Degradation and detoxification of azo dyes with recombinant ligninolytic enzymes from Aspergillus sp. with secretory overexpression in Pichia pastoris

Ligninolytic enzymes, including laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP), have attracted much attention in the degradation of contaminants. Genes of Lac (1827 bp), MnP (1134 bp) and LiP (1119 bp) were cloned from Aspergillus sp. TS-A, and the recombinant Lac (69 kDa), MnP (45 kDa) and LiP (35 kDa) were secretory expressed in Pichia pastoris GS115, with enzyme activities of 34, 135.12 and 103.13 U l−1, respectively. Dyes of different structures were treated via the recombinant ligninolytic enzymes under the optimal degradation conditions, and the result showed that the decolourization rate of Lac on Congo red (CR) in 5 s was 45.5%. Fourier-transform infrared spectroscopy, gas chromatography–mass spectrometry analysis and toxicity tests further proved that the ligninolytic enzymes could destroy the dyes, both those with one or more azo bonds, and the degradation products were non-toxic. Moreover, the combined ligninolytic enzymes could degrade CR more completely compared with the individual enzyme. Remarkably, besides azo dyes, ligninolytic enzymes could also degrade triphenylmethane and anthracene dyes. This suggests that ligninolytic enzymes from Aspergillus sp. TS-A have the potential for application in the treatment of contaminants.

-Incubation time at each temperature is not reported in the thermostability study (2.5.1). It can be assumed that it is the time of the activity assay so two effects are being measured simultaneously and it is not a stability study per se. -It would be important to report how much activity is added to the reaction system (2.6) in 0.5mL of enzyme solution in each case, or how many mg of protein and the specific activity of the sample.
-In 2.6 the term "concentration" is used to express Decolorization but it is not explained how it is calculated. Was absorbance correlated with a standard? -In 2.7, what reaction volume was used for the analysis of the degradation products? -Why was the pepper seed bioassay selected for the toxicity study? A reference is needed for this study (2.9). <u>Results</u> -It cannot be deduced molecular mass of a protein from an SDS-PAGE unless it can be said it is monomeric based on literature or another complementary study (See in 3.2 first paragraph). How were the bands corresponding to the enzymes of interest selected from among all the bands seen in the electrophoresis in fig.1d? One could refer to the literature, for example by citing reference 33 to say that the selected band corresponds to the laccase, but not make the deduction backwards, as was discussed. In addition, in that review several native enzymes are shown, which one is referenced in particular?  Table 4.
Supplementary_material -The legend in figure S1 must be corrected, see the order of the three enzymes results. -In figure S4 the legend should be corrected too, because the result shown is not from the recombinant enzymes.

Decision letter (RSOS-200688.R0)
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Dear Dr Xu:
Title: Degradation and Detoxification of azo dyes with recombinant ligninolytic enzymes from Aspergillus sp. with secretory overexpression in P.pastoris Manuscript ID: RSOS-200688 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: Comments to the Author: This research contributes to a deeper understanding of the degradation mechanism of three ligninolytic enzymes. While the conclusions are supported by the data, a serious problem with the English needs to be solved. In many cases it is difficult to understand what is meant and can lead to misunderstandings that affect the validity of the presented results. Finally, it is highly recommended to place the sequences of the ligninolitic enzymes and weel as the Aspergillus spp. in some repository.
Degradation of dyes by ligninolitic enzymes is not new, and the authors mentioned some papers related to this topic. However, the Ms showed interesting results. But I have some queries to be answered: -Are the sequences of the 3 enzymes publically available in some website? Since the authors isolated the genes of the 3 lignolitic enzymes from an Aspergillus spp.
-The aim of the work was to degrade dyes partially or totally, means potential use at large scale. So, how stable are the plasmids in P. pastoris? (How many generations? How many batches? Did the authors try to scale up the proccess? -In a complex mixture of chemical pollutans (e.g. more than one toxic molecule commonly available in tannery wastewaters, how the authors are able to combine the activities of 3 enzymes? -Why the toxicity assays was performed in pepper seeds? Since Congo red is considered carcinogenic,and the result showed (decolorization, %) were from 45 to 38%? Some common mamalian cell line should be tested (e.g. normal human fibrosblasts).

Reviewer: 2
Comments to the Author(s) A linguistic and grammatical revision of all work is strongly recommended. Abstract: -The term "specific activity" is misused because activity per mg protein was not reported. -The term "decolorization ratio" was used instead of "rate". Materials and Methods -The meaning of the acronym for microbiological culture media should be written.
-According to the description of the study of pH effect on enzyme stability (2.5.1), stability is measured for 30 min and then the substrate was added. If the pH is not changed to the reference value of activity assay, activity is being measured at different pHs in each case. In this way, two studies overlap (stability/optimum pH) and should be carried out separately. Some details of the procedure are probably missing to help to understand it.
-Incubation time at each temperature is not reported in the thermostability study (2.5.1). It can be assumed that it is the time of the activity assay so two effects are being measured simultaneously and it is not a stability study per se.
-It would be important to report how much activity is added to the reaction system (2.6) in 0.5mL of enzyme solution in each case, or how many mg of protein and the specific activity of the sample.
-In 2.6 the term "concentration" is used to express Decolorization but it is not explained how it is calculated. Was absorbance correlated with a standard? -In 2.7, what reaction volume was used for the analysis of the degradation products? -Why was the pepper seed bioassay selected for the toxicity study? A reference is needed for this study (2.9). Results -It cannot be deduced molecular mass of a protein from an SDS-PAGE unless it can be said it is monomeric based on literature or another complementary study (See in 3.2 first paragraph). How were the bands corresponding to the enzymes of interest selected from among all the bands seen in the electrophoresis in fig.1d? One could refer to the literature, for example by citing reference 33 to say that the selected band corresponds to the laccase, but not make the deduction backwards, as was discussed. In addition, in that review several native enzymes are shown, which one is referenced in particular? We hope that you as well as the reviewers will find the revised version suitable for publication. Thanks a lot again for your time and concern to our work. We are looking forward to hearing from you soon.

Yours sincerely Xiaolin Xu
The following is the point-by-point response to the Reviewers' Comments RSC Associate Editor:

Comments to the Author:
This research contributes to a deeper understanding of the degradation mechanism of three ligninolytic enzymes. While the conclusions are supported by the data, a serious problem with the English needs to be solved. In many cases it is difficult to understand what is meant and can lead to misunderstandings that affect the validity of the presented results. Finally, it is highly recommended to place the sequences of the ligninolitic enzymes and weel as the Aspergillus spp. in some repository.
A: Thank you for your support and encouragement. We have revised some of the unreadable parts of the manuscript with the help of a professional language editing service. We have provided certifies of English language editing (at the end of this Appendix A document). We uploaded the sequences of the ligninolitic enzymes from Aspergillus sp. as part of the supporting data in submission system when we submitted the manuscript.
We sincerely considering that the sequences of enzymes in repository, and we have A: Many thanks to reviewer for pointing out our work. Considering the stability, the multi-copy integration, high expression, plasmid pPIC9K and pichia pastoris were selected as the expression system. Results verified that plasmids and recombinant enzyme activity could be detected after 5 generations, and enzyme activity could still maintain 80%-90%. It showed great stability of plasmid in recombinant yeast GS115.
We will continue to modify these three ligninolytic enzymes gene to improve its activity and yield. Therefore, the following experiments can be consider using fermentation tank to produce recombinant enzymes in a large scale.

Q3:
In a complex mixture of chemical pollutants (e.g. more than one toxic molecule commonly available in tannery wastewaters, how the authors are able to combine the activities of 3 enzymes? A: Thank you for your support and encouragement to our work. The combined with three ligninolytic enzymes showed greater degradation ability than the single enzyme did. In facing of complex dye wastewater, these three enzymes could be co-immobilized with appropriate materials, such as sodium alginate, chitosan. We have also tried immobilized enzymes, and the experimental data were not shown in this article. The results showed that the immobilized enzyme could maintain the enzyme activity and recycle easily. It could be one effective method to degrade complex dye wastewater using combined enzymes. and Solanum lycopersicum seeds to detect toxicity of dye. It can be seen that the toxicity of dyes to plants weakened after degradation. Therefore, to make it more accurate, we adjusted the "Toxicity Assay" to "Phytotoxicity Assay" (In 2.8; page 5, line 2). Once COVID-19 is over, we will try to use some common mammalian cell line cells for toxicity assay.
The decolorization rate of Congo red mentioned by reviewers was 45%-38%, which was the decolorization rate within a short period of time, while the decolorization product after 24 hours was used for the phytotoxicity assay. Through our previous experiments, the Congo red decolorization rate could reach 90% at 24 h. According to the comments of reviewers, we have added the decolorization rate of CR at 24h in the supplementary material ( Figure S4 Special thanks to you for your comments.

Reviewer: 2 Comments to the Author(s)
A linguistic and grammatical revision of all work is strongly recommended.
A: Thank you for your support and encouragement. We have already revised some of the linguistic and grammatical in the manuscript with the help of a professional language editing service. We have provided certifies of English language editing (at the end of this document).

Q1:
The term "specific activity" is misused because activity per mg protein was not reported.
A: Thank you very much for your support and encouragement to our work. We have modified "specific activity" as "enzyme activities" in abstract. (Page 2, line 27) Q2: The term "decolorization ratio" was used instead of "rate".
A: Thank you for correcting us. We have modified this expression throughout the text according to the comment.

Q3:
The meaning of the acronym for microbiological culture media should be written. Q4: According to the description of the study of pH effect on enzyme stability (2.5.1), stability is measured for 30 min and then the substrate was added. If the pH is not changed to the reference value of activity assay, activity is being measured at different pHs in each case. In this way, two studies overlap (stability/optimum pH) and should be carried out separately. Some details of the procedure are probably missing to help to understand it.
A: Thank you very much for your support and encouragement to our work. The experiment 2.5.1 was designed to explore the optimal pH of the recombinant enzymes, and it is more appropriate to describe as the optimal pH. We have changed "Effect of pH and temperature on activity and stability" to "Effect of pH and temperature on activity". We have modified the materials and methods to explore the optimal pH for the recombinant enzymes, which is "To evaluate the optimal pH for recombinant enzymes using ABTS, MnSO4, and veratryl alcohol as substrates, the enzymatic reaction was performed in buffers of different pH values (1,3,5,7,9)". (Page 4, line 30-32) Q5: Incubation time at each temperature is not reported in the thermostability study (2.5.1). It can be assumed that it is the time of the activity assay so two effects are being measured simultaneously and it is not a stability study per se.
A: Thank you very much for your support and encouragement to our work. Our experiment 2.5.1 was designed to explore the optimal temperature for the recombinant enzymes. In 2.5.1, (Page 4, line 33-34) we have modified "thermostability" as "the optimum temperature". The statements were corrected as "The optimum temperature of the recombinant enzymes was determined by monitoring the change in activity at different temperatures (25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C)". In the optimum temperature experiment, the change factor was the temperature at which the activity assay was detected.
Q6: It would be important to report how much activity is added to the reaction system (2.6) in 0.5mL of enzyme solution in each case, or how many mg of protein and the specific activity of the sample. Q9: Why was the pepper seed bioassay selected for the toxicity study? A reference is needed for this study (2.8).
A: Pepper, an economic crops, is an important plant grown on a large scale in Xinjiang.
Given that Xinjiang has the largest textile industry in China, and the potential contamination on irrigation system and water bodies by the dye effluent, this research detected the impact of dye wastewater on pepper. The growth of pepper showed that the length of leaf, shoot, root and weight of pepper were obviously different between dye and dye degradation products treatment. Results showed that adding degradation products could reduce the growth stress of pepper compared with adding dye solution.
According to the literature, P. Aravind et al. phyto-toxicity were analyzed on seeds of Vigna radiate. N. Asses et al. the used of Zea mais and Solanum lycopersicum seeds to detect toxicity of dye. It can be seen that the toxicity of dyes to pepper weakened after degradation. Therefore, we have adjusted the "Toxicity Assay" was to "Phytotoxicity Assay" in 2.8 (Page 5, line 2). We have added reference 45 to results and discussion 3.6 (Phytotoxicity Assay; Page 12, line 52) Reference: