From the two preliminary growth experiments it seems that the strain of L. crispatus I am using is able to breakdown glycogen and use it for growth. If this observation holds (I am currently repeating it a third time, running the starch assay again and running the supernatants on HPLC to measure lactate production), it poses lots of questions for what this means in practice. Although the results have not been robust, it looks like starch-degrading activity is only present when L. crispatus was grown on glycogen and not when the cultures were grown on glucose. This immediately makes me think of carbon catabolite repression: the mechanism where bacteria in the presence of glucose shut down the expression of enzymes metabolizing other, less preferential, carbon sources.
My colleague Jurgen Haanstra posed an alternative hypothesis in the hallway yesterday. This doesn’t necessarily have to be regulation at the transcriptional level. This can also just be caused by product inhibition on the enzyme level! (thanks Jurgen, I really appreciate it). A few minutes later he sent me this paper from 1986 (cause that’s how he rolls): Glucose feedback inhibition of amylase activity in Aspergillus sp. and release of this inhibition when cocultured with Saccharomyces cerevisiae.
Here, they were unable to measure amylase activity in the supernatants of this fungus, but when they dialysed the enzymes (i.e. replacing the liquid, while retaining the enzyme) amylase activity was back. If this phenomenon is also going on with the glycogen-degrading enzymes of L. crispatus this could not only explain why I am not seeing activity in glucose grown wells, but also why in the starch assay untill now only about half of the starch was degraded. If glucose accumulates, enzyme activity will seize. Pretty basic biochemistry actually. (Hundred years ago someone could have been doing the experiments I’m doing now, nonetheless it is pretty exciting. And as far as I am aware, pretty novel too.)
So, I am first going to simply add some glucose to the starch assay and also other sugars such as galactose and maltose. Maltose is also a breakdown product of glycogen/starch metabolism, so perhaps this will also provide some inhibition. Will keep you updated!
One Reply to “Thoughts about product inhibition of amylase.”
I just came across this blog and I’m adding it to my RSS feed. It conveys very nicely the feeling of doing research at the lab and I kind of miss that since I finished my Ph.D.
I was part of a workshop organized at the Lorentz center by Jurgen and he is a great guy, we had lots of fun at the dinner of the course. Keep up the good work!