Thermostable tyrosine phenol-lyase of Symbiobacterium sp. SC-1: gene cloning, sequence determination, and overproduction in Escherichia coli

Protein Expression and Purification
S G LeeM H Sung

Abstract

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%.

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Jan 1, 1975·Advances in Applied Microbiology·H Yamada, H Kumagai
Jun 18, 1971·Biochemical and Biophysical Research Communications·H EneiH Yamada
Apr 27, 1993·Biochemistry·A A AntsonK S Wilson

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