The protease of Bacillus pumilus treated with EMS was studied. Enzyme production has improved. Also, increased enzyme activity accompanied a decreased Km value.
How can this be explained?
It seems that you have identified a mutant with higher affinity for the particular substrate you use in your assay than the wild-type. It would be interesting to compare the Kms of the wt and mutant for a variety of substrates. Was kcat affected by the mutation?
kcat had improved.
Thank you for your suggestion. I shall try the same with other substrates too.
I also intend to study the changes in the primary structure of the enzyme as well s its gene.
Lucky you.. As explained by Adam, this is what normally expected by mutuation, either random or site directed to enhance affinity of substrate by reducing Km and increasing the activity. However, many a times we do not get the activity significantly higher. in principle, the mutuation has changed the amino acid in the active site pocket, leading to the changes in the affinity or Km value.
Adam's suggestion will be quite helpful. You will be able to explain your mutant activity when you estimate its physiological efficiency.
You have obtained a mutant with high affinity for particular substarate. Try another relevant substrate to check the Km. How about Kcat?Did U check the partioning of your substrate?
one has to know what substrates you are using .
the compounds show hydrophobicity and hydrophilicity these will play the role in binding the substrate to the active site . so you can study the partion coefficient of the compounds and can plan the substrates with high V max and low Km.
protease activity in also in biphasic medium
All the above answers are interesting for further experimental characterization of the Bacillus protease. Please to see my answer in the attachment.
dear Dr. Sangeetha and all, I have one question. what is the best producing strain's of protease reported or used in the industry? what is the titre of enzyme Units per ml of the broth? in comparison with the prevailing technology, how does the present mutant fares.
If we make a random mutation, there are various possible outcomes. There may be no detectable effect in our chosen activity assay (in which case we won't know there has been a mutation); There may be an increase in activity in our assay. If this is confirmed with the purified enzyme, it could reflect a decrease in Km, an increase in Vmax, or both. If there is a decrease in Km, then your ability to detect that improvement will depend on the conditions of your assay. Thus if, as is often the case, the standard assay uses a nice, high substrate concentration, and if that concentration is near-saturating for the enzyme, the assay will be insensitive to improvement in Km. At lower substrate concentrations you will pick up improvement either in Vmax OR in Km. And it is quite possible to have mutants which 'improve' Km but decrease Vmax or alternatively increase Vmax but at the same time increase the Km. For practical applications, it is of course important to sort this out, and, as other respondents have said or implied, the pattern with one substrate may be quite different with another. With proteases it is often an artificial chromogenic substrate that is used for standard assay, and the kinetics for that artificial substrate will be be affected in quite different ways from the kinetics for the physiological protein substrate. It keeps us enzymologists in business!
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