Synthesis of Linoleic Acid 13-Hydroperoxides from Safflower Oil Utilizing Lipoxygenase in a Coupled Enzyme System with In-Situ Oxygen Generation
Round 1
Reviewer 1 Report
Line 97 From “flowrate” to “flow rate”
Line 109 From “ultraturrax” to “homogenizer (IKA® Ultraturrax®)”.
Line 178. Is there any influence of co-existing glycerol, which increases accompanied with the progress of the lipase-catalyzed hydrolysis. Does glycerol work as inhibitor? Enhancer? or no effect?
Line 254. When the reviewer sees Figure 5, there is severe forming which would lower the efficiency of the mixing of reaction broth. Is the retention of oxygen in the bubble is necessary to enhance the efficiency of LOX? Have the authors tried the reaction with the addition of antifoam agent such as emulsified silicone oil?
Line 345. Please clearly describe the scaled-up reaction conditions with how many millimole of the triglyceride, workup and extraction procedure, and the isolated yield of the 13-hydroproxy acid derived from triglyceride. The readers would anticipate to realize the advantage and practicability of the newly proposed combined method.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript addresses the topic of green bioprocesses, developing an enzymatic cascade suitable for one-pot transformation of safflower oil, rich in linoleic acid, to 13-hydroxyperoxyoctadecadienoic acid, with good yield and regioselectivity. The presented results are original, and, at my best knowledge, such an efficient and scalable enzymatic cascade was not yet reported, although the considered reactions (hydrolysis of triglycerides by lipases, utilization of soybean lipoxygenases to obtains 13-hydroperoxides, and utilization of catalase for in situ generation of oxygen) are well documented. The manuscript is well written, and I consider it appropriate for publication, with only a few recommendations that I suggest, as it will follow.
- The Introduction part is appropriate, but there still are some recent articles discussing a possible lipase-lipoxygenase cascade which I consider that should be included and discussed. Particularly, a very recent article (maybe published after the finalization of this manuscript) reporting the biocatalytic transformation of soybean oil into fatty acid hydroperoxides using lipase and LOX should be cited (https://doi.org/10.1016/j.compositesb.2021.109091). The article of Lowe et al. (https://doi.org/10.1002/advs.201902973), although considering the plant oil derived linolenic acid peroxidation by LOX as the first biocatalytic step of a biocatalytic cascade (without special highlight on the oil hydrolysis), could be also of interest.
- The structural formula of safflower oil, as presented in Scheme 1 is not appropriate (with the unspecified R radical). I recommend replacing it by a pictogram of the oil.
- Figure 2a is not relevant, since the HLB dependence of HPODE yield is not significant (as the authors specified in the manuscript). Anyway, relative values of about 200% are not common (probably they were calculated against the reaction without surfactant but is not specified in the legend). I recommend moving it to the Supplementary material. Instead, Table S1 could be moved to the main text, as contains all the emulsifiers tested and justifies the selection of the "green" surfactant and its concentration. Maybe the values at 9% and 12% surfactant concentration could be omitted, because, in my opinion, the increase of the surfactant concentration above 6% is not justified.
- I also recommend rethinking the scaling of Figure 2b. In the present situation it is difficult to follow the surfactant effect at linoleic acid concentrations between 0-10 mM, when the influence of the surfactant is important in relation to the enzyme/substrate ratio.
- The lipase screening, as presented in Figure 3a, is a little bit deceptive, because the authors used the same amount from every commercial enzyme preparation (5%, w/v), without taking into account that the effective enzyme content is highly different, also containing other compounds added during the formulation of the preparation, to increase its stability. It would have been more accurate to assay the protein content of each lipase and use the same protein amount in every experiment (supposing that this protein is mainly lipase). Another possibility is to express the catalytic efficiency of each lipase as micromoles linoleic acid produced by 1 mg enzymatic protein in 1 min, instead of conversion.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
In this manuscript, lipases were screen for hydrolysis of safflower oil. The lipase from pseudomonas fluorescens was combined with lipoxygenase to obtain >80% hydroperoxides. The catalase was used for oxygen production with H2O2 dosage. It was found that foam was significantly reduced. The work is interesting and well organized. However, there are two issues that need to be presented more clearly.
1) It is known that H2O2 has some damage or inactivation on the enzymes. The detailed effect of H2O2 on the 3-enzymes should be given to elucidate that the possibility of the cascade enzymatic reactions.
2) The ratio of the 3 enzymes should be investigated to make the process economical.
3) If possible, the docking model may give some clear explanation on the lipase screening.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
All the issues have been solved in the revised manuscript, and it could be published at the present form.
