FIBROBACTER-SUCCINOGENES S85 POSSESSES AT LEAST 9 DISTINCT GLUCANASE GENES

The construction of genomic libraries of Fibrobacter succinogenes S85 in lambda-Dash, lambda-DashII, and pUC19, and the screening of recombinant clones for carboxymethylcellulose hydrolysis yielded 38 glucanase clones. These clones along with a collection of 10 glucanase clones in pUC8, were compared by restriction fragment and Southern hybridization analyses. Seven distinct glucanase clones (pCe14, pCe15, and pCe17 in pUC8, pCe16 in pUC19, and LCel8, LCel10, and LCel12 in lambda-Dash) were identified, which were nonhomologous to previously studied cel3, lichenase, and cellodextrinase genes from F. succinogenes S85. Specific activities of the encoded enzymes expressed in Escherichia coli were higher for carboxymethyl cellulose, barley beta-glucan, and lichenan, and lower for acid-swollen cellulose, laminarin, and xylan. The enzymes were predominantly acidic, with pIs between 3.5 and 5.2, with the exception of the pCe16 enzyme, which was basic with a pI of about 8.4. As determined by zymogram analysis after SDS-PAGE, the LCel8 enzyme was 97 kDa, the pCel10 enzyme exhibited components of 70 and 45 kDa, and the pCel12 enzyme components were 69 and 65 kDa. These seven new glucanase clones, along with the cel3 and lichenase genes, indicate that F. succinogenes S85 possesses at least nine distinct endoglucanase genes.