Round 1

Reviewer 1 Report

In this manuscript, Shang et al developed two types of surface displayed levansucrases for levan biosynthesis. After conforming that the designed fusion protein was successfully displayed on the surface of yeast, levansucrase activity and optimal experimental conditions was measured. The group found that the properties of the displayed enzyme did not alter much compared to the free enzyme. Both systems were tested for levan biosynthesis under optimal conditions and the authors reported high yield of levan, higher than most of the previously reported catalysts.

The paper is well structured and clearly written and the necessary control experiments were presented. Therefore, the paper can be published in the presented form. 

Author Response

Responses to the reviewers’ comments:

Summary: In this manuscript, Shang et al developed two types of surface displayed levansucrases for levan biosynthesis. After conforming that the designed fusion protein was successfully displayed on the surface of yeast, levansucrase activity and optimal experimental conditions was measured. The group found that the properties of the displayed enzyme did not alter much compared to the free enzyme. Both systems were tested for levan biosynthesis under optimal conditions and the authors reported high yield of levan, higher than most of the previously reported catalysts.

Response: Thanks for the summary, it correctly summarized the major points of our work.  

Comments: The paper is well structured and clearly written and the necessary control experiments were presented. Therefore, the paper can be published in the presented form.

Response: We really appreciate the time and effort you dedicated to providing feedback on our manuscript. Thank you very much for your positive comments.

Reviewer 2 Report

Summary: The product of levansucrase activity is a polysaccharide called levan. Levan is used in cosmetics, foods, and chemicals. Low molecular weight levan is preferred for certain applications. The goal of this work was to express levansucrase enzyme from the bacteria Zymomonas mobilis in a system that makes it cost effective to reuse the enzyme multiple times. Levansucrase was anchored to the surface of yeast as a fusion protein. Two types of fusion protein were made in opposite orientations, so that either the C-terminus or the N-terminus of levansucrase was free. Both types produced high levels of levan, though the C-terminal free orientation produced a higher yield of low molecular weight levan. Both types could be reused at least 6 times. Thermal stability of the anchored levansucrase was higher than the thermal stability of free levansucrase.

1. Please cite the following work. A Hill, L Chen, A Mariage, JL Petit, V de Berardinis, S Karboune. Discovery of new levansucrase enzymes with interesting properties and improved catalytic activity to produce levan and fructooligosaccharides   Catalysis Science & Technology 9(11), 2019   DOI:10.1039/C9CY00135B

2. Figure 1 panel d. Please correct typing error. C-mcy Tag should be C-myc-Tag

Author Response

Responses to the reviewers’ comments:

Summary: The product of levansucrase activity is a polysaccharide called levan. Levan is used in cosmetics, foods, and chemicals. Low molecular weight levan is preferred for certain applications. The goal of this work was to express levansucrase enzyme from the bacteria Zymomonas mobilis in a system that makes it cost effective to reuse the enzyme multiple times. Levansucrase was anchored to the surface of yeast as a fusion protein. Two types of fusion protein were made in opposite orientations, so that either the C-terminus or the N-terminus of levansucrase was free. Both types produced high levels of levan, though the C-terminal free orientation produced a higher yield of low molecular weight levan. Both types could be reused at least 6 times. Thermal stability of the anchored levansucrase was higher than the thermal stability of free levansucrase.

Response: Thanks for the summary, it correctly summarized the major points of our work. 

Comment #1: Please cite the following work. A Hill, L Chen, A Mariage, JL Petit, V de Berardinis, S Karboune. Discovery of new levansucrase enzymes with interesting properties and improved catalytic activity to produce levan and fructooligosaccharides Catalysis Science & Technology 9(11), 2019 DOI:10.1039/C9CY00135B

Response: We sincerely appreciate the valuable comments. We read the paper carefully, the reference and appropriate texts were added in the Results and Discussion section (2.4, Line 199-203), as following.

“The difference in the yield of two systems may be due to different anchoring motifs, besides the location of His-tags. A. Hill et al. found that the levansucrase from Vibrio natriegens with the His-tags located at the N-terminal (VnLS-N) appeared a better transfructosylating efficiency than the C-terminal form (VnLS-C), therefore the VnLS-N can produce levan more efficiently than VnLS-C [30].”Consequently, the numbers of the latter references were automatically added by 1. 

Comment #2: Figure 1 panel d. Please correct typing error. C-mcy Tag should be C-myc-Tag.

Response: Thank you very much for pointing this typing error. We have corrected it and updated Figure 1.