & Nutr, Taichung 433, Taiwan
关键词
N-carbamoyl-L-amino acid amidohydrolase; Bacillus kaustophilus; N-carbamoyl-L-homophenylalanine; thermostable enzyme; phylogeny;
D O I
10.1007/s00203-003-0524-9
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A thermostable N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) gene composed of an 1,230-bp ORF encoding a 44.3-kDa protein was cloned from the thermophile Bacillus kaustophilus CCRC11223. This L-N-carbamoylase contained six cysteine residues that form three disulfide bridges. The purified L-N-carbamoylase was stringently L-specific and exhibited high activity in the hydrolysis of N-carbamoyl-L-homophenylalanine. N-carbamoyl derivatives of beta-alanine, beta-amino-isobutyric acids, L-tryptophan, and D-specific amino acids were not recognized as substrates. The L-N-carbamoylase required the divalent metal ions Mn2+, Co2+, and Ni2+ for increasing activity. The pH and temperature optima of the enzyme were pH 7.4 and 70 degreesC, respectively. This enzyme was completely thermostable at 50degreesC for 36 days in the presence of D- and/or L-specific substrates. Phylogenetic analysis of the available amino acid sequences of N-carbamoyl and N-acyl amino acid amidohydrolases from the three main kingdoms of life showed that they can be divided, into four distinct families. The B. kaustophilus enzyme could be classified into the family Of L-N-carbamoylases and some beta-ureidopropionases, but did not hydrolyze beta-ureidopropionates.
引用
收藏
页码:250 / 257
页数:8
相关论文
共 24 条
[1]   NEW METHOD FOR DISULFIDE ANALYSIS OF PEPTIDES [J].
ANDERSON, WL ;
WETLAUFER, DB .
ANALYTICAL BIOCHEMISTRY, 1975, 67 (02) :493-502
[2]  
[Anonymous], 1983, COLD SPRING HARBOR L
[3]   Two amino acid amidohydrolase genes encoding L-stereospecific carbamoylase and aminoacylase are organized in a common operon in Bacillus stearothermophilus [J].
Batisse, N ;
Weigel, P ;
Lecocq, M ;
Sakanyan, V .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1997, 63 (02) :763-766
[4]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[5]  
BURKHARD W, 1999, J BIOTECHNOL, V68, P101
[6]   Identification of the open reading frame for the Pseudomonas putida D-hydantoinase gene and expression of the gene in Escherichia coli [J].
Chien, HR ;
Jih, YL ;
Yang, WY ;
Hsu, WH .
BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 1998, 1395 (01) :68-77
[7]  
DOI RH, 1983, RECOMBINANT DNA TECH, P162
[8]   MICROBIAL CONVERSION OF DL-5-SUBSTITUTED HYDANTOINS TO THE CORRESPONDING L-AMINO-ACIDS BY PSEUDOMONAS SP STRAIN-NS671 [J].
ISHIKAWA, T ;
WATABE, K ;
MUKOHARA, Y ;
KOBAYASHI, S ;
NAKAMURA, H .
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 1993, 57 (06) :982-986
[9]   MICROBIAL CONVERSION OF DL-5-SUBSTITUTED HYDANTOINS TO THE CORRESPONDING L-AMINO-ACIDS BY BACILLUS-STEAROTHERMOPHILUS NS1122A [J].
ISHIKAWA, T ;
MUKOHARA, Y ;
WATABE, K ;
KOBAYASHI, S ;
NAKAMURA, H .
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 1994, 58 (02) :265-270
[10]   FACTORS AFFECTING THE ISOLATION OF CCC DNA FROM STREPTOMYCES-LIVIDANS AND ESCHERICHIA-COLI [J].
KIESER, T .
PLASMID, 1984, 12 (01) :19-36