Showing papers by "Alan Collmer published in 1983"

Cloning and Expression of a Thermomonospora YX Endocellulase Gene in E. coli

Abstract: A set of E. coli transformants (2,500) containing plasmid pBR322 with Thermomonospora YX DNA inserts larger than 3 kb was prepared. The transformants were screened directly for the ability to hydrolyze carboxymethyl cellulose. Two colonies exhibiting cellulase activity were found. One colony contains a plasmid (pD365) that contains a 7.6 kb insert, including the entire gene coding for the major endoglucanase activity of Thermomonospora YX. The other colony (D315) had two plasmids in it: pD316 contains a 8.5 kb insert which includes at least 80% of the major endoglucanase gene; pD315 contains a 6.6 kb insert and does not express cellulase activity. Restriction maps were prepared for each plasmid and the endoglucanase gene was localized to a 2.3 kb SalI fragment in pD316 and to a 3.7 kb EcoRI fragment in pD365. These fragments were subcloned into plasmids containing active promoters regulated by the lactose repressor. The new strains each made about 50 times more cellulase than the original strains. E. coli transformants containing plasmids pD316, pD365 and their derivatives secrete about 30% of their endoglucanase activity into the medium, 30% into the periplasmic space, while 40% remains in the cytoplasm. Thermomonospora YX DNA restriction digests were fractionated by agarose gel electrophoresis and the fragments which hybridized to pD315 and pD316 were detected by nitrocellulose blotting techniques. The results indicated that each plasmid contains a single contiguous segment of Thermomonospora YX DNA and that the pD315 and pD316 inserts are linked in the Thermomonospora YX genome.