Molecular cloning and characterization of the xylose isomerase gene from a thermophilic Bacillus species.
The gene (xylA) encoding a thermostable xylose isomerase has been isolated and characterized from a thermophilic Bacillus species for the first time. The xylA open reading frame of 1323 bp encoded a protein containing 441 amino acids with a calculated molecular weight of 50,176. The amino acid sequence of this protein showed 76% homology to xylose isomerase isolated from Bacillus subtilis and contained all the important catalytic domains of the enzyme. The gene complemented the xyl-5 mutation and produced a functional enzyme constitutively in Escherichia coli. The crude cell-free extract of E. coli recombinants exhibited xylose isomerase activity over a wide range of temperatures from 60 to 100 degrees C with an optimal enzyme activity of 10.4 Units/mg protein at 85 degrees C. This optimal temperature was one of the highest reported so far for thermostable xylose isomerases. The recombinant enzyme was found to be a tetramer with each subunit having molecular weight of 50,000.
Liao
W X
WX
Department of Biochemistry, Faculty of Medicine, National University of Singapore.
Earnest
L
L
Kok
S L
SL
Jeyaseelan
K
K
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D015342:DNA Probes; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase