Enhancing the Thermostability of a New Tannase Through Rational Design and Site-Directed Mutagenesis: A Quality Improvement Strategy for Green Tea Infusion

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The  manuscript describes work to develop and characterized new tannase to improve tea quality without negatively impacting sensory attributes. The manuscript does a good job of describing the determination of which modifications to make and how the new tannase were produced. The manuscript also describes the characterization of both the native tannase as well as the new mutants. The thermal stability as well as activity of both the wild type as well as the mutants is explored and described. All the results and conclusions presented were supported by data collected and literature.  The manuscript was well written and I support the acceptance of this manuscript.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

The article (beverages-3658908) titled “Enhancing the Thermostability of a New Tannase Through Rational Design and Site-Directed Mutagenesis: A Quality Improvement Strategy for Green Tea Infusion” presents comprehensive research on the innovative thermophilic tanase, TanPL1, which is produced by expressing it heterologously in Yarrowia lipolytica. This represents a significant step towards the industrial application of enzymes in the food industry.

The results of planned design and changes to genes (mutagenesis) are interesting. These led to the creation of the MuTanPL1 mutant, which can handle heat better and works best at 60°C. Researchers have achieved impressive results by using this enzyme to process green tea infusion: they have significantly increased the content of free catechins and gallic acid, improved the infusion's sensory properties and increased its antioxidant activity.

These findings underscore the immense potential of MuTanPL1 in the beverage sector, particularly with respect to enhancing the quality and health-promoting properties of tea products. Furthermore, the paper emphasises the significance of protein engineering methodologies in enhancing the functionality of enzymes.

However, the article does not provide a detailed analysis. It does not analyse the molecular mechanism. This mechanism is responsible for improving the thermal stability of TanPL1 after mutagenesis. The authors have announced that this aspect will be discussed in a subsequent article, which has left some readers dissatisfied but also generated interest in further research.

The article is a valuable addition to the development of enzymatic biotechnology and its applications in the food industry. The results presented are well documented. The research approach is innovative and well-founded.

I have a question about this article. What is the reason for the absence of the results of the antioxidant activity of tea samples tested using three methods against DPPH? Were these results provided, or not? If they are done, would it be worth having them added?

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

I appreciate the authors' effort in addressing the topic of improvement new tannase with an entitled “Enhancing the Thermostability of a New Tannase Through Rational Design and Site-Directed Mutagenesis: A Quality Improvement Strategy for Green Tea Infusion”; however, I recommend clarifying many aspects in Section (Introduction, Material and Method, and Results &Discussion) to ensure the manuscript is transparent and clear.

The manuscript should fulfill the following comments:

1- L 17, The aim of the study given in abstract is lack and insufficient. It should be expanded to include the exact purpose and full context of the study's purpose.   

2- L 66, Is there another treatment to reduce tannin levels except from enzymatic treatments even on a small scale? If so, authors should give the other treatment procedures in the text.

3- L 90, Currently, Is a commercial tannese use in tea industry? Authors should mention about the commercial types of this enyzme used in food industry. 

4- L 153, why was the His60 Ni purification type chosen? Is it food-grade method in terms of contaning Ni residue? 

5- L 109, Yarrowia lipolytica is explained as food-grade from GRAS or FDA? It should be clarified in the text.

6- L 127, Why was Escherichia coli DH5α chosen as the vector? What ensures expression of tannase with Escherichia coli DH5α?  

7- L 150, Escherichia coli DH5α primarily produces proteins intracellularly."After 84 h ....... the best extracellular tannase 150 activity,....." It was written as an extracellular tannase, can the authors explain this difference?  

8- L 288, On what basis were these fungal organisms selected? Which organism was chosen as the reference? In addition, What is the similarity rate of these sequences?

9-  L 491, why was the title of "Treatment of Green Tea Infusion with Tannase MuTanPL1" explained in Results & Discussion chapter?  If it is given in this chapter, the authors should support it by comparing different studies for the applicaiton of green tea treatment?

10- L 597, For the sensory analysis, some taste attributes have been examined however, is there any change in the color of tea because of this treatment? It has not been clarified in the text. 

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript is of a good quality overall, and focuses on an interesting topic, however certain aspects of the manuscript are not very clear and need minor improvement:

1) Section 2.2 doesn't read well, the authors have just stated each software and what was it used for. Instead, this section should read like a method with step-wise descriptions of what has been done in chronological order. Imagine someone want to replicate this, what would be the first step (and onwards)?

2)  Table 3 is very confusing and incorrectly labelled. Please check the column titles for the EGCG. There are a lot of results in this table and hard for the reader to understand the trends. Convert this table into multiple bar charts.

Author Response

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Reviewer 5 Report

Comments and Suggestions for Authors

This manuscript describes the identification and engineering of a novel thermostable tannase enzyme with potential applications in commercial tea processing. The authors describe the identification of a novel tannase enzyme from reported genetic data and its comparison to known tannases, the expression of the novel tannase using a food-grade organism and subsequent purification/characterization, determination of optimal conditions for tannase activity/stability, characterization of tannase substrate specificity/products, successful rational engineering of the novel tannase to increase thermostability,  and practical application of the engineered enzymes in tea processing. The introduction section provides valuable context as to the importance of tannase enzymes, but does not contextualize the described study in the context of previous work in the field (see comment below). The materials and methods section is sufficiently detailed such that this work could be repeated in another laboratory. The experiments are well designed and described, and the results may be of importance in industrial tea processing. This study would be interest to the enzyme engineering and beverage processing communities among others, and the minor issues described below should be addressed.

The introduction section does not contextualize the present study with respect to previous work that has been done in tannase engineering. The authors describe a wide variety of industrial applications for tannases, do any of those commonly utilize engineered tannases? Have any previous tannase engineering studies been reported? Have any targeted improvements in thermostability? If so, this should be addressed in the introduction.

The introduction section would benefit from a scheme showing the general chemical structures of tannase substrates and products.

The manuscript does not clearly explain why the four specific mutants chosen for further investigation were selected. Why 4 and not 6 or 3 or another number? This section requires more detail/explanation.

The supplementary information format (header, etc.) is taken from the journal “marine drugs”, not from “beverages”

Author Response

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