subtilis has six extracellular proteases including neutral protease A, subtilisin (also known as alkaline protease), extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B (8) In view of the fact that the protease production is limited, protease deficient B.
subtilis 168, it was decided to inactivate the aprE gene of this bacterium encoding one of the major extracellular alkaline serine proteases, Subtilisin E, by site directed mutation of this gene using homologous recombination techniques.
To quantitatively compare the protease activity of wild and mutant strains deficient in active subtilisin E, the Protease Fluorescent Detection Kit (Sigma-Aldrich, Germany) based on the proteolytic hydrolysis of a fluorescein isothiocyanate (FITC)-labeled casein-substrate was used according to the manufacturer's protocol.
subtilis 168 would not further be able to produce active subtilisin E and finally would decrease the protease activity.
Subtilisin E is one of typical examples which functions similarly (15) .
The effects of different carbon sources on subtilisin (Pr1) like protease activity by M.
High levels of subtilisin (Pr1) activity were observed in cultures supplemented with diamondback moth cuticle or chitin plus 1% glucose, and lower levels were observed in cultures containing 1% glucose, 1% GlcNAc or when diamondback moth cuticle and chitin were used in combination with 1 % GlcNAc whereas a high subtilisin (Pr1) activity was observed from the supernatant having 1% diamondback moth cuticle as a sole carbon source.
The subtilisin like Pr1 from the mushroom pathogen Trichoderma harzianum is induced also by chitin in fungal cell wall preparations and is repressed in the presence of casein or BSA (Geremia et al., 1993).
Regulation of cuticle degrading subtilisin proteases fron the entomopathogenic fungi, Lecanicillium spp: implications for host specificity.
They ended up with a subtilisin that was 256 times more active in DMF than the original enzyme.
Arnold and Chen found it advantageous to use test-tube evolution because they didn't know much about how solvents inactivate subtilisin. Despite this lack of knowledge, test-tube evolution allowed them to create the enzyme they wanted.
With subtilisin, the researchers were lucky to observe a stepwise progression of the protein product toward an enzyme that could function in the hostile environment of organic solvents.