Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease

Round 1

Reviewer 1 Report

Paper with some interest, but that needs a deep rewritten and improved discussion, as they have some conceptual mistakes.  Many of the problems start by the poor introduction.

In general, English must be revised; some sentences are hard to understand.

Abstract

First sentences of abstract are more adequate for introduction.

“Elastase strain K; an organic solvent tolerant protease wAS used”

“with a cross-linking time of 2 hours” If they did not reduce the CLEAs, I guess they really emant a glutaraldehyde treatment time of 2 h, as crosslinking may continue after eliminating the free glutaraldehyde.

Indicate the substrate

Give the data on stabilization and activity at different pH values compared to the free enzyme, the same with organic solvents, etc

“with particle 26  distribution of 1497nm”.?? Tis may be the average diameter, no the distribution.

Introduction

In am not sure that enzymes may be considered unexpensive  in a general way, although it is true that prices at industrial level are going down.

Organic solvents shift equilibrium in the direction od synthesis, not hydrolysis (see old paper from Kasche regarding thermodynamically controlled synthesis). Other point is that may be necessary to use them to increase the substrate solubility.

There are many enzymes that are quite stable in some solvents, although I agree that areas contaminated by organic solvents are prone to contain microorganism that  has extracellular enzymes stable in these media. This needs to be better explained.

Many enzymes are stable in only 25% organic solvents (e.g., some of the most used lipases)

“Physical approach of enzyme 59

“ immobilization involve MANY weak interactionS between enzyme molecules”

The group in the support is better called active group than crosslinking reagent, that suggest a bifunctional reagent.  Crosslink agent involve a multifunctional reagent, in some cases may be the case when a previously adsorbed enzyme is treated with a bifunctional agent, but in miost cases the support is actiovated  and have just one reactive group.

A proper immobilization may permit to greatly improve the enzyme stability, via multipoint or multisubunit immobilization, or generation of a favorable microenvironment.  Moreover, if properly designed, it may alter enzyme purity, resistance to inhibitors or chemicals, and also may alter enzyme selectivity, specificity. There are many reviews on this matters (e.g., Sheldon, Lafuente, Bilal), use some of these and improve the role of immobilization in the enzyme  biocatalysts design.

“cross- linking agent to form covalent bonding between enzyme molecules IN AGGREATED FORM,” Other strategies may be to produce copolymers, for example, but that is different to CLEAs.

CLEAs save the support, but as it can retain 90% water, making the enzyme loading similar to that of a good propous support (around 100-150 mg/ml). Please avoid excessive appraisal of the technique.

Some points are no treated in the introduction.

First, the CLEAs may be hard to manage at industrial level due to the fragility.  Basket or vortex reactors have been proposed. Other author trap the CLEAs in more resistant materials (e.g., Lentikats), or add magnetic nanoparticles.  Second, diffusion problems may be huge, due to the high volumetric activity and narrow pores. Finally, as stated in abstract, the crosslinking step may be problematic, mainly when the enzyme is poor in surface Lys. Solutions are the use of a feeder  rich in primary groups(albumin or other  proteins, pEI) or to aminate the enzyme. Please, enlarge the introduction adding these topics and some references.

Remark likely used of proteases in biocatalysis. In the case of proteases, used in proteolysis, there some added problems: the substrate is of a similar size to that of the enzyme and that can add some additional problems (there are some reviews on this topic). And immobilization prevent autolysis, that way enzyme stability may increase without really improve enzyme rigidity

Finally, some discussion on glutaraldehyde may be added. There is a recent review from Barbosa than may be of interest in this point.

Results

“is insufficient to crosslink most of the enzyme MOLECULES”

“and as a result many residual??? enzymes were discarded” I do not see their meaning.

I recommend to check the old Monsan paper regarding  to glutaraldehyde activation and the most recent review from Barbosa (and perhaps some papers cited there).  The concentration of glutaraldehyde just determine the modification of the amino groups of the enzyme, using this low concentraitons and mil conditions most modifications should give amino-glutaraldehye, that are very reactive with amino-glutaraldehdye but no so much with amino groups, and less at mild pH. That way, this needs to be much better explained.

2.2 Change by glutaraldehyde modification time and use previous ideas in discussion. How they explain to have 55% activity recovery if before they have 60%? Mainly, considering the very small mistakes showed in the figures.

BSA and starch can not be considered crosslinking additive. BSA may be names better as protein feeder, starch is used to have larger pores, I can not see a usual rola as enzume protective agant except for starch-degrading enzymes.  In introduction this should be already presented. BSA decreases the  protease loading and that decrease the diffusional limitations. Moreover, as a rich Lys protein, may help to have a better crosslinking. Use references.

This lysine-rich surface prevents excessive cross-linking between enzyme molecules thus 120

 making the structure of CLEA to be less compacted?? It should be more the opposite.

Figure 4 shows the effect of T on enzyme activity, not the optimal temperature.

Any explanation for the lower activity at 70ºC of the CLEAs? Remark the much higher activity at 60ºC, in fact giving specific activity at 60ºC the activity of he immobilized enzyme may be even higher than that of the free enzyme.

Explanation, re3ported in many papers and reviews, should be a rigidification of the enzyme structure by multipoint immobilization, and prevention of autolysis.

Define how thermal stability is determined. Improve explanations

Figure 5. identical problems, again at very drastic pH the behavior of free enzyme is better.

Elastase strain K has a wide range of pH stability profile?? Rewrite

Figure 7 shows the effect of the incubation of the enzyme on organic solvents on the activity

“ Acetonitrile, ethanol, benzene and xylene displayed activity enhancement of 164%, 172%, 189

 152% and 133% respectively.” They have not activity

Using the free enzyme, enzyme aggregation cannot be discarded to explain the results.

Did they suggest that starch is not released and remains in the CLEA? This should increase the diffusion problems, it is easier to think that they are released. In the operationalk stability, they control the initial and final amount of CLEA? CLEA lost may be a explanation of the results (as it can break on very small particles that may be washed way).

2.10 distribution and average diameter are not the same concepts, although both have interest,

Increase in the size of the aggregates confirm the formation of CLEA 227

 superstructure.????

I do not find informative FTIR spectra, they try to say too much from scarce differences, comparing different things. The CLEA with additives should have as referenbce a sample with these aditives, but I think that this may be fully deleted, there is not real information.

 

Revise materials and method English and style

 

Add references on the topics indicated, and use that in a improved discussion.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Razib et al. reports a biochemical characterization of Cross Linked Enzyme Aggregates (CLEA) of elastase strain k in order to obtain an organic solvent tolerant protease useful for biotechnological applications. Although the manuscript is technically sound the following items have to be considered.

Maximum recovery activity reported in Fig. 2 is roughly 70% after two hrs and not 55% as reported in the text. The recovered activity without additive reported in Fig. 3 (about about 60%) is incongruent with that reported in Fig. 2 (about 70%), even in the light of the good statistic reported. Explain well the whole paragraph 2.5 (Optimum pH of CLEA-elastase). At which pH has been assayed the activity after the different pH treatments? Try to give at least a tentative meaning of the small peak at in the CLEA-elastase DLS panel. Without the mention of how many replicates have been carried out, the standard error/deviation reported in each graph cannot be evaluated. Extensive language editing is required (see the non comprehensive list below).

 

Language editing

Substitute the term “pallet” with “pellet” throughout the manuscript. Undefined abbreviations should be avoided in the Abstract. Lines 83-84, change the phrase “Glutaraldehyde is a highly …….. present in the mixture” with “Glutaraldehyde is a highly reactive protein cross-linking compound acting even at very low concentration in the mixture.” Lines 90-91, change the phrase “… , the number of cross-linking is insufficient ……….. discarded during the washing process.” With “…, the cross-linking is insufficient as many residual free enzyme molecules would be discarded during the washing process.” Line 119, change “molecules” with “protein”. Line 121, change “compacted” with “compact”. Line 131, add “activity” after “was”. Line 142, add “was” after “CLEA-elastase”. Line 143, delete “threshold”; change “until” with “to”. Lines 189-190, Change the phrase “Acetonitrile, ethanol, benzene and …….. respectively.” With “In particular, in the presence of acetonitrile, ethanol, benzene and xylene the activity become 164%, 172%, 
152% and 133%, respectively, of that measured in the absence of organic solvents.” Line 197, add “which” after “DMSO”. Line 203, add “the” after “Generally”. Line 232-237, change the entire paragraph as follows “As shown in figure 11 the FTIR spectra of free elastase, CLEA-elastase, and CLEA-elastase with additive, were similar. However, a close-up analysis of the 1600 – 1700 cm-1 region (C=C or C=N bonds), reveals an increment of the FTIR signal for CLEA-elastase sample compared to free elastase. This behaviour would suggest the formation of additional linkage after cross-linking, beside those occurring during cross-linking of amino group of the enzyme with glutaraldehyde .” Line 281, change “the” with “it”. Line 327, delete “and thermal stability”.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Tnglish nedds further attention, and the explanations amd discussion more related to the reviews cited now in the paper.

Proteass problams have no references, glutaraldehyde cycles are nit mentioned in the glutaraldehyde,..

Plese, re-revise the paper.

Author Response

We thanked the reviewer for the comment to improve the manuscript.

"English needs further attention, and the explanations and discussion more related to the reviews cited now in the paper."

As suggested by the reviewer, some grammatical error has been corrected.

"Protease problems have no references, glutaraldehyde cycles are not mentioned in the glutaraldehyde,.."

As suggested by the reviewer, more explanation on the glutaraldehyde cycles have been included in te discussion. Explanation on this can be read between lines 296 - 304. "Glutaraldehyde which is used as the protein cross-linking reagent reacts actively with the amino group of the enzyme forming CLEA as a result of a Schiff base reaction [22]. In fact, there are many proposed reactions of glutaraldehyde since the reaction in aqueous environment were affected by the pH of the solution, presence of active amino group and its ability to form many conjugates [22,38]. The reactive group in glutaraldehyde are in equilibrium between its polymeric and monomeric configuration. Reaction in acidic or neutral condition involve formation of Schiff base and nucleophilic attack on the amino group of the protein. On the other hand, reaction of glutaraldehyde in alkaline condition underwent two mechanisms; formation of Schiff base and Michael addition of C-C bonds forming the linkages [38]." Protease problems have been briefly mentioned in the introduction between lines 40 - 41. "Protease for instance are susceptible to oxidation and autolysis when kept over a period of time [3]."

Round 3

Reviewer 1 Report

Some references ssems to be repeated, but that may be solved during editing.

Author Response

Thank you for the comments to improve this manuscript.

Reviewer 1 commented:

"Some references ssems to be repeated, but that may be solved during editing."

-repeated references has been removed and edited accordingly. Some formatting error has been edited as well.