β-Glucosidase: Progress from Basic Mechanism to Frontier Application

Comments 1: [belongs to cellulase]

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have modified [belongs to cellulase familly]

“[updated text in the manuscript if necessary]” line 26

Comments 2: [in plants, microorganisms and microorganisms [1,2].]

Response 2: Agree. We have modified. [ in plants, microorganisms and animals]

“[updated text in the manuscript if necessary]”  line 34

Comments 3: [It can be produced by Candida peltata [6], Trichoderma reesei [7] 38 and Aspergillus niger [8] in eukaryotic microorganisms.]

Response 3: Agree. We have modified. [It can be produced by eukaryotic microorganisms: Candida peltata [6], Trichoderma reesei [7]  and Aspergillus niger [8] ]

“[updated text in the manuscript if necessary]”  line 40-41

Comments 4: [all contain]

Response 4: Agree. We have modified. [contain]

“[updated text in the manuscript if necessary]”  line 45

Comments 5: [Different sources of β-glucosidases have different  structures and enzymatic properties,]

Response 5: Agree. We have modified. [β-glucosidases from different sources have different   structures and enzymatic properties,]

“[updated text in the manuscript if necessary]”  line 46-47

Comments 6: [;in the feed]

Response 6: Agree. We have modified. [. In the feed]

“[updated text in the manuscript if necessary]”  line 50-51

Comments 7: [; in agriculture,]

Response 7: Agree. We have modified. [. In agriculture,]

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Comments 8: [and acid and alkali]

Response 8: Agree. We have modified. [and acid or alkali]

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Comments 9: [( GH )]

Response 9: Agree. We have modified. [(GH)]

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Comments 10: [in GH1]

Response 10: Agree. We have modified. [of GH1]

“[updated text in the manuscript if necessary]”  line 102-103

Comments 11: [observed in the GH9 enzyme]

Response 11: Agree. We have modified. [observed for the GH9 enzyme]

“[updated text in the manuscript if necessary]”  line 123

Comments 12: [a water molecule ]

Response 12: Agree. We have modified. [water molecule ]

“[updated text in the manuscript if necessary]”  line 141-142

Comments 13: [The reverse hydrolysis reaction ( or reverse hydrolysis reaction ) catalyzed .. ]

Response 13: Agree. We have modified. [The reverse hydrolysis reaction catalyzed…]

“[updated text in the manuscript if necessary]”  line 39-41

Comments 14: [the disaccharide yield of almond β-glucosidase increased 151 with the increase of glucose]

Response 14: Agree. We have modified. [The reverse hydrolysis reaction catalyzed…]

“[updated text in the manuscript if necessary]”  line 155

Comments 15: [the disaccharide yield of almond β-glucosidase increased 151 with the increase of glucose ]

Response 15: Agree. We have modified. [the disaccharide yield  synthetized by β-glucosidase increased  with the increase of glucose]

“[updated text in the manuscript if necessary]”  line 159-162

Comments 16: [of substrate it can accept]

Response 16: Agree. We have modified. [of accepted substrate ]

“[updated text in the manuscript if necessary]”  line 204

Comments 17: [β-glucosidase in different fields.]

Response 17: Agree. We have modified. [β-glucosidase for different fields.]

“[updated text in the manuscript if necessary]”  line 214

Comments 18: [Bacillus subtilis and Escherichia coli in bacteria, Aspergillus niger and Aspergillus oryzae in fungi, and Saccharomyces cerevisiae in]

Response 18: Agree. We have modified. [Bacillus subtilis and Escherichia coli in bacteria, Aspergillus niger and Aspergillus oryzae in fungi, and Saccharomyces cerevisiae in]

“[updated text in the manuscript if necessary]”  line 220-221

Comments 20: [an acidic rang]

Response 20: Agree. We have modified. [acidic rang]

“[updated text in the manuscript if necessary]”  line 245

Comments 21: [Saccharomyces cerevisiae]

Response 21: Agree. We have modified. [Saccharomyces cerevisiae]

“[updated text in the manuscript if necessary]”  line 296

Comments 22: [line 285,289,294,311,509 E.coli]

Response 22: Agree. We have modified. [E.coli]

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Comments 23: [302,305, 306 Lactobacillus fermentum]

Response 23: Agree. We have modified. [Lactobacillus fermentum]

“[updated text in the manuscript if necessary]”  line 318、321、322

Comments 24: [Moniliophtora perniciosa expressed in E.coli cells.]

Response 24: Agree. We have modified. [Moniliophtora perniciosa expressed in E.coli cells.]

“[updated text in the manuscript if necessary]”  line 323

Comments 25: [A.thermocellus]

Response 25: Agree. We have modified. [A.thermocellus]

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Comments 26: [Microbacterium dextranolyticum]

Response 26: Agree. We have modified. [Microbacterium dextranolyticum]

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Comments 27: [Cyamopsis tetragonoloba]

Response 27: Agree. We have modified. [Cyamopsis tetragonoloba]

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Comments 28: [Chlamydia fusarium]

Response 28: Agree. We have modified. [Chlamydia fusarium]

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Comments 29: [Alteromonas sp.]

Response 29: Agree. We have modified. [Alteromonas sp.]

“[updated text in the manuscript if necessary]”  line 538

Comments 30: [Scutellaria baicalensis]

Response 30: Agree. We have modified. [Scutellaria baicalensis]

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Comments 31: [Polygonum cuspidatum]

Response 31: Agree. We have modified. [Polygonum cuspidatum]

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Comments 32: [KöTZLER M P, ROBINSON K, CHEN H-M.]

Response 32: Agree. We have modified. [Kötzler M P, Robinson K, Chen H-M.]

“[updated text in the manuscript if necessary]”  line 871

Comments 33: [ZAYULINA K S, ELCHENINOV A G, TOSHCHAKOV S V]

Response 33: Agree. We have modified. [Zayulina K S, Elcheninov A G, Toshchakov S V]

“[updated text in the manuscript if necessary]”  line 875

Comments 34: [GUO JH, LU HY, HONG JF.]

Response 34: Agree. We have modified. [Guo J H, Lu H Y, Hong J F.]

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Comments 35: [SHEN FF, MIU TT, ZHANG]

Response 35: Agree. We have modified. [Shen F F, Miu T T, Zhang X X]

“[updated text in the manuscript if necessary]”  line 953

Comments 36: [In table 2 italic for species names of plants  Repetitions]

Response 36: Agree. We have modified.

“[updated text in the manuscript if necessary]”  line 254

Comments 37: [In table 2 italic for species names of plants  Repetitions]

Response 37: Agree. We have modified.

“[updated text in the manuscript if necessary]”  line 254

Comments 38: [Lines 197-198 β-glucosidase is a widely distributed enzyme that exists in a variety of organisms, including plants, animals and microorganisms.and Line 256-259  

β-glucosidase is a kind of enzyme that can catalyze the hydrolysis of glycosidic bonds  and decompose complex carbohydrates into glucose or other monosaccharides. It is  widely found in microorganisms, plants and animals, and plays a key role in many fields  such as food processing, pharmaceutical synthesis and biomass energy development. Repetitions]

Response 38: Agree. We have modified and delete the corresponding content.[β-glucosidase is a kind of enzyme that can catalyze the hydrolysis of glycosidic bonds and decompose complex carbohydrates into glucose or other monosaccharides. It plays a key role in many fields such as food processing, pharmaceutical synthesis and biomass energy development.]

“[updated text in the manuscript if necessary]”  line 271-274

Comments 39: [Ammonium sulfate precipitation is and old method ,  maybe  to precisely described?]

Response 38: Agree. We have modified.[Ammonium sulfate salting-out : This is a traditional protein separation technology, commonly used in the extraction process of enzymes such as β-glucosidase [69].]

“[updated text in the manuscript if necessary]”  line 380-381

Comments 40: [Methods of proteins purification too precisely described! Importance of paper must be focused on beta-galactosidase not methods!]

Response 40: Agree. We have modified and add some content to further illustrate.[The extraction and purification technology of β-glucosidase is a key step to realize its industrial application. First, it is necessary to break the cells containing β-glucosidase by physical or chemical methods, release the intracellular enzyme, and then extract and purify the β-glucosidase. The ' purification method ' of β-glucosidase is not a simple subsequent processing, but a core link that determines the final commercial enzyme yield. By 1 optimizing the precipitant type and saturation, 2 shortening the desalination / dialysis time, 3 finely designing the chromatographic gradient and flow rate, 4 and collaborative optimization of the fermentation stage (host selection, induction conditions, glycosylation engineering ), the traditional < 20% enzyme activity yield can be increased to 25% -30% or even higher, directly amplifying the economic benefits of each batch of fermentation. On the contrary, if the purification scheme is improper, even if the fermentation enzyme activity is high, it may lead to the dilemma of ' high activity and low yield ' due to early mass inactivation or miscellaneous protein co-precipitation.At present, the purification technology of β-glucosidase mainly includes traditional extraction methods and efficient purification methods.]

“[updated text in the manuscript if necessary]”  line 362-376

Comments 41: [Chapter 7. Future prospect Is a repetition of part on genetic engineering, proteins purification and immobilization. In my opinion the description of biosynthesis of enzyme in bioreactors, on large scale, was not included, missed in such review paper. ]

Response 41: Agree. We have modified and add some content to further illustrate.[7.3. Regulation strategy of β-glucosidase biosynthesis

In addition to genetic engineering, protein purification and immobilization. By regulating the medium composition, inducer addition and metabolic flow during fermentation, the synthesis efficiency of β-glucosidase can be further improved. The current mainstream regulation strategies include carbon source optimization, inducer regulation and metabolic flux redistribution.

Carbon source is the core substrate of strain growth and enzyme synthesis, and the induction effect of different carbon sources on β-glucosidase synthesis is significantly different. Traditional studies have found that cellulose carbon sources (such as microcrystalline cellulose, straw hydrolysate)are efficient inducers of β-glucosidase, and their decomposition products (cellobiose) can activate the expression of enzyme genes. The easy-to-use carbon sources such as glucose inhibit enzyme synthesis through CCR. In recent years, the optimization strategy has focused on the design of ' mixed carbon source ' : glucose (growth carbon source) and microcrystalline cellulose (induced carbon source) are mixed in a certain ratio (such as 1:3), which not only ensures the rapid growth of the strain in the early stage (glucose provides energy), but also induces enzyme synthesis through cellulose in the later stage. In addition, the use of agricultural waste (such as corncob, bagasse) as a cheap carbon source can further reduce the fermentation cost and provide feasibility for industrial production[].

The addition of exogenous inducers can specifically activate the promoter of β-glucosidase gene and improve the efficiency of enzyme synthesis. At present, the commonly used inducers include small molecule glycosides (such as cellobiose, salicyloside) and non-glycosides (such as sodium benzoate, Tween-80).Small molecule glycosides : cellobiose is the most effective inducer, but its cost is high ; as an alternative inducer, when the concentration of salicyloside was 0.5 g/L, the β-glucosidase production of T.reesei increased by 1.2 times, and the cost was only 1/5 of cellobiose[]. Non-glycosides : Tween-80 as a surfactant can increase cell membrane permeability and promote enzyme secretion. When 0.1 % Tween-80 was added to the fermentation, the enzyme yield increased from 25 U/mL to 30 U/mL ; sodium benzoate relieves the inhibition of glucose on enzyme synthesis by inhibiting the activity of CCR-related proteins, which is suitable for the mixed carbon source system containing glucose[].

The timing of the addition of inducers is also crucial : the addition of inducers at the later stage of the logarithmic growth phase ( fermentation 12-16 h ) can avoid the early inducers being consumed by the strain as a carbon source, so that the enzyme yield is 20 % higher than the ' initial addition '.

By regulating the concentration of nutrients such as nitrogen source and phosphorus source, the metabolic flow of the strain can be changed, and the carbon source is preferentially used for β-glucosidase synthesis, rather than cell proliferation. For example, under the condition of nitrogen limitation (e.g.,the concentration of ammonium sulfate decreased from 2 g/L to 1 g/L ), the cell biomass of Aspergillus niger decreased by 15 %, but the production of β-glucosidase increased by 25 %. The reason is that nitrogen limitation inhibits cell division and makes carbon flow more to enzyme synthesis ; phosphorus source limitation (e.g., the concentration of potassium dihydrogen phosphate decreased from 0.5 g/L to 0.2 g/L) further increased enzyme production by inhibiting energy metabolism (reduced ATP synthesis) and reducing the conversion of carbon sources to energy storage substances such as glycogen.

In addition, metabolic flux redistribution can also be achieved by adding metabolic intermediate product inhibitors (such as fluoroacetic acid inhibiting the tricarboxylic acid cycle), but the concentration of inhibitors needs to be strictly controlled to avoid excessive inhibition leading to strain death.]

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Comments 1: [Line 33：“microorganisms” is repetitive.]

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have modified[in plants, microorganisms and animals]

“[updated text in the manuscript if necessary]”line 34

Comments 2: [Line 80: With website data support, the number should be clear and should not be a vague value..]

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have modified[As of July 2025, the CAZymes database contains 190 glycoside hydrolase ( GHs ) families. Polysaccharide lyase ( PLs ) family : 44. Carbohydrate esterase ( CEs ) family : 20. Glycosyltransferases ( GTs ) family : 138. Auxiliary active enzyme ( AAs ) family : 17. Carbohydrate binding modules ( CBMs ) : 106.]

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Comments 3: [As mentioned in Line 117: “but the GH1, 2, 3, 4, 5, 30, 39 and 116 families follow the retention mechanism,” and in Line 118: “The retention mechanism of GH1, GH2, GH3, GH5, GH30, GH39 and GH116 family includes two consecutive steps of glycosylation and deglycosylation.” Is GH4 omitted? Does its retention mechanism also include the two consecutive steps of glycosylation and deglycosylation?]

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have modified[ but the GH1,2,3,4,5,30,39 and 116 families follow the retention mechanism. [16] The retention mechanism of GH1, GH2, GH3, GH4, GH5, GH30, GH39 and GH116 family includes two consecutive steps of glycosylation and deglycosylation [17].]

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Comments 8: [Lines 255-259：This sentence appears repeatedly in the article, using exactly the same expression each time, which may make the reader feel redundant rather than emphasized, thus weakening theasiveness of the argument to some extent. It is suggested that the author could consider diversifying this core expression.]

Response 8: Thank you for pointing this out. Thank you for pointing this out. We agree with this comment. Therefore, we have modified [In the process of biosynthesis, if the pH value and temperature of the reaction system deviate from the optimum conditions of β-glucosidase, the enzyme activity may be reduced, which will affect the synthesis efficiency. When the enzyme is used to hydrolyze cellulose to produce glucose, too high temperature or pH discomfort will inhibit the enzyme activity, slow down the hydrolysis rate of cellulose, lead to a decrease in glucose production and hinder the biosynthesis process. ]

Comments 9: [Line 228: In Tables 1 and 2, the “Activator” and “Inhibitor” columns for some strains are blank. Is this because the β-glucosidase of these strains has no relevant activators or inhibitors, or because no relevant research has been conducted?]

Response 9: Thank you for pointing this out. Thank you for pointing this out. We confirm that there is no relevant research in the references.

Comments 10: [Line 297-303: The enzyme activity units in Line 297 and Line 303 are inconsistent. Please check the entire text and standardize the formats.]

Response 10: Thank you for pointing this out. Thank you for pointing this out.Therefore, we have modified.

“[updated text in the manuscript if necessary]”line 313、534、537、

Comments 11: [Lines 224–225: It is mentioned that “the optimum pH for microbial β-glucosidase is 5–”, but the optimum pH for Candida adriatica in Table 1 is 8.2, is it out of the range? Please check.]

Response 11: Thank you for pointing this out. Thank you for pointing this out.Therefore, we have modified.

“[updated text in the manuscript if necessary]” in Table1

Comments 12: [Please check and ensure that all references are complete and formatted consistently (e.g., references 22, 52, 63)]

Response 12: Thank you for pointing this out. Thank you for pointing this out.Therefore, we have modified.