Enhancement of alkaline protease production in Bacillus circulans using plasmid transformation

Abstract

Plasmid transformation is an efficient and crucial biotechnological tool that enables the enhancement of many important microbial characters that would be beneficial in a lot of industrial, agricultural and environmental applications. In the present study, five Bacillus species (B. subtilis, B. cereus, B. alvei, B. circulans and B. pumilus) were investigated. They were isolated from agricultural soils of different local arid environments of the Kingdom of Saudi Arabia, identified and characterized for their plasmid content. The main objective of the present study was to enhance the production of alkaline protease in Bacillus circulans (the recipient strain) through plasmid transformation from B. subtilis (the donor strain). All the tested Bacillus strains successfully produced unique multiple (3, 4 and 5) spontaneous antibiotic resistant mutants against chloramphenicol, neomycin, rifampicin, streptomycin, kanamycin and tetracycline and all of which were mutated to Rif r strains. B. pumilus showed the highest resistance against five of the six tested antibiotics while both of B. alvei and B. circulans showed the lowest resistance to only three of the tested antibiotics. Results revealed that B. subtilis was the best among the tested species concerning the production of alkaline protease (90.2 U/ml) while B. pumilus was the lowest in activity (40.3 U/ml). Screening of plasmid content revealed the presence of one or two mega indigenous plasmids in all the tested species. The four transformant strains BC 1 , BC 2 , BC 3 and BC 4 resulting from plasmid transformation exhibited significant increases in the activity of alkaline protease and recorded 2.31-to 3-fold increases compared to the parent B. circulans cells and 2.11-to 2.75-fold increases compared to the donor cells of B. subtilis. They also acquired antibiotic resistance to tetracycline and chloramphenicol that was completely absent in the parent cells of B. circulans. Results revealed that plasmid transformation among the tested Bacillus spp. is a powerful technique that can be efficiently exploited to enhance alkaline protease production in the transformed Bacillus spp. compared to their wild strains and we recommend using the improved transformant strains for commercial and industrial purposes.

High-level expression of alkaline protease using recombinant Bacillus amyloliquefaciens

“…The increase in productivity of the alkaline protease by the transformants may be attributed to the donor plasmid harboring the apr gene. In this context, previous reports were in support of these findings (Jorgensen et al, 2000;Sabir et al, 2009), indicating that the increase in gene copy number enhances the enzymatic production. The maximum crude enzyme activity of alkaline protease in B. licheniformis CICIM B5102 was 3,505 U/ml after 96 h, whereas that of the recombinant B. amyloliquefaciens strain D was 4,926 U/ml after 60 h. More transformants were selected for the shaker flask fermentation test to obtain higher protease production.…”

Bacterial Production of Protease-A Preliminary Study

“…from fish which was capable of producing protease enzyme. Sabir et al (2009) reported that, B.subtilis was the best protease producer among the tested strains. In the present study, all the bacterial species isolated had a better potentiality of degrading market fish wastes.…”