Facile One-Pot Immobilization of a Novel Thermostable Carboxylesterase from Geobacillus uzenensis for Continuous Pesticide Degradation in a Packed-Bed Column Reactor

Round 1

Reviewer 1 Report

Presentation of the work: although the manuscript was generally well organized , some sections require further revision .

On pag1-line 36 (introduction section) add more references if available.

Figure 2 (section 2.4)  is not clear.

Figure S3(section 2.5) .  the SEM figure of enzyme before and after immobilization are made at different magnification. Why? Could this  be    the cause of great difference between the two images? 

Table 2 (section 2.5) It is not clear. the authors report that " Na, Co and Mn did not significantly affect enzyme activity" and that "Zn  significantly reduced enzyme activity" How can to be that Co has a residual activity of 92.7, Na 94.5 and Zn of  96.1? Please, explain this. In addition please define the acronyme CTAB 

figure 5: it is confused since it is not reported in the text the meaning of the inserts. In particular must be increased visibility of the chromatogram 

line 224 (section 2.7) the authors report table S1 but probably the correct table is table S2. The authors could also eliminate this table putting the data in the text.

General consideration:

-The authors must define the acronimes when they first appear, even if they are well known: for example AOX (line 50 pg2) ; PBR (line 68 pg 2); SDS (line 158 pg 6), CTAB (line 159 pg 6), SEM (line 168, pg 6)

The pH and the immobilized activity of 1x-Est741M in the enzymatic reaction are never reported .

It is necessary more information about the analysis of malathion The authors report that standard malathion was used as the control group.It is not clear if  the authors have determined the recovery of the standards after  n-hexane extraction, centrifugation and evaporation.   Has a calibration curve been made for the quantitative analysis?

The authors report that 1x-Est741 is able to degradate 3 pyrethroids but in the text is only displayed the percentage removal of bifenthrin.

Author Response

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Reviewer 2 Report

The authors described the expression of a new carboxylesterase gene (est741) from Geobacillus uzenensis as new possibility for pesticide degradation. This carboxylesterase showed an increased temperature stability by site-directed mutagenesis and is expressed furthermore in P. pastoris. The enzyme was immobilized on an epoxy resin, which could be reused in 10 batches with a residual activity of 72%. Furthermore it was implemented in a packed bed reactor and could be used successfully in the degradation of pesticides in synthetic wastewater.

All in all the topic is quite interesting, but there are still some points, which have to be revised:

1. There are several typos and sentences, which are not easy to understand:
   1. Line 63-64: ‘Another important advantage of the resin catalysts is suitable to continuously degradation pesticide.’
   2. Line 43-44: ‘Of these, malathion contains two carboxyl esters bonds.’
   3. Line 144: ‘By structure analysis, the mutation site M160K on the surface of the enzyme molecule.’
   4. Line 229-232: ‘The results showed that when 2.5 g of immobilized enzyme was charged into a 10 mL column reactor, the concentration of bifenthrin was 500 mg/L, reaction temperature was 60 °C, pump speed was 0.7 mL/min (optimization data is not displayed), and bifenthrin removal was 90.4%.
   5. Line 181-184, 189-190: Pleases rewrite the discussion here. This sounds as you worked with a lipase or amidase.
   6. Line 149: ‘at high temperatures’
   7. Line 388/375: This sounds as you want to re-dissolve the organic solvent.
   8. The introduction of abbreviations is missing.
   9. ‘Packed-bed reactor’ vs. ‘packed bed reactor’
   10. Line 216 ‘malathion’, Line 174-175
2. The immobilization part needs to be revised:
   1. Line 52-60: Immobilization is not protein engineering. That are two different ways to modify biocatalysts.
   2. Line 54: Thermostability is for most enzymes important
   3. Line 63: Not only immobilized enzymes can degrade pesticides.
   4. Line 67-68: ‘Packed-bed reactor is a kind of practical and efficient reactor, showing high degradation efficiency and long reaction time.’

                These are no properties of a PBR. You can have long reaction                        times in a batch reactor as well and the degradation efficiency                      depends on the catalyst. This sentence you should rewrite.

3. Line 108-109: ‘After purification, Est₇₄₁ and 108 Est_741M showed moderate specific activity of 460 U/mg…’

           …moderate in comparison to what enzyme? Here a comparison is                missing.

4. Table 2: Did you combine the best results of organic solvent, ions and surfactant for one experiment, e.g. in the PBR?
5. Line 168-171: Do you have any information about the mechanical influence? That the surface is not smooth anymore, could be caused by the immobilization procedure, as well. Do you have a control?
6. Line 187: At this point you should compare T_1/2 also with Est_741M to clarify the improvement by immobilization.
7. You stated that you have rather an esterase than a lipase. (Line 133) But you are sometimes writing about a lipase (Line 359, 182). Please clarify.
8. What is the storage time of the immobilized enzyme between the batches and overall during the recycling measurements?
9. You mixed up the tables in the supporting information. Table S2 in the SI is S1 in the manuscript.
10. Line 236: Why was the operation stability not ideal? In line 239 you write, that you have a good operation stability. Please clarify.
11. 5: Why do the solutions have different turbidities? The quality of the HPLC Chromatograms is not good.
12. Line 316: Did you used the optimal reaction system? Or only optimal temperature? Please clarify.
13. Line 399-340: ‘and a packed bed column reactor was designed to maximize the biocatalytic activity for potential industrial applications’         An increasing of the activity is not possible by the usage of a PBR!
14. Line 323-325: T_1/2 methodology should be shifted to the part before additive usage.
15. Line 366: What means ‘After the reaction was completed…’? Did you pumped the reaction solution in a cycle until you finished the reaction? Otherwise you can calculate a residence time for your system and make a suggestion for further investigations.

          What is the maximum degradation of the substrate? Is there a                    limitation or did you reach the maximum of the yield?

Author Response

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Reviewer 3 Report

The manuscript entitled “Facile One-pot immobilization of a novel thermostable carboxyesterase from Geobacillus uzenensis for continuous pesticide degradation in a packed bed column reactor” shows the cloning, mutation, production, and immobilization of an esterase enzyme that shows activity against some pesticides such as malathion, bifenthrin, fenpropathrin, and fenvalerate. In my opinion, this paper cannot be published in the way it is showed and written. Consider total modification of the presented work and resubmission. I found this paper difficult to read and follow. It is presented using a sketchy style, without links between sentences, ideas, and arguments. I strongly suggest the authors to rewrite the paper in a more smoothy and readable style.

Moreover, the experimental part is not clearly explained. For example, the part regarding the selection of the residues to modify is completely missing. Authors should explain why you chose to exchange Met160 by a Lys. The removal of a Met can have a strong effect on the tertiary structure of the protein. For the reader is also misleading if you name the mutant enzyme Est741M. Using the general convention, the name should be EstM160K.

Important experimental details are missing such as immobilization yields, specific immobilization conditions (buffer, pH, concentrations)

Also, Figure captions should be revised, I think. For example, in Figure 5 “A schematic of the packed bed bioreactor for the biodegradation of bifenthrin”, besides being bad written, makes no reference to the different panels that are shown in the Figure.

Author Response

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Round 2

Reviewer 1 Report

The paper is now in a satisfactory form.  The chromatogram  shown in figure 5 could be improved. 

Author Response

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Reviewer 3 Report

Authors have substantially improved the manuscript following the reviewer´s suggestions. I think it can be accepted now.

Author Response

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