Thermostable tyrosine phenol-lyase of Symbiobacterium sp. SC-1: gene cloning, sequence determination, and overproduction in Escherichia coli.
During the screening for tyrosine phenol-lyase-producing thermophiles, we isolated an obligatory symbiotic thermophile, Symbiobacterium sp. SC-1, which grew only in coculture with Bacillus sp. SK-1. A gene encoding thermostable tyrosine phenol-lyase (TPL) was cloned from the genomic DNA of the Symbiobacterium sp. SC-1 and the nucleotide sequence of the TPL structural gene was determined. The gene consists of 1374 base pairs encoding a polypeptide of 458 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 52,196 Da. The structural gene of TPL was amplified by PCR, blunt-ended, and ligated into the NcoI-HindIII site of plasmid pTrc99A to construct an expression vector for the overproduction of the thermostable TPL. The level of thermostable TPL production was about 15% of the total soluble proteins of Escherichia coli extract. The enzyme was purified to homogeneity from the E. coli extract with an overall yield of 48%.
Lee
S G
SG
Microbial Conversion RU, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon, Korea.
Hong
S P
SP
Choi
Y H
YH
Chung
Y J
YJ
Sung
M H
MH
D000595:Amino Acid Sequence; D001407:Bacillus; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014447:Tyrosine Phenol-Lyase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014447:Tyrosine Phenol-Lyase