Studies for Xylose isomerase and beta-Xylosidase from thermus caldophilus GK24

Abstract

Extremely thermophilic bacterium, Thermus caldophilus GK24 is a strain belonging to genus Thermus. It seems likely an ancient line that predates the development of distinct Gram-positive and Gram-negative microorganisms. Because enzymes from thermophilic organisms are designed to "take the heat", many researchers believe they may one day be useful as industrial catalysts for the production of fuels, pharmaceuticals, foods industry, and specialty chemicals, among other applications. Especially in carbohydrates field, thermostability gives advantages in substrates solubility by increasing the reaction temperatures as well as no need of cooling. Thermus caldophilus GK24 produces xylose isomerase. Activity screening for xylose isomerase was carried out with other enzyme using glucose and crude extract. As it was difficult to detect the isomerization process, analysis of isomerization reaction was achieved by HPAEC. Reaction mixture with fructose was also analyzed and the peak pattern was found to be identical regardless of kind of substrate or concentration of substrate. The gene encoding xylose isomerase (xylA) was cloned from Thermus caldophilus GK24 and the DNA sequence was determined. The gene xylA encodes the enzyme xylose isomerase (XI) consisting of 387 amino acids (calculated Mr of 44, 749). There was a partial xylulose kinase gene overlapping 4 bp at the end of xylose isomerase gene. The xylose isomerase gene was stably expressed in E. coli and purified by a procedure of heat treatment at 90℃ for 10 min followed by a column chromatography of DEAE-Sephacel. The molecular mass of the purified enzyme was estimated to be 45 KDa on the SDS-polyacrylamide gel electrophoresis, suggesting that molecular mass of the cloned xylose isomerase is absolutely the same as that purified from the donor bacterium. However, molecular mass of the cloned xylose isomerase was 185 KDa in native condition, indicating that the xylose isomerase was consisted as homermeric tetramer. ...