Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli.
A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined. The sequence was found to contain two large open reading frames. Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids. We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein. The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography. The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein. Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein. The possible involvement of this domain in interaction with the yeast cell wall is discussed.
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Section of Molecular Genetics, National Research Council of Canada, Montreal, Québec.
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Bastien
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009618:Nocardiaceae / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D004693:Glucan Endo-1,3-beta-D-Glucosidase