CLONING AND EXPRESSION OF THE GENE FOR XYLOSE ISOMERASE FROM THERMUS-FLAVUS AT62 IN ESCHERICHIA-COLI

The gene encoding xylose isomerase (xylA) was cloned from Thermus flav us AT62 and the DNA sequence was determined. The xylA gene encodes the enzyme xylose isomerase (XI or xylA) consisting of 387 amino acids (c alculated Mr of 44,941). Also, there was a partial xylulose kinase gen e that was 4 bp overlapped in the end of XI gene. The XI gene was stab ly expressed in E. coli under the control of tac promoter. XI produced in E. coli was simply purified by heat treatment at 90 degrees C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purif ied enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electr ophoresis. However, Mr of the cloned XI was 185 kDa on native conditio n, indicating that the XI consists of homomeric tetramer. The enzyme h as an optimum temperature at 90 degrees C. Thermostability tests revea led that half life at 85 degrees C was 2 mo and 2 h at 95 degrees C. T he optimum pH is around 7.0, close to where by-product formation is mi nimal. The isomerization yield of the cloned XI was about 55% from glu cose, indicating that the yield is higher than those of reported enzym es. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn2+, Co2+, and Mg2+ are requ ired for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.