The Immobilization of β-Galactosidase on Glass Fiber Rolls

Round 1

Reviewer 1 Report

The authors presented an interesting study of galactosidase immobilization on glass fibre rolls.

There are several questions.

 1)       In the title of the article, the authors state "a new method". What do the authors mean? Indeed, in this work, the authors use a well–known method of immobilization - covalent binding.

2)       In the experimental part, the authors did not provide information on how the amount of the immobilized enzyme was determined. How complete was the immobilization? When comparing data on the effects of temperature and pH, the authors should be sure that they are comparing samples with the same amount of immobilized enzyme.

3)       When the authors discuss the conversion of lactose using an immobilized enzyme, they do not show data on the conversion of lactose using a native enzyme in the same amount and under the same conditions.

4)       In Figure 5, the authors presented data on the effect of the concentration of the GA solution used in the immobilization of the enzyme on the conversion of lactose. At the same time, the optimal GA concentration was not determined. The decrease in the concentration of GA had a positive effect on the conversion of lactose. But it is possible that with a further decrease in the concentration of GA (0.1%, 0.01%), the conversion of lactose would be even higher. After all, it is known that with an excessive content of GA, aldehyde molecules bind to each other, and not to the amino group of the enzyme. Moreover, it is necessary to take into account that, according to the results of the BET, the support -  glass fibre rolls have a very small and non–porous surface. Therefore, the value of the optimal concentration of GA is very important.

5)       I am thinking that there is no need to present Figure 4, it does not carry information.

6)       For Figures 7-9, it is necessary to indicate the process conditions in the caption, as it is done for Figure 6 (for example).

7)       Line 324. How did the authors determine the absence of enzyme leakage?

8)       For all the data presented in Figures 5-11, it is necessary to indicate the experimental error. How many times has each experience been repeated?

Comments for author File: Comments.pdf

Author Response

The authors response is in the attached file.

Best regards,

Darja Pečar

Reviewer 2 Report

In this manuscript the authors show that they can successfully immobilize b-galactosidase on glass fibre rolls and optimized the process by varying the concentrations of the reagents (APTES and glutaraldehyde) used. Next, they tested the activity of the immobilized enzyme in a continuous laboratory reactor with the conversion of lactose into glucose and galactose. They optimized this process by varying the reaction temperature, pH and flow rate of the substrate solution. Finally, they showed that the set-up could be used at least 3 times without loss of activity.

The immobilization of proteins by glutaraldehyde is not very specific. This could have a negative effect on the activity of the immobilized enzyme. Therefore, the authors should have compared the activity of the immobilized b-galactosidase with the activity of the enzyme in solution. Furthermore, they should explain why at lower concentrations of the immobilization reagents better results are obtained than at higher concentrations of these reagents.

The results show that the chosen reactor set up was not optimal. As the authors mention at higher flow rates some of the substrate flowed through the small holes in the middle of the roll. Hence, a good comparison of the results obtained at the different flow rates is not possible. For this a better experimental set up is needed. Another indication is that the maximal conversion reached is only 56%. Therefore, the authors should indicate how the experimental set up can be improved to make it more attractive for potential industrial applications.

In the final experiment in par. 3.2 the re-usability of the immobilized b-galactosidase glass fibre rolls is tested. Despite optimal conditions the obtained conversion yields are only 20% instead of the expected 50-60%. Please explain these findings.

The manuscript contains a large number of figures. Many of the these (especially figures 5 to 11) can be combined into 1 or 2 larger figures.

Minor comments

1.     The authors do not mention the thickness of the glass fibre sheets used.

2.     In Materials and Methods it is mentioned that a purified industrial b-galactosidase is used for the experiments. More details should be given about the nature of this enzyme.

3.     In some cases in par. 2.3 and 2.5 ml should most likely be ml.

4.     In par. 3.2 ‘At lower flow rates’ should probably read ‘At higher flow rates’.

5.     In par. 3.2 ‘less than 10% at pH = 8’ should probably read ‘10% less at pH = 8’.

6.     Yellow is not a very visible color in the figures. Please consider changing this.

Author Response

The authors response is in the attached file.

Best regards,

Darja Pečar

Author Response File: Author Response.pdf

Reviewer 3 Report

Despite the fact that a large number of works are devoted to methods of immobilization and selection of carriers for immobilization of β-galactosidase, the manuscript “A new method for immobilization of β-galactosidase on glass fiber rolls” contains new scientific results.

Authors have developed a method to immobilize the β-galactosidase on special glass fiber rolls. Glutaraldehyde was used as a non-specific cross-linking agent for the covalent binding of β-galactosidase on modified glass fibers. High immobilization efficiency, enzyme activity and stability were obtained. The optimal reaction temperature, substrate flow rate and pH were found. The activity and stability of the enzyme entrapped on the glass fiber rolls remained almost unchanged during reuse, which is a good prospect for potential industrial applications.

Major notes:

The authors should compare the parameters they obtained (immobilization efficiency in %, optimal pH, temperature, hydrolysis time, reusability) for β-galactosidase immobilized on glass fiber rolls with the results obtained by other authors when immobilizing β-galactosidase on other types of supports. Additionally, Km, Vmax and Kcat should be determined.

In addition, because glutaraldehyde was used as a non-specific cross-linking agent and the immobilized enzyme is planned to be used for the food industry, it is necessary to prove the absence of its toxicity at least according to literature data.

Technical notes:

Line 90: Aspergillus oryzae should be italicized

Line 127: in SiO2 and Al2O3 the numbers should be subscripted

Line 140: section title 2.3. Immobilization needs to be moved to the same page as the section itself

Line 143: “20 cm x 60 cm” should be replaced with “20×60 cm”

Lines 159, 234, 308, 309, 311: there is a strange symbol before “-galactosidase”, it should be replaced with the greek letter β

It is advisable to combine Figures 5 and 6 into one figure, enter the designations A and B

It is advisable to combine Figures 7, 8 and 9 into one figure, enter the designations A, B, C

Minor editing of English language required

Author Response

The authors response is in the attached file.

Best regards,

Darja Pečar

Author Response File: Author Response.pdf

Reviewer 4 Report

The study on which this article is based has the twofold aim of investigating the use of glass fibres as a support for immobilising the enzyme β -galactosidase, and of optimising the process and evaluating its efficiency. The experimental design is quite clear, but a little dispersed on some points. However, there is no discussion of the applicability of this new technology. Therefore, the following observations and suggestions are made:

-        Lines 40-41: The sentence “β-galactosidases are enzymes that convert lactose into glucose and galactose” is a repetition that could be deleted.

-        Lines 42-53: It could be improved if the discussion of lactose intolerance is moved at the begin of the introduction (between lines 29 and 30).

-        Lines 105-107: Are glass fibres waste? From what has been understood, this research not only provides a valid alternative to lactose hydrolysis, but also reuses waste materials and therefore, given the cost effectiveness, underlines the applicability in the industrial field. Adding a brief description of the difficulties in reusing glass fibres could support the thesis.

-        Section 2.2.: This is partly a repetition of what is explained in lines 108-110. Therefore, this part needs to be improved by providing some additional information on the techniques used for support characterisation or removed.

-        Section 3.1.: The glass fibres were not characterised after preparation? This must be specified in the experimental procedure and the choice must be justified.

-        Section 3.2.: Given the large number of considered parameters, I suggest adding a table summarising the main points of the experimental design at the beginning of the results and discussion in section 3.2 to facilitate the reading of the results.

-        Conclusion: The discussion of the results lacks comparisons with other immobilization methods. Then broaden the conclusions, also underlining the innovativeness of the technology under consideration.

-        Bibliography:

Reference 20 - Delete the repetition of "doi:".

References 8, 11,12,13,14, 21, 27, 32- add DOI.

The year of all references must be in bold.

Author Response

The authors response is in the attached file.

Best regards,

Darja Pečar

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have revised the article in accordance with the comments that were made.

Reviewer 3 Report

Authors took into account most of my comments. I think the article can be published

Reviewer 4 Report

I think the authors have made the necessary corrections. The paper is well structured and the topic interesting. I therefore recommend acceptance for publication.