Hi there,

Is there any B. subtilis strain deficient for extracellular proteases which you could consider as an host for your lipase production? In this review, you should find some of interesting strains:

http://www.sciencedirect.com/science/article/pii/S0167488904000837

Hope it helps.

Hi Karthikumar, according to this paper, B. pumilus proteinases have a chymotrypsin-like specificity:

 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166261/pdf/bjm-45-389.pdf

Since the lipase also has serine proteinase-like active site, any covalent inhibitors of serine proteinases that target the active site Ser or His will probably also inhibit your lipase.

In lieu of generating a proteinase resistant lipase (as the above paper demonstrates), you have some options. 1) include non-competitive inhibitors of chymotrypsin-like proteinases in your culture, like L-Tyr, L-Phe, L-Trp. 2) Add aprotinin or soybean trypsin inhibitor. (They also inhibit chymotrypsin quite well). 3) do cultures in serum containing media. Serum has a lot of trypsin/chymotrypsin inhibitors, but I don't know what they will do to your lipase.

Please keep me informed, I am really curious about this lipase!

you can add any protease inhibitor to your solution which in turn will not affect your lipase activity

Dear Prof. Dominique Liger and Prof. Steingrimur Stefansson, thank you for your article link. Both articles are very useful. I try option 1, addition of inhibitors of chymotrypsin-like proteinases in culture.

You can add the famous inhibitor coktail containing PMSF....etc. but try to use reversable inhibitors just to be able to remove after purification 

or the lipase gene from thr producer bacteria cloned into the vector pET-15b and  expressed in Escherichia coli strain BL21 (DE3) or expressed by the Escherichia coli-Bacillus subtilis shuttle expression vector pSUGV4 in the protease-deficient strain B. subtilis WB600.

I recommend my first answer for you as it is very simple for begainers

If your protease has trypsin-like specificity then benzamidine can be used as reversible inhibitor. It's easily removable by dialysis/affinity column. PMSF is an irreversible inhibitor.

Use some protease inhibitor to inhibit according to the type of protease in order to inhibit proteolytic activity in the medium.

you can also you Chymostatin and Leupeptin kocktail for reversiable inhibition do not use PMSF because it is irreversable inhibitor and it will be difficult to  remove from the active site

Hi Karthikumar, I forgot another option you can do. You can add excess protein, like BSA or gelatin, to your culture as a competitive substrate for the proteinases.

However, this will only work well if you have a purification procedure for your lipase that is independent of extraneous proteins.

You have to check to nitrogen content in the media also..lesser nitrogen in media may also give less protease activity.

I used PMSF although my lipase has a catalytic Ser residue and I did not see any loss in lipase activity while proteases (trypsin and proteinase K) were inhibited. So it also depends whether PMSF is able to bind in the active site of your lipase. PMSF undergoes autohydrolysis so after quite short incubtation time it won't be active anymore.