Some of the fungal endoglucanases are giving me better CMC'ase activity in succinate and citrate buffer. However activity measurement in acetate as buffering media is bringing 30% reduction. Is acetate destabilizing or inhibitory? Any references or explanation on the same is highly appreciated
Hi there,
I guess it is due to the actual ionc strength generated by your buffer system. At fixed pH and fixed buffer concentration, acetate (monoacid) will generate less ionic strength than both succinate (diacid) and citrate (triacid). Your enzyme activity is surely dependent on ionic strength. For more info:
https://en.wikipedia.org/wiki/Ionic_strength
of course my dear, because enzyme activity depended on ionic strength of buffer , therefore we make some experiment by using many buffer to selected the best depend on enzyme activity ....good luck.
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Thanks for the answers. But this effect is being extended even in the saccharification process and we are getting higher yields of total RS and glucose when experiments are being conducted in citrate rather than acetate at same pH and with same ionic strengths (50mM).. Kindly answer Dr. Liger and Dr. Jabbar...
Hi again,
50mM citrate at pH 4.75 (I guess you are working around the pK of acetic acid) is generating more ionic strength than 50mM acetate at the same pH because this pH corresponds to the pK of second acidic function of citrate (ie. the first being already totally deprotonated) whereas it is the unique pK of acetate. Citrate will on average bear 1.5 negative charges and acetate only 0.5.
I am really very very thankful for your answer Dr. Liger.... We are working in pH range of 4.0 to 4.5.
Thanks Alberto for your answer. We are trying here different buffers such as citrate, acetate, citrate phoshate, succinate, some good buffers and are also trying to assess enzymatic saccharification of acid pretreated bagasse by adjusting pH with liquor ammonia. The main intent is that the enzyme cocktail with which we are working should be robust enough to generate RS and glucose effectively without any inhibitory effect of some buffering species. With Aspergillus extracts, we are getting huge reductions in RS release when changing buffer from citrate to acetate but thats not the case with Penicillium cellulase.
these two fungi have different types of cellulases, and the production of b1-4 exo and endo glucanases is different too, that is why you do not get the same results, although both fungi have a high production of these enzymes, you should also see if cellobiose accumulates in the medium because it is an inhibitor (Inhibition by product) of the enzymatic activity specifically of the b glucosidase.
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