Advances in Chitosanase Research: From Structure and Function to Green Biocatalytic Production of Chitooligosaccharides

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review provides a comprehensive overview of chitosanase research, covering catalytic mechanisms, structure–function relationships, microbial sources, heterologous expression, kinetic properties, protein engineering and green bioprocessing strategies. The  review   highlights the potential of chitosanases for industrial-scale chitooligosaccharides production. The manuscript is well-organized, with extensive references supporting, and addresses a topic in green biocatalysis and sustainable bioprocessing. The manuscript is suitable for publication after revisions. Some areas require refinement to enhance clarity. 

 

Figure 1 is informative but could be improved with clearer labeling and a more professional layout. And is there a type of exo-chitosanase that cleaves glucosamine monosaccharides? It needs to be clearly stated in the text.

Section 2.2: This section is dense and highly technical. Consider adding a summary table comparing key structural features and substrate preferences across different GH families to improve readability. Furthermore, this part of the text should be written in segments according to different GH families or divided into sub-sections.

The Section 3 would benefit from a brief comparative summary highlighting the advantages and limitations of different expression hosts (E. coli, B. subtilis, P. pastoris) for recombinant chitosanases preparation.

The recognition mechanism and catalytic ability of chitosanases for chitosan sugar chains with different acetylation degrees are the potential focus of chitosanase research. The authors should summarize and elaborate on the research progress of the above-mentioned related research.

It would be helpful to include a paragraph discussing the limitations of current engineering approaches for chitosanases.

The Conclusion should address unresolved questions and could be more forward-looking.

Author Response

Reviewer #1: This review provides a comprehensive overview of chitosanase research, covering catalytic mechanisms, structure–function relationships, microbial sources, heterologous expression, kinetic properties, protein engineering and green bioprocessing strategies. The review highlights the potential of chitosanases for industrial-scale chitooligosaccharides production. The manuscript is well-organized, with extensive references supporting, and addresses a topic in green biocatalysis and sustainable bioprocessing. The manuscript is suitable for publication after revisions. Some areas require refinement to enhance clarity.  

Figure 1 is informative but could be improved with clearer labeling and a more professional layout. And is there a type of exo-chitosanase that cleaves glucosamine monosaccharides? It needs to be clearly stated in the text.

Author response: “Exo-β-D-glucosaminidases are exo-type chitosanases that release GlcN monomers from the non-reducing ends, and have been identified in only a few microorganisms, such as Aspergillus fumigatus, Aspergillus spp., Trichoderma reesei, Penicillium funiculosum, Nocardia orientalis, and Thermococcus kodakaraensis.” We have stated in the text in lines 97-100.

Section 2.2: This section is dense and highly technical. Consider adding a summary table comparing key structural features and substrate preferences across different GH families to improve readability. Furthermore, this part of the text should be written in segments according to different GH families or divided into sub-sections.

Author response: Thank you for your suggestion. In this review, key structural features and substrate preferences of chitosanases are thoroughly discussed and organized by GH family within the text. We have separated the content into distinct segments according to different GH families (Lines 147, 172, 210, 226 in the manuscript) to improve readability. This narrative presentation allows for a detailed and continuous discussion of each family’s properties and effectively conveys the differences across GH families without requiring an additional summary table.

Section 3 would benefit from a brief comparative summary highlighting the advantages and limitations of different expression hosts (E. coli, B. subtilis, P. pastoris) for recombinant chitosanases preparation.

Author response: Line 343. We added a brief comparative summary of the advantages and limitations for E. coli, B. subtilis, P. pastoris expression hosts.

Table 1. Advantages and limitations of common heterologous expression systems for recombinant chitosanases.

Expression Host system	Advantages	Limitations	References
E. coli	Rapid growth; high cell-density cultivation; low-cost media; ease of genetic manipulation	Protein overexpression may impose metabolic burden; limited folding of complex proteins	[42]
Bacillus subtilis	Nonpathogenic; genetically well-characterized; strong protein secretion; suitable for industrial fermentation	Expression efficiency is strain-dependent; extracellular proteases may degrade recombinant proteins	[43]
P. pastoris	Eukaryotic folding and glycosylation; high cell-density fermentation; efficient secretion of active enzymes	Requires methanol induction; relatively long cultivation times; higher cost and operational complexity	[44]

 

The recognition mechanism and catalytic ability of chitosanases for chitosan sugar chains with different acetylation degrees are the potential focus of chitosanase research. The authors should summarize and elaborate on the research progress of the above-mentioned related research.

Author response:

We appreciate the reviewer’s comment. The section has been revised to clarify that our study focuses on how chitosanases recognize chitosan chains with varying degrees of acetylation and how this recognition influences their catalytic efficiency. Specifically, chitosanases interact with both GlcN and GlcNAc residues within the substrate-binding cleft, and their activity is modulated by the degree of acetylation (DA). Generally, a lower DA (GlcN-rich chains) favors stronger binding and higher catalytic rates, whereas higher DA (GlcNAc-rich chains) can reduce enzymatic activity due to steric and electronic effects. We have revised the text accordingly in lines 141-146.

 

It would be helpful to include a paragraph discussing the limitations of current engineering approaches for chitosanases.

Author response: we added a paragraph discussing the limitations of current engineering approaches for chitosanases in Lines 729-737, which read as follows:

Despite these advances, current engineering strategies for chitosanases still face several challenges. Directed evolution requires extensive screening efforts, while rational and semi-rational design approaches remain limited by incomplete understanding of structure-function relationships, often resulting in trade-offs between activity, stability, and product specificity. Moreover, most engineered variants are optimized under laboratory conditions, which may not reflect industrial settings and thus limit scalability and robustness. These limitations highlight the need for integrated strategies combining computational prediction with experimental validation to generate application-oriented chitosanases.

The Conclusion should address unresolved questions and could be more forward-looking.

Author response: We have rewritten conclusion section in Lines 1036-1069 as suggested by you, which reads as follows:

Chitosanases are essential biocatalysts for converting chitosan into value-added COSs, whose structure-dependent bioactivities support broad applications in agriculture, food, biomedicine, and environmental biotechnology. Over the past decade, significant advancements have been made in understanding the catalytic mechanisms and structure-function relationships of chitosanases across various GH families, particularly GH8, GH46, GH5, and GH75. The identification of active-site architectures and subsite configurations has clarified the molecular basis for substrate selectivity and product specificity, laying the groundwork for targeted enzyme engineering. Consequently, an increasing number of chitosanases with desirable catalytic features have been discovered, enabling their significant contribution to biotechnological processes, particularly in the production of functional COS. High-purity COS can be achieved through appropriate separation and purification techniques. In addition, as key biological functions of COS are being unveiled, their applications are expanding rapidly.

Despite these advancements, several challenges remain. The detailed molecular mechanisms underlying the endo-/exo-activity switching, substrate transglycosylation, and product pattern control are still not fully understood, particularly in underexplored GH families. In addition, discrepancies often exist between in vitro biochemical characterization and in vivo functional relevance, highlighting the need for integrative approaches that couple structural biology with systems-level analysis. Another unresolved issue lies in scaling laboratory findings to industrially robust processes, where enzyme stability, reusability, and cost-effectiveness must be rigorously addressed under complex feedstocks and processing environments. Furthermore, the diversity of COS structures has not yet been systematically linked to their bioactivities, leaving gaps in the rational design of COS-based products with predictable functionality.

Future directions should therefore prioritize the discovery of novel chitosanases from extremophilic or uncultured microorganisms using metagenomic, combined with machine learning-guided mining to predict functional diversity. Systems and synthetic biology strategies offer opportunities to engineer optimized expression hosts and metabolic pathways for sustainable production. Integrating continuous bioprocessing with advanced separation technologies may enhance scalability, while elucidating COS structure-activity relationships will be essential for their rational deployment in food, biomedical, and packaging applications. Achieving these goals will require interdisciplinary collaboration across enzyme science, computational modeling, materials chemistry, and process engineering.

 

 

Thank you once again for your comments and suggestions. We greatly appreciate your time, effort and critical comments help us improve the paper.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript authored by Oanh Thi Kim Nguyen et al. reviews the catalytic mechanisms, structure-function relationships, microbial production possibilities, kinetic characteristics, and protein engineering strategies related to chitosanases, as well as the possibilities to optimize the processes leading to chito-oligosaccharides (COSs) catalyzed by these enzymes. It is a truly comprehensive work, addressing a very actual topic, well organized and written, which, in my opinion, could arouse scientific interest. Therefore, I recommend it for publication, with only a few remarks and recommendations, as it will follow.

1. Page 7, Figure 2. The figures must be self-explanatory. Please add to the caption of Figure 2 some information to facilitate its understanding without searching in the text.
2. Section 3.2 and throughout the manuscript. The chitosanase activities are expressed in U/mL or U/mg, but I am not sure that 1 unit of enzymatic activity was calculated identically in all cited source works (most probably as μmol/min), since different authors could, in principle, use different formulas. Please verify and specify it somewhere in the manuscript. It will allow the readers to compare the different reported results without a possible misunderstanding. It should be also specified what “specific activity” exactly means, since it could be also different in various reports.
3. Section 4.1. “Chitosanases differ not only in their preference for fully or partially deacetylated polymers but also in their acceptance of powder or colloidal chitosan forms”. I think that it is an essential characteristic of a chitosanase considering the possible applications because it is hard to believe that colloidal chitosan can be the most appropriate substrate for large scale utilization. Based on the literature, could the authors explain the possible substrate preference toward powdered chitosan forms as a criterion for the selection of the enzyme for a designed application?
4. Section 6 focuses on the microbial production of chitosanases using fermentation on low-cost substrates and stabilization of the enzyme through immobilization. Since the production of COSs is the ultimate goal of such a bioprocess, the enzymatic hydrolysis using native and immobilized chitosanases should be also discussed (maybe as a distinct subchapter 6.3). A scheme should be also useful, including a pretreatment step to obtain powdered (or colloidal?) chitosan, the hydrolysis step, a separation step of the COS product (also indicating its further applications in food, agriculture, biomedicine, cosmetics), and the reuse of the enzyme. I think that it would be more intuitive for the readers to understand the importance of the subject addressed by this review.

Author Response

Reviewer #2: The manuscript authored by Oanh Thi Kim Nguyen et al. reviews the catalytic mechanisms, structure-function relationships, microbial production possibilities, kinetic characteristics, and protein engineering strategies related to chitosanases, as well as the possibilities to optimize the processes leading to chito-oligosaccharides (COSs) catalyzed by these enzymes. It is a truly comprehensive work, addressing a very actual topic, well organized and written, which, in my opinion, could arouse scientific interest. Therefore, I recommend it for publication, with only a few remarks and recommendations, as it will follow.

1. Page 7, Figure 2. The figures must be self-explanatory. Please add to the caption of Figure 2 some information to facilitate its understanding without searching in the text.

Author response: Figure 2 presents representative three-dimensional structures of chitosanases from different GH families (GH2, GH8, GH46, GH75, GH80), with their respective PDB codes labeled beneath each structure. We have also updated the figure title to “Three-dimensional structures of chitosanase” and included the corresponding PDB codes, which can be accessed from https://www.rcsb.org/structure, it can read as follow:

Figure 2. Three-dimensional structures of chitosanase with the corresponding PDB codes from https://www.rcsb.org/structure

 

2. Section 3.2 and throughout the manuscript. The chitosanase activities are expressed in U/mL or U/mg, but I am not sure that 1 unit of enzymatic activity was calculated identically in all cited source works (most probably as μmol/min), since different authors could, in principle, use different formulas. Please verify and specify it somewhere in the manuscript. It will allow the readers to compare the different reported results without a possible misunderstanding. It should be also specified what “specific activity” exactly means, since it could be also different in various reports.

Author response: Thank you for your comment. we have added a footnote below the Table 2 (lines 413-415) in the revised manuscript defining both “one unit activity” and “specific activity” to prevent possible misunderstandings across different reports.

 

3. Section 4.1. “Chitosanases differ not only in their preference for fully or partially deacetylated polymers but also in their acceptance of powder or colloidal chitosan forms”. I think that it is an essential characteristic of a chitosanase considering the possible applications because it is hard to believe that colloidal chitosan can be the most appropriate substrate for large scale utilization. Based on the literature, could the authors explain the possible substrate preference toward powdered chitosan forms as a criterion for the selection of the enzyme for a designed application?

Author response: Thank you for your question. Chitosanases indeed display differential substrate preferences not only regarding the degree of acetylation but also the physical form of chitosan. While many studies mainly employ soluble or colloidal chitosan for laboratory preparation, powdered chitosan represents a more relevant substrate for large-scale or industrial processes due to its lower cost, higher availability, and direct applicability in bioconversion. Enzymes that can efficiently hydrolyze powdered chitosan therefore hold greater promise for practical applications, particularly in producing COS at scale. Supporting this, a recent study showed that ball-milled powdered chitosan increased COS and GlcN yield by 10.8-fold (14.5 μmol/mL), demonstrating a practical and environmentally friendly strategy for industrial-scale processing.

Su, H.; Sun, J.; Guo, C.; Jia, Z.; Mao, X. Heterogenously‐expressed chitosanase combining a green ball milling method for enzymatic degradation. Food Bioeng. 2022, 1, 37-46.

 

4. Section 6 focuses on the microbial production of chitosanases using fermentation on low-cost substrates and stabilization of the enzyme through immobilization. Since the production of COSs is the ultimate goal of such a bioprocess, the enzymatic hydrolysis using native and immobilized chitosanases should be also discussed (maybe as a distinct subchapter 6.3). A scheme should be also useful, including a pretreatment step to obtain powdered (or colloidal?) chitosan, the hydrolysis step, a separation step of the COS product (also indicating its further applications in food, agriculture, biomedicine, cosmetics), and the reuse of the enzyme. I think that it would be more intuitive for the readers to understand the importance of the subject addressed by this review.

Author response: As suggested by you we have added a subsection on the bioprocess of COS production in line 963-1034 which reads as follows.

6.3.  Bioprocess of COS production

Shrimp and crab are important aquatic food products and also serve as raw materials for some processed seafood items. However, their shell waste accounts for a significant proportion of their total weight, generating significant quantities of waste annually. In the past, much of this waste was simply discarded, causing environmental pollution, or processed into fishmeal and fish feed, resulting in limited economic benefits. Effectively utilizing this waste to create high-value products would not only address the waste problem but also enhance its economic value. With proper separation and purification, approximately 20–35% chitin can be extracted from shrimp and crab shell waste, or from squid sheaths. The production of chitin generally involves deproteinization, which removes proteins from shells, and demineralization, which eliminates calcium carbonate and other minerals. Subsequent deacetylation of chitin, usually achieved by alkali treatment, yields chitosan. Using these materials to prepare chitin and chitosan can significantly increase the value of these wastes [104].

Chitosan has the potential to lower blood cholesterol, fight tumors, and enhance immunity, but its application in food applications still faces numerous challenges. First, chitosan has poor water solubility and dissolves only in acidic solutions, which can alter the pH, flavor, and texture of foods and make it unsuitable for neutral foods. Second, its solution viscosity is high, making it unsuitable for low-viscosity foods. Furthermore, chitosan can agglutinate proteins, potentially causing them to coagulate or precipitate, affecting food quality. Chitosan's high molecular weight makes it difficult to disperse in highly viscous or solid foods. In addition, chitosan has an astringent taste and can form a film in the mouth, affecting taste. These characteristics limit the widespread use of chitosan in food. However, chitosan hydrolysates have a lower molecular weight, resulting in reduced viscosity and increased water solubility. When hydrolyzed to a certain degree, chitosan does not coagulate or precipitate with food proteins and does not present astringency issues. Furthermore, even lower molecular weight oligosaccharides offer a refreshing sweetness, potentially making chitosan suitable for food processing.

COS, composed of D-glucosamine linked via β-1,4 bonds, are obtained from chitosan via chemical or enzymatic hydrolysis. Their water solubility and low molecular weight offer advantages for commercial applications. COS are important in food and agriculture, and they also have applications in healthcare. COSs have been reported to prevent tumor growth, treat asthma, improve bone strength, and prevent malaria, and can be used as gene delivery vectors in gene therapy [105]. The biological activities of COS include antibacterial, antifungal, antiviral, antitumor, antioxidant, immunomodulatory, fat-depleting, blood pressure-control, and cholesterol-lowering proper-ties [106]. COS can be produced using either acid hydrolysis or enzymatic hydrolysis. Ac-id hydrolysis of chitosan using hydrochloric acid is the most widely used method. This method offers advantages of low cost and ease of operation. However, acid hydrolysis requires high temperatures and large amounts of acid, which can cause environmental pollution and energy consumption. Acid hydrolysis is a random hydrolysis process, making the degree of hydrolysis difficult to control, and the products are mostly GlcN. Enzymatic hydrolysis, on the other hand, utilizes chitosanase to hydrolyze chitosan. Due to the enzyme's specificity, the hydrolysis reaction is easy to control and relatively fast, saving time and reducing environmental pollution, making it a preferred method for hydrolyzing chitosan.

Microbial chitosanases can be used to produce COS from chitosan at relatively high concentrations. Through endo-enzymatic activity, chitosanase hydrolyzes chitosan, particularly highly deacetylated chitosan, to form COS with (GlcN)₂, (GlcN)₃, and (GlcN)₄ as the main products. However, large-scale application is limited because chitosanases are highly specific to chitosan and remain costly to produce. This makes strategies for enzyme recovery and reuse particularly important for industrial COS production. Immobilization of chitosanase can significantly improve its thermal and operational stability. For example, the immobilized chitosanase prepared by adsorption retained 70% of its activity after six hydrolysis cycles [100]. Partial hydrolysis of chitosan (PHC) in a batch reactor significantly reduced viscosity, facilitating substrate flow and enhancing productivity in the packed-bead reactor with immobilized chitosanase. This dual-reactor system represents a promising strategy for continuous COS production and can be applied to other viscous biopolymers [107]. On the other hands, the ultrafiltration membrane reactor enabled simultaneous hydrolysis and product separation, preventing over-degradation of chitooligosaccharides and improved yield and selectivity. It also helped in retaining enzyme’s activity for repeated use, reducing production costs, while allowing continuous operation with controlled molecular weight distribution. Kuo et al. [108] used crude chitosanase from Bacillus cereus NTU-FC-4 to hydrolyze chitosan to produce COS in a membrane reactor. The main COS produced in the reactor were (GlcN)₂, (GlcN)₃, (GlcN)₄, (GlcN)5, and (GlcN)6. The main drawback of ultrafiltration membrane reactors for COS production is membrane fouling, this leads to reduced permeability and decreased productivity [109]. Jeon and Kim used a dual reactor system combining an enzyme-packed column and a UF membrane reactor that enabled continuous COS production from chitosan, with 5 ml/min PHC providing optimal efficiency and minimal fouling [110]. Bioprocesses for COS pro-duction are now available, but they remain challenging to fully exploit. Scaling up purification methods to an economically acceptable level is another major obstacle.

 

Thank you once again for your comments and suggestions. We greatly appreciate your time, effort and critical comments help us improve the paper.

Author Response File: Author Response.pdf