Cloning and expression of endo-beta-1,3-glucanase gene from Flavobacterium dormitator in Escherichia coli and characterization of the gene product.
The beta-1,3-glucanase (1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKU beta G1 (8.2 kb), showed a high beta-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the periplasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-beta-1,3-glucanase II and a nonlytic endo-beta-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-beta-1,3-glucanase I was also formed by a proteolytic digestion of endo-beta-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-beta-1,3-glucanase II was coded for in the cloned gene.
Nagata
S
S
Department of Agricultural Chemistry, Kochi University, Japan.
Maru
I
I
Ishihara
F
F
Misono
H
H
Nagasaki
S
S
D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007150:Immunohistochemistry; D007526:Isoelectric Point; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D004693:Glucan Endo-1,3-beta-D-Glucosidase