Optimized Recombinant Expression and Characterization of Collagenase in Bacillus subtilis WB600

Round 1

Reviewer 1 Report

This work is focused on the recombinant expression of collagenase in B. subtilis and the subsequent optimization of expression and production conditions. The authors verified the influence of the most critical parameters on growth and moreover characterized the product. This paper is suitable for publication in Fermentation with minor revisions.

Below are some questions and the detail revision points for the authors' consideration.

1)      The authors in the introduction could better argue the importance of producing collagenase for commercial purposes.

2)      How many replicates were performed for assay? Specify it in the materials and methods

3)      The images have a poor resolution the authors should insert images with better quality.

4)      In figure 5 indicate the standard deviation

5)      Why did the authors monitor the stability over time at different pH and temperature for only 120 min?

6)      In paragraph 3.6.3 the authors analyzed the temperature parameter concluding that at 35°C they get the maximum activity (2444.3 u/mL). How do the authors explain that in paragraph 3.6.2 they obtained a better activity at 37 °C of 2746.7 U/mL (keeping 260 rpm and pH 7 constant)?

7)      In paragraph 3.6.4 insert the pH, temperature and stirring used.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript by  Zhu et al.  reports cloning and over-expression of the enzyme collagenase in B. subtilis. The recombinant protein was purified and tested for its activity both in vitro and in vivo. In particular, this study analyzed a lot of factors that may affect the expression of the recombinant protein from the pP43NMK-col vector in Bacillus subtilis. Thus, the Authors state that the best conditions to obtain large amounts of this enzyme have been found. I am not totally convinced of that. In fact, few in vivo experiment are not, in my opinion, correctly interpreted and conclusions could be different from those drawn by the Authors.

Furthermore, several points should be addressed.                                   

-        In the text is not mentioned the organism from which the over-expressed col gene derives. In Materials and Methods is reported only the GenBank cod. (CP011686.1, locus tag: AB13_2557). According to this code, the collagenase gene belongs to Bacillus velezensis. Reasonably, B. velezensis is not evolutionally far away from B. subtilis but the choice of this sequence should be explained.

-        More details should be provided about the techniques (site-directed mutagenesis, artificial genes etc.) used to obtain the optimized DNA sequence by Sangon Biotech.

-        Indications about the X-axis should be added in Figure 2ab.

-        A low quality SDS-PAGE is shown in Figure 3b. The problem is that lane 1 is overloaded as compared with lane 2. Thus, all bands are much stronger in lane 1 than in lane 2 making very hard to compare the two samples. A better gel should be provided and the band of 35 kDa indicated.

-        The pattern of collagenase activity as a function of pH is quite strange (Fig. 5a). The enzymatic activity drops down from 20 to 40 min. Then the enzyme suddenly resumes increasing its function at 60 min but this activation is poorly dependent of pH (excluding pH = 11). Though less pronounced, a similar trend at 40 min is observed also in Figure 5b. This point deserves some explanations.

-        The major criticism of this study concerns the optimization of collagenase activity related to growth of B. subtilis WB600/pP43NMK-col. The measures of the enzymatic activity were plotted in Figures 7 and 8 without taking into account cell density that significantly changes in the different growth conditions tested. The results could be in many cases diverse if the normalization for cell density would be applied. For example, making a rough normalization, it  might result that: (i) the best fructose concentration is 5-10 g/L (Fig. 7b); (ii) the best nitrogen source is gelatin (Fig. 7c); (iii) the best temperature is 30°C (Fig. 8c). The optimized conditions should be reviewed and beside the absolute values, the collagenase activity should be expressed as a function of Abs_{(600 nm)}.

- Why is the enzymatic activity so low (one third as compared with other assays) in the nitrogen sources experiment (Fig. 7c)?

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx