Cloning of genes encoding fibrolytic enzymes from Bacillus isolates and determination of the potential use of recombinant bacteria as silage inoculants

In this thesis study, Bacillus strains that produce CMCase enzymes active under low pH conditions have been isolated from soil samples. Among the isolates, Bacillus sp. KED3 was selected for further studies and was found to be closely related to Bacillus amyloliquefaciens with 99.63% similarity based on 16S rRNA sequencing analysis. The gene responsible for the production of the bifunctional enzyme with CMCase and lichenase activities was cloned in E. coli JM109 and Lactobacillus plantarum using the pUC18 and pUC18rA vectors, respectively. Molecular genetic analyses confirmed the cloning of the bifunctional gene in both bacteria, although recombinant enzyme activity was observed in E. coli but not in L. plantarum. Zymogram analysis calculated the molecular weight of the enzyme as 31.78 kDa. The bifunctional enzyme's CMCase activity reached its maximum level at pH 5.0, while its lichenase activity peaked at pH 6.0. The optimum temperature for both enzyme activities was determined to be 60°C. MnCl2, CaCl2, CuSO4, MgCl2, and CoCl2 each at a concentration of 5 mM variously enhanced CMCase activity, whereas SDS and EDTA inhibited it. CaCl2, CuSO4, and MgCl2 enhanced lichenase activity at varying rates, while MnCl2, CoCl2, SDS, and EDTA inhibited it. Thermal stability analyses showed that both enzymes completely retained their activities after 15 minutes of incubation at 40°C, but activity losses began above 60°C. Both enzyme activities in B. amyloliquefaciens reached maximum levels 24 hours after inoculation, while in recombinant E. coli, CMCase activity peaked at 48 hours and lichenase activity at 96 hours. The addition of recombinant E. coli to pearl millet silage affected the pH, dry matter (DM), crude cellulose (CC), neutral detergent insoluble fiber (NDF), and acid detergent insoluble fiber (ADF) contents. The addition of recombinant bacteria resulted in a decrease in silage pH and crude cellulose content, while analyses at 21, 28, and 35 days showed a reduction in the dry matter content of the silage, with partial increases in ADF and NDF contents.