Screening and Characterization of Bacillus thuringiensis Stock Cultures from Middle Tennessee for Production of Chitinase Enzyme
Chitinases are secreted by Bacillus thuringiensis strains (Bt). They serve as an alternative to chemical pesticides. Chitin products obtained by the action of chitinases on chitin polymer for different purposes such as clinical, industrial, and pharmaceutical Bt is a Gram-positive soil bacterium, with a genome size of 2.4 to 5.7 million base pairs. Bt strains are used in this research because it is a safer bacterium for humans and the environment. The spatial occurrence of this organism is not restricted and has been isolated worldwide from many habitats, including soil, stored-product dust, insects, and deciduous and coniferous leaves. Sixty-Six strains of B. thuringiensis used in this research were collected isolates from Middle Tennessee. PCR amplification was used to obtain the chitinase gene from Bacillus thuringiensis isolates and analyze by agarose gel electrophoresis. We found some strains have chitinase gene with both primers used in this research, and some strains just produced chitinase gene with degenerate primer. The sequence aligned with corresponding sequences of both primers specific and degenerate from the database by using BLASTX and the sequences compared with the sequence in the data gene bank. Bacillus thuringiensis strains were screened on colloidal-chitin agar medium. Then, after incubation for 72h at 30°C, Chitinase production was determined by the zones that hydrolysis produced. Chitinase production was determined by zones of hydrolysis produced after 72, 96 and 120 h of incubation at 37°C. After four days of incubation, 41 bacteria isolates obtained from Middle Tennessee were selected as chitinase-producing bacteria based on the clear zones of hydrolysis produced. The result with both primers concludes that Bt 8 does not produce the chitinase gene and, it is not secreted in the colloidal chitin agar plate. Bacillus thuringiensis strains will be very important for chitinase production in biotechnology and biocontrol of fungal pathogens and harmful insects.
Rasha Hadi Alsamadani,
"Screening and Characterization of Bacillus thuringiensis Stock Cultures from Middle Tennessee for Production of Chitinase Enzyme"
ETD Collection for Tennessee State University.