Abstract:
Genes for endo-glucanase (Egl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotea, were cloned in pUC18 in its Sac1 cloning site and transformed to E. coli. Six clones showed yellow zones of hydrolysis on carboxy methyl cellulose (CMC) plates with Congo red, in liquid culture, and on native polyacrylamide gel electrophoresis activity gels. They belonged to three distinctly different groups. Three representative E. coli clones carrying recombinant palsmids were designated pRM2, pRM13 and pRMC28. The genes were located on 1.1, 2.1 and 4.6 kb fragments respectively. Their location was obtained by deletion analysis which revealed that 0.65, 1.1 and 3.2 kb fragments were essential to code for EglA, EglB and EglC, respectively and conferred intracellular production of endoglucanase on E. coli. Expression of the Egl genes resulted in hyperproduction of endo-glucanase in all clones. The secretion occurred into the periplasmic fractions. The endo-glucanases produced by recombinant E. coli resemble the native endo-glucanases with respect to temperature optima, pH optima, and effect of metal ions on enzyme activities but differ with respect to location of the enzyme and some other enzyme properties. The cloned genes can be used as selection markers for introducing recombinant plasmids in wild strains of E. coli.