Review of 'Production and characterization of cyclodextrin glycosyltransferase from Bacillus.sp isolated from Cuban Soil.'

The manuscript by Kárel Hernández Sánchez, Marlene María Martínez Mora and Héctor Luis Ramírez, addresses the identification, purification and biochemical characterization of a cyclodextrin glycosyltransferase isolated from a Bacillus strain from Cuban soil, including determination of specific activity of the enzyme, pH profile, temperature profile and stability of the enzyme.

The relevance of the work is the discovery of a basophilic CGTase with an important production of γ-cyclodextrin (a third of the production of α-CD or β-CD). The results shown are convincing, however the manuscript is full of imprecisions and a trend to over interpret the results. I point out some of these:

Figure 1, shows the elution profile from the gel filtration chromatography. In this figure two major bands are detected by absorbance at 280 nm, the smaller, between 25 and 40 mL is the one with higher specific activity. However the authors mention that they used the fractions with higher specific activity eluting between 10 and 20 mL. It is not clear to this reviewer how did the authors interpreted specific activity.

In the first paragraph of the Results and Discussion section, the authors say that they purified the enzyme with a 66.5 % yield and a 1.49 purification factor. According to the volumes, enzymatic activity and protein concentration reported in Table I, the protein yield should be 65 % and a purification factor of 4. This should be corrected, both in the text and in the table I.

Figure 2 shows the pH profile of the enzyme. The first point with this figure is that the Y axis is labeled as % Residual activity, instead of Relative activity. It is not clear if the authors mislabeled the Y axis? Or exchanged figure 2 with figure 3? The second point is that the authors mention that there are two activity maximum for the enzyme, one at pH 6.2 and the other at pH 7.1. This seems to be data over interpretation. The activity in figure 2 shows, within experimental error, a plateau between pH 5.5 and 9. Another concern about these results is that phenolphthalein´s color is sensitive to pH. As a matter of fact, it is used as a pH indicator with a pKa of 9.3. At low pH is uncolored. I understand that the activity assay is based on the ability of β-cyclodextrin to form a colorless inclusion complex with this dye. It is not clear to this reviewer how this assay can be used to measure cyclodextrin production at low pH where phenolphthalein becomes colorless.

Figure 3 (if indeed is Figure 3), shows that activity can be recovered after incubation of the enzyme between pH 5 and 11. The authors incorrectly assume that the enzyme can be applied in all this pH range. The activities were measured at neutral pH after preincubating the enzyme for 24 hours at the different pHs.

In the discussion concerning figure 4, the authors should not confuse the residual activity profile, which is what they did measure, with the temperature activity profile (that is shown in figure 5).

In the discussion of Vmax and Km results, the authors mention that the enzyme has “higher affinity for the substrate”. They do not mention against what they are comparing. Other enzymes previously reported? In the same section, units for substrate inhibition seem to be wrong. It should say g/L instead of L/g, unless they are reporting association constant, but usually Ki is reported as dissociation constant.

Figure 6, the data shows that after 3 hours of reaction the approximate ratio of CDs is 1.0:1.0:0.3 for α, β, and γ-CD, respectively, not the 1 to 1 ratio the authors claim for α and γ-CD products. Another point with this figure is the units used for concentration of products. What is m(x-CD)/mg? Is it expressed based on molarity units or mass units?

The manuscript is plagued of grammar errors. I will list some of them:

Last line in the abstract section should say “with a significant yield of γ-CD” instead of “which is the significant yield of γ- CDs”

First line in the Introduction section should say “The increasing interest on alkaliphilic Bacillus sp.“ in place of “The increasing interest to the alkaliphilic Bacillus sp.”

Third line, second paragraph Introduction section, delete “Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.9)” it is repeated twice.

Third paragraph of the introduction section “The industrial production of CGTase became attractive” instead of “The industrial production of CGTase was made attractive”

Same paragraph fourth line “Therefore, a CGTase that synthesizes…” instead of “ A CGTase that synthesizes…”

Last paragraph of introduction section, remove “by us”

Screening and isolation section, remove “Medium was incubated at 37°C.”

Section CGTase production and purification, it should say “medium was filtered across diatomaceous earth”, instead of “medium was across diatomaceous earth”

Same section, it should say “was concentrated by rotoevaporation at 30°C.” instead of “was concentrated with rotoevaporation at 30°C.”

Same section, it should say " applied to a Sephadex chromatography gel G-50 column (15 X 3.76) using buffer Tris/HCl pH 8.0 20 mM as mobile phase.”, instead of “applied to Sephadex chromatography gel G-50 (15 3.76) with mobile phase buffer Tris/HCl pH 8.0 20 mM.”

In the Cyclizing activity of CGTase section, it should say “form a colorless inclusion complex” , instead of “form a colourness inclusion complex”

Last sentence of the Determination of Kinetic parameters it should say “The data” instead of “The dates”

Page 3, paragraph 2, second sentence, it should say “The CGTase obtained was highly stable between pH 5.0 and 11.0 maintaining 84% and 80% residual activities, respectively. This property permits to apply this enzyme in a wide range of pH.”, instead of “The CGTase obtained was highly stable at pH 5.0 and 11.0 maintaining 84% and 80% residual activities, respectively. This property permit to apply this enzyme in a wide range of pH.”

Page 3, column 2 line 3, remove “the”

Page 3, column2 paragraph 2, it should say “were measured at different temperatures in the” instead of “were measured at the temperature in the”

Page 3, column2 paragraph 3, line 1, “using” instead of “by”

Page 3, column2 paragraph 4, line 6, “is shown in Figure 6.” Instead of “was shown in Figure 6.”

Conclusions section last line, it should say “efficient CD production with significant yield of γ-CDs” instead of “efficient CD production which is the significant yield of γ-CDs”