Lee S G, Hong S P, Choi Y H, Chung Y J, Sung M H
Microbial Conversion RU, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon, Korea.
Protein Expr Purif. 1997 Dec;11(3):263-70. doi: 10.1006/prep.1997.0792.
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%.
在筛选产酪氨酸酚裂解酶的嗜热菌过程中，我们分离出一种专性共生嗜热菌，共生杆菌属SC-1菌株，它仅在与芽孢杆菌属SK-1菌株共培养时才能生长。从共生杆菌属SC-1菌株的基因组DNA中克隆到一个编码耐热酪氨酸酚裂解酶（TPL）的基因，并测定了TPL结构基因的核苷酸序列。该基因由1374个碱基对组成，编码一个含有458个氨基酸残基的多肽；酶亚基的分子量估计为52,196道尔顿。通过PCR扩增TPL的结构基因，将其平端连接到质粒pTrc99A的NcoI-HindIII位点，构建用于过量表达耐热TPL的表达载体。耐热TPL的产量约占大肠杆菌提取物总可溶性蛋白的15%。该酶从大肠杆菌提取物中纯化至均一，总产率为48%。
