Improvement of cellulose degradation by cloning of endo-ß-1, 3-1, 4 glucanase (bgls) gene from Bacillus subtilis BTN7A strain.

The aim of this study is to construct a new recombinant strain able to degrade cellulose efficiently. The endo-ß-1, 3-1, 4 glucanase (bgls) gene was cloned from Bacillus subtilis BTN7A strain by using PCR technique. The specific primers of bgls gene were deduced. Optimization of PCR mixture and program were identified. The nucleotide sequence of bgls was placed in the public domain (GenBank accession number KM009051.1). The obtained bgls DNA was cloned with pGEM®-T Easy Vector. The recombinant plasmid designated as Bgls-NRC-1 was transformed into E. coli DH5α. The successful cloning of the bgls gene was tested either by PCR or by evaluating its expression in its new bacterial host. The bgls gene was expressed efficiently in E. coli and the enzyme activity of the transformant was compared to the enzyme activity of the donor bacterial strain. The new constructs produce much higher enzyme yields than the donor bacterial strain, they produce about 29% and about 57% higher cellulase specific activity at 37 °C and 55 °C respectively. Optimization of cellulolytic activity of the new recombinant strain were described. The effect of minimal medium supplemented with CMC or cellulose, or complete medium (LB) on bgls expression were tested, the order of cellulase activity production was CMC27.2 > cellulose 21.9 > LB 19.8 U/mg protein, respectively at 24 h. CMC was proved to be the best medium for cellulase production. Results also showed that double the initial inoculum resulted in more cellulase activities in all media.

Optimization and molecular identification of novel cellulose degrading bacteria isolated from Egyptian environment.

Cellulase producing bacteria were isolated from both soil and ward poultry, using CMC (carboxymethylcellulose) agar medium and screened by iodine method. Cellulase activity of the isolated bacteria was determined by DNS (dinitrosalicylic) acid method. The highly cellulolytic isolates (BTN7A, BTN7B, BMS4 and SA5) were identified on the basis of Gram staining, morphological cultural characteristics, and biochemical tests. They were also identified with 16S rDNA analysis. The phylogenetic analysis of their 16S rDNA sequence data showed that BTN7B has 99% similarity with Anoxybacillus flavithermus, BMS4 has 99% similarity with Bacillus megaterium, SA5 has 99% homology with Bacillus amyloliquefaciens and BTN7A was 99% similar with Bacillus subtilis. Cellulase production by these strains was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, different volumes of media, aeration rate and carbon source. The cellulase specific activity was calculated in each case. In conclusion four highly cellulolytic bacterial strains were isolated and identified and the optimum conditions for each one for cellulase production were determined. These strains could be used for converting plant waste to more useful compounds.