Physiological and Genetic Improvement of Egyptian Cellulolytic Microorganisms for Biotechnology Applications

Abstract

Cellulose, a long linear polymer consisting of glucosyl units connected by β-1,4-glycosidic linkages, is the most abundant biomass produced on Earth as a renewable bioresource. The aim of this study was to obtain high production of cellulases from wild types and genetically modified local bacterial strains. In this study soil, ward poultry and sea grasses samples were used for cellulolytic bacteria isolation and screened for their cellulase production. Four bacterial isolates were selected as cellulase producers promising strains and identified using morphological, biochemical tests and 16S rDNA sequencing. These strains were identified and named as Bacillus amyloliqueficans subsp. plantarum SA5, Bacillus megaterium BMS4, Bacillus subtilis subsp. subtilis BTN7Aand Anoxybacillus flavithermusBTN7B. Their 16S rDNA sequencing were placed in public domain (ID: KC438369, KC429572, KC438368 and KC429571, respectively). Optimum conditions for cellulases production of these strains were determined including pH, aeration, medium volume, incubation period and carbon source. Two cellulases genes (i.e., endo-β-1,3-1,4 glucanase (bgls) and endo-β-1,4 glucanase (egls)) were isolated using PCR technique, cloned in pJET1.2/blunt and pLUG®-Multi TA cloning vectors, transferred to E. coli DH5α, and sequenced. The donor of these genes was Bacillus subtilis subsp. subtilis 168. Confirmation of the existence of bgls and egls genes in the recombinant plasmids (pJET-bgls-nrc1 and pJET-egls-nrc1) was done by colony PCR and their orientation was detected using restriction enzymes and PCR amplification. Bglsgene orientation was found to be clockwise in Ferm1 and Ferm 2 transformants while counter clockwise in Ferm3 and Ferm4 transformants. Egls gene was counter clockwise in all tested transformants.