TAILIEUCHUNG - Báo cáo Y học: Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis Gene cloning and protein characterization

Random PCR mutagenesis was applied to theThermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylAgenes were cloned and expressed in Escherichia coliHB101 using the vector pGEMÒ-T Easy, and their physicochemical and catalytic properties were determined. | Eur. J. Biochem. 269 157-163 2002 FEBS 2002 Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis Gene cloning and protein characterization Anna Lonn1 Mark Gardonvi1. Willem van Zvl2. Barbel Hahn-Haaerdal1 and Ricardo Cordero Otero2 1 Department of Applied Microbiology Lund University Sweden department of Microbiology University of Stellenbosch Matieland South Africa Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions E372G V379A M-1021 E372G F163L M-1024 and E372G M-1026 . The wildtype and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM -T Easy and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was w which is similar to the wild-type enzyme. Compared with the wild-type the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants kcat for D-xylose compared with the wild-type enzyme at 60 C but they did not show any increase in catalytic efficiency kcat Km . For D-glucose both the kcat and the kcat Kn values for the mutants were increased compared with the wild-type enzyme. Furthermore the mutant enzymes exhibited up to 255 times higher inhibition constants Ki for xylitol than the wild-type indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures. Keywords xylose isomerase cold adaptation random mutagenesis Saccharomyces cerevisiae xylose fermentation. The use of ethanol from renewable raw materials is an attractive alternative for meeting increasing global demand for liquid fuels because its combustion does not .

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