P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules
, Vlada B. Urlacher 2, Frank Hollmann 1,* and Gonzalo de Gonzalo 3,*Round 1
Reviewer 1 Report
My comments and remarks are given in the attached file.
Comments for author File: 
Comments.pdf
Author Response
We are most grateful to the referee's efforts and criticism that helped us to improve the quality of the manuscript.
Please see the file enclosed for a detailed answer.
Author Response File: Author Response.pdf
Reviewer 2 Report
Gonzalo and coworkers present in this contribution the use of dual functional small molecules to perform oxidation of styrene or thioanisole in a H2O2-dependant manner. Three mutants of cytochrome P450 monoxygenases were used as biocatalysts, and conversions and enantiomeric excesses were determined by chiral GC.
Firstly, the organic additives were synthesized according to known procedures. They are characterized by the presence of an imidazole ring linked, via a lipophilic spacer (C5 or C6), to an aminoacyl residue (Phe, Ile, Met). Even if the procedure were described, it would have been interesting to precise the enantiomeric excess for all DFSMs since saponification under basic solution may induce racemization. Moreover, could you precise the molecular role of the imidazole ring and the aminoacid?
Three mutants of P450BM3 were produced, all having the mutation F87A previously identified to increase the enzymatic activity.
For the oxidation of styrene, the authors have shown that the reaction was more efficient in the presence of Im-C6-Phe additive, at pH 8, and with low concentration in oxidative agent for the F87A mutant. Lower efficacy has been noticed with the double mutants.
Starting from the thioanisole, the best results were obtained once again with the simple mutant.
However, in both cases, ee values were moderate.
Interestingly, a double biocatalytic system was proposed to limit the concentration of H2O2. Very interesting results were thus obtained, even if ee were still moderate. Finally, oxidation of styrene has been observed in neat media using BuOOH as an oxidative agent in the presence of Im-C5-Ile (and not the Phe derivatives).
Experimental part as well as materials are well described.
In conclusion, this paper is really interesting is suitable for publication in Catalysts, providing that (i) the molecular role of the additive and (ii) ee of DFSMs, are given.
Author Response
We are most grateful to the referee's efforts and criticism that helped us to improve the quality of the manuscript.
Please see the file enclosed for a detailed answer.
Author Response File: Author Response.pdf
