Thermostable glycerol kinase from Thermus flavus:: cloning, sequencing, and expression of the enzyme gene

The thermostable glycerol kinase (EC 2.7.1.30) gene from Thermus flavus was cloned and expressed in Escherichia toll DH5 alpha. An open reading frame of 1488 bp for the glycerol kinase gene (glpK) starting with an ATG methionine codon was found, which encodes a protein of 496 amino acid residues whose calculated molecular weight is 54,835. The amino acid sequence of T. flavus glycerol kinase is 80.6% and 64.1% identical with those of Bacillus subtilis and E. coli. Transformants of E. coli DH5 alpha harboring plasmid pGYK12 with a 1505 bp chromosomal DNA fragment containing the T. flavus glycerol kinase gene showed about 23.8-fold higher glycerol kinase activity than T. flavus. (C) 1998 Elsevier Science B.V.