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1) The activities reported in table 1 are by mg. It is not clear if it is mg of CLEA or mg of protease. In the last case, there is no explicit explanation in the experimental part about how the weight of protease in the p-CLEAs or in the CLEas was obtained. No report of procedure to know about remaining protease in the supernatant of the preparation or in the washing steps. The initial amount was 5 mg of papain in 10 mls.
I recommend the authors to be very careful with the methods of detection of enzyme amount as protein quantification. Further material
Bradley J.S.C. Olson and John Markwell
Current Protocols in Protein Science (2007) Supplement 48 ; 3.4.1 to 3.4.29 by John Wiley & Sons, Inc.
2) It is known today that several protein detection methods have several problems in its application to protease/other enzymes concentration-especially if stirring is involved and buffer is present.It is included the parameter Ew as enzyme amount in page 4,but it is not clear how this amount was obtained- if it is protease. IF the reported weight is of CLEA and no mass balance was done to know about how much protease is present in the CLEA, this should be clearly stablished. One way to do it is to measure activity in supernatants and washings,assuming similar activity of the free enzyme and of the remaining not-precipitated and not cross-linked enzyme.
3) Taking into account the above, the authors presented in Table 1 several results associated to their CLEAs. Without the knowledge on the total amount of protease, they do not know per mg of it the actual activity. However, the comparison is pertinent between CLEAs.Without knowledge on the protease amount present in both CLEAs what is not pertinent is the comparison with the free enzyme.
4) With these ideas in mind, how do they know if the lower activity in the case of the CLEA in table 1 is due to the presence of the starch and a "dilution" effect of the starch. Because it is not clear if the activity reports are presented by mg of enzyme or mg of CLEA, this is not known. BTW, if it is by mg of enzyme, how the protease amount in CLEAs was determined?
5) IF the porous structure is different, the authors could try to perform a BET Area determination for the CLEA and the p-CLEA to give further support to their assumptions. They also could perform the complete N2 isotherm to know about the pore distribution.