p. 19 "Procerain, a stable cysteine protease […] was found to have a molecular mass of 28.8KDa and an isoelectric point of 9.32." The original paper (Dubey, VK and Jagannadham, MV (2003) Phytochemistry, 62, 1057-1071) should also be referenced here, in addition to the citation on the end of the following paragraph.
p.33 The protease assay should be described before the first time its referenced (i.e. before the “Effect of Substrate Concentration and Protease Activity of Isolate” section.
The data on Figs 1 to 8 would be much more readable in tabular form. The data in Figs 4-8 are from single measurements in each strain, or they averages from different cultures from each strain?
Figs 9-14 would be more easily interpreted if they were presented as "effect of temperature in each strain" (i.e. one graph per strain, showing protease production at different temperatures, in a total of three graphs) instead of the current presentation of 6 graphs with three strains eache, which force the reader to move back and forth in order to understand which temperature is best for each organism, and which set of conditions is most favourable.
Figs 15-18 should benefit from a similar presentation (i.e., by strain, instead of "by innoculum size")
Other minor points:
Figs 25-35: The xx axis are labeled “enzyme concentration” instead of urea /CaCl2, NaCl, etc… concentration. Are those concentrations final concentrations or (as implied by the description in the Methods section) the concentrations of the stocks added to the enzyme?
In the description of the protease assay “The bottles were then allowed to stand for one hour at 2oC to allow undigested protein to precipitate. The mixture was then centrifuged at 10,000rpm at 4oC for 5 minutes.” What do you mean by “mixture”? Is it the precipitate or the supernatant? I assume the supernatant is meant, but that is not clear. Why was the absorbance measured at 660 nm? That wavelength is often used to measure turbidity, but I would guess that degraded protein would not yield much turbidity. Indeed, Kunitz (1947) J. Gen. Phys. 30, 291-310 (DOI: 10.1085/jgp.30.4.291 ) measures degraded protein at 280 nm instead. In the protocol, Kunitz 1946 is cited but is not listed in the references.
Other Major points:
The data on figs 9-18 are extensively described verbally, which is redundant as proper graphs tell the story much more effectively. An attempt to discuss, e.g. why the inoculum size seems to be important for the pH optimum observed, is unfortunately lacking.
Figs 19-20f: Trying to obtain Km and Vmax values from kinetic data which obviously do not follow Michaelis-Menten kinetics is not correct. The Lineweaver-Burke plots themselves are too different from the straight lines expected from pure enzymes with a Michaelis-Menten mechanism. The extracts obviously contain several different proteases, as that is the most simple explanation for the presence of multiple activity maxima.
Figs 23-35 are quite hard to read, due to the abundance of data sets. I am not, however, quite convinced of their relevance, since inhibitors that only act at concentrations >0.1 mol/L are (at best) very weak inhibitors. I also cannot understand the rationale behind the investigation of the effect of pure amino acids on the protease activities. Again, there is no attempt at a discussion of the results, or to ascertain their relevance.
Overall, while the report has some interesting data, the presentation is unfortunately deficient, and conclusions/discussions are too sparse.
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