Cloning and expression in Escherichia coli of the gene for an Arthrobacter beta-(1----3)-glucanase.
When inserted in the correct orientation at the BamHI site of plasmid YRp7, an 8.6-kilobase BamHI fragment of Arthrobacter sp. strain YCWD3 DNA gave Escherichia coli HB101 cells harboring the recombinant plasmid pBX20 the ability to lyse bakers' yeast cell walls or bakers' yeast glucan in agar medium. An extract of the transformed E. coli cells contained an endo-beta-(1----3)-glucanase with the same activity pattern as that of glucanase I produced by Arthrobacter sp. strain YCWD3. Although part of the glucanase activity was contributed by apparently defective molecules, two protein species were found which had high lytic activity on yeast cell walls and adsorbed to microcrystalline cellulose, and both had a single constituent polypeptide with a molecular weight of about 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these properties the protein species were indistinguishable from those glucanase I protein species of Arthrobacter sp. strain YCWD3 which we believe are nearly the intact molecule. We conclude that the cloned fragment of Arthrobacter sp. strain YCWD3 DNA contains the structural gene for glucanase I. A recombinant plasmid obtained by subcloning a PstI fragment of pBX20 into pBR322 caused the transformed E. coli cells to produce apparently defective glucanase molecules only. This observation serves as additional supporting evidence for our conclusion.
D001173:Arthrobacter / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D043326:Glucan 1,3-beta-Glucosidase; D005959:Glucosidases / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005959:Glucosidases; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase