Applications of Enzymatic Protein Crosslinking Systems in Gel Construction and Functional Evaluation

Enzymatic protein crosslinking can regulate the interconnection mode among protein molecules under mild conditions and is an important method for the construction of functional gels. This system is applicable to the development of food gels, biomedical hydrogels, active substance delivery carriers, and composite network materials.

Keywords: enzymatic protein crosslinking; transglutaminase; laccase; tyrosinase; peroxidase; protein gel; hydrogel; functional evaluation

1 Technical basis

1.1 Principles of enzymatic crosslinking

(1) Reaction sites

Transglutaminase mainly acts on glutamine residues and lysine residues; laccase, tyrosinase, and peroxidase mainly participate in the oxidative crosslinking of phenolic hydroxyl groups, tyrosine residues, or phenol-modified structures.

(2) Network formation

After proteins are moderately unfolded, reactive groups are exposed and participate in enzymatic reactions. As the number of crosslinking points increases, protein molecules gradually transform from loose aggregates into a continuous network, and the pore size distribution, network density, and phase structure of the gel change accordingly.

(3) Structural reinforcement

Enzymatic crosslinking can strengthen the interconnection among protein molecules and improve the ability of gels to resist external mechanical damage and water migration. When the degree of crosslinking is moderate, the gel usually exhibits higher storage modulus, mechanical strength, and water-holding capacity.

1.2 Technical advantages

(1) Mild conditions

Most enzymatic reactions can be carried out in aqueous phase and under neutral or weakly acidic/alkaline conditions, causing relatively little damage to protein nutritional value, active components, and composite ingredients.

(2) Tunable structure

By changing enzyme addition level, reaction time, pH, temperature, protein concentration, and auxiliary components, the gelation rate, gel strength, elasticity, water-holding capacity, and release behavior can be regulated.

(3) Application compatibility

Enzymatic crosslinking can be used synergistically with thermal induction, ionic induction, polysaccharide compounding, polyphenol oxidation, and physical field treatment, and is suitable for structural design in multicomponent gel systems.

2 Major enzyme systems and crosslinking pathways

2.1 Transglutaminase system

(1) Reaction mechanism

Transglutaminase can catalyze the formation of ε-(γ-glutamyl)lysine isopeptide bonds between glutamine residues and lysine residues in proteins. This bond has high stability and can significantly enhance the interconnection strength among protein molecules.

(2) Applicable substrates

Whey protein, casein, gelatin, soy protein, pea protein, and myofibrillar protein can all serve as substrates. The number of reactive residues, conformational flexibility, and degree of site exposure in protein molecules affect crosslinking efficiency.

(3) Structural role

The transglutaminase system can improve the continuity of the gel network and reduce large pores and syneresis. Moderate crosslinking helps enhance hardness, elasticity, sliceability, freeze-thaw stability, and processing tolerance.

2.2 Oxidase systems

(1) Laccase system

Laccase can catalyze the oxidation of phenolic hydroxyl groups to generate radical intermediates, followed by coupling reactions. Tyrosine residues in proteins can participate in dityrosine crosslinking; after the addition of polyphenols, protein–polyphenol–protein bridged structures can also be formed.

The laccase system is suitable for protein–polyphenol composite gels, edible films, active packaging materials, and functional food gels. After polyphenol participation, the gel can simultaneously obtain structural enhancement, antioxidant activity, and interfacial stabilization functions.

(2) Tyrosinase system

Tyrosinase can catalyze the hydroxylation of tyrosine and further oxidize it to quinone intermediates. Quinone structures can react with amino groups, sulfhydryl groups, or phenolic hydroxyl groups in proteins to form complex crosslinked networks.

This system is suitable for gels based on gelatin, silk fibroin, collagen, whey protein, and some plant proteins. When oxidation reaction activity is high, enzyme dosage, oxygen supply, and reaction time need to be controlled to avoid excessive protein oxidation, deepened color, and gel embrittlement.

2.3 Peroxidase system

(1) Reaction mechanism

Horseradish peroxidase can catalyze the oxidation of phenolic hydroxyl groups in the presence of hydrogen peroxide, allowing phenol-modified proteins or composite materials to rapidly crosslink into gels. This system has a fast reaction rate and is suitable for injectable hydrogels, in situ gelation, cell encapsulation, and local drug delivery systems.

(2) Key points of control

Hydrogen peroxide concentration is the key control factor in this system. Too low a concentration results in insufficient crosslinking, whereas too high a concentration may cause oxidative damage to proteins, inactivation of active substances, or decreased cytocompatibility.

3 Gel construction strategies

3.1 Single-protein gels

(1) Whey protein gels

Whey protein has good nutritional value and gel-forming ability. After enzymatic crosslinking, the interconnection among protein molecules is enhanced and the gel network becomes more continuous, improving hardness, elasticity, storage modulus, and water-holding capacity.

(2) Gelatin gels

Gelatin has good film-forming ability and biocompatibility, but natural gelatin gels have limited thermal stability and mechanical strength. Enzymatic crosslinking can improve the anti-swelling ability, thermal stability, and mechanical support performance of gelatin gels, making them suitable for hydrogel carriers, wound dressings, and cell encapsulation materials.

(3) Plant protein gels

Soy protein, pea protein, and wheat protein often have problems such as insufficient solubility, non-uniform structure, and limited gel strength. Enzymatic crosslinking can promote molecular rearrangement of plant proteins and improve network continuity, making it suitable for plant-based meat products, composite protein gels, and high-protein foods.

3.2 Composite protein gels

(1) Animal protein–plant protein composite systems

Animal proteins usually have good gelation and emulsifying properties, while plant proteins have advantages in resource availability and nutritional structure. After compounding the two, transglutaminase can promote heterologous protein crosslinking and improve problems such as loose plant protein networks, insufficient elasticity, and poor water-holding capacity.

(2) Milk protein–gelatin composite systems

Milk proteins can provide structural strength, while gelatin can improve flexibility and film-forming ability. After enzymatic crosslinking, this system can reduce the thermal reversibility of gelatin and improve structural retention after heating, making it suitable for dairy gels, gel desserts, and nutritional soft-gel systems.

3.3 Protein–polysaccharide composite gels

(1) Regulatory role of polysaccharides

Polysaccharides can regulate protein gel structure through thickening, water retention, electrostatic complexation, and steric hindrance. Polysaccharides such as sodium alginate, pectin, chitosan, and sodium carboxymethyl cellulose can improve water retention, network support performance, and freeze-thaw stability.

(2) Participation of crosslinkable polysaccharides

Some polysaccharides, after modification with phenolic, amino, or carboxyl groups, can directly participate in enzymatic crosslinking. Phenol-modified polysaccharides can be used in peroxidase or laccase systems; amino groups in chitosan can participate in quinone intermediate reactions, improving gel film-forming ability and adhesive performance.

3.4 Protein–polyphenol composite gels

(1) Functional enhancement

Polyphenols contain multiple phenolic hydroxyl groups and can form radicals or quinone structures under the action of oxidases, and then undergo covalent or non-covalent interactions with proteins. An appropriate amount of polyphenols can improve the antioxidant activity, interfacial stability, and antimicrobial potential of gels, and enhance the protein network through a bridging effect.

(2) Formulation limitations

When the addition amount of polyphenols is too high, it may lead to excessive protein aggregation, deeper gel color, increased bitterness and astringency, and non-uniform structure. Protein–polyphenol gels need to control polyphenol concentration, oxidation rate, and system pH to avoid simultaneous occurrence of structural enhancement and quality deterioration.

4 Process parameters and structural regulation

4.1 Substrate factors

(1) Protein source

Different proteins differ in amino acid composition, molecular weight, conformational flexibility, and degree of site exposure, which ultimately determine the gel network type and crosslinking strength. Milk proteins are usually suitable for transglutaminase crosslinking; gelatin has more flexible chain segments and is suitable for hydrogel and biocompatible network construction; plant proteins have relatively complex structures and often require heat treatment, ultrasound, homogenization, or pH-shift treatment to promote exposure of reactive sites.

(2) Protein concentration

When protein concentration is too low, molecular contact is insufficient and a stable network is difficult to form; when concentration is too high, system viscosity increases and mass transfer is limited, and local non-uniform crosslinking may occur. A moderate concentration is usually more favorable for forming a uniform gel with certain strength.

4.2 Reaction conditions

(1) Enzyme addition level and reaction time

Insufficient enzyme addition results in inadequate crosslinking, slow gel formation, and low strength; excessive enzyme addition may cause rapid local crosslinking, forming large aggregates or brittle networks. Extending reaction time usually increases crosslinking density, but excessive reaction reduces flexibility and digestive accessibility.

(2) pH and temperature

pH affects enzyme activity, protein charge state, and protein solubility. Temperature affects enzymatic reaction rate and protein conformational changes. In process design, the enzyme activity range should be matched first, while avoiding uncontrollable protein denaturation or enzyme inactivation.

(3) Ionic strength

Salt ions can change electrostatic interactions and hydration state among protein molecules. Moderate ionic strength helps regulate network pores and gel elasticity; excessively high ionic strength may induce salting-out, large aggregates, and structural non-uniformity.

4.3 Table of parameter effects

Control factor	Key control point	Structural effect	Key focus of functional evaluation
Protein source	Amino acid composition, molecular flexibility, site exposure	Determines crosslinking efficiency and network skeleton type	Gel strength, digestibility, nutritional retention
Protein concentration	Basic density for network formation	Increased concentration can enhance gel strength, but may reduce uniformity	Hardness, elasticity, syneresis rate
Enzyme addition level	Crosslinking rate and degree of crosslinking	Low level causes insufficient crosslinking; high level easily causes local over-crosslinking	Gelation time, brittleness, rheological stability
Reaction time	Network development stage	Prolonged reaction increases crosslinking density, but may reduce flexibility	Elastic recovery, compression performance
pH	Enzyme activity and protein charge state	Changes protein solubility, aggregation behavior, and pore size	Network uniformity, water-holding capacity
Temperature	Enzyme activity and protein conformational change	Moderate temperature increase promotes reaction, excessive temperature causes inactivation or denaturation	Process compatibility, thermal stability
Ionic strength	Electrostatic shielding and hydration state	Affects aggregation scale and water distribution	Freeze-thaw stability, water-holding capacity
Auxiliary components	Polysaccharides, polyphenols, lipids, mineral salts	Form composite networks or regulate phase behavior	Release performance, antioxidant activity, sensory quality

5 Functional evaluation system

5.1 Rheological evaluation

(1) Gelation kinetics

Time sweep can reflect the gel formation process. An increase in storage modulus indicates that an elastic network is gradually forming, and the relationship between storage modulus and loss modulus can be used to determine the gel point and network development rate.

(2) Viscoelastic stability

Frequency sweep and strain sweep can be used to evaluate gel network stability. Stable gels usually show a storage modulus higher than the loss modulus and are insensitive to frequency changes. Injectable gels also need to be evaluated for shear thinning, self-recovery, and in situ gelation time.

5.2 Texture and mechanical evaluation

(1) Food gel evaluation

Food gels should focus on hardness, elasticity, adhesiveness, cohesiveness, and chewiness. Hardness is related to cutting stability, elasticity reflects recovery ability after stress, and cohesiveness and chewiness affect gel mouthfeel and processing compatibility.

(2) Biomaterial evaluation

Biomaterial gels should focus on compressive modulus, fracture strain, elastic recovery, and fatigue stability. Compressive modulus affects tissue support capacity, fracture strain reflects flexibility, and fatigue stability is used to evaluate structural retention under repeated stress conditions.

5.3 Microstructural evaluation

(1) Pore structure

Scanning electron microscopy can be used to observe gel pore size, pore wall thickness, and pore connectivity. Networks with smaller and more uniform pore size distribution are usually favorable for improving water-holding capacity and mechanical stability.

(2) Phase distribution state

Confocal laser scanning microscopy is suitable for observing the spatial distribution of proteins, polysaccharides, or lipid components in composite gels. Obvious phase separation reduces gel continuity and increases the risk of syneresis and fracture.

(3) Surface morphology

Atomic force microscopy can be used to analyze gel surface roughness, microdomain aggregation, and local network density, and is suitable for comparing microstructural differences under different crosslinking strengths.

5.4 Molecular interaction evaluation

(1) Transglutaminase system

A decrease in free amino group content, an increase in high-molecular-weight aggregates, and changes in electrophoretic band migration can be used to determine the degree of isopeptide bond crosslinking and protein polymerization state.

(2) Oxidase systems

Dityrosine fluorescence, phenolic hydroxyl consumption, sulfhydryl changes, and the degree of protein carbonylation can be used to evaluate oxidative crosslinking strength and the risk of excessive oxidation.

(3) Composite systems

Protein–polysaccharide or protein–polyphenol gels can be analyzed by infrared spectroscopy, fluorescence spectroscopy, particle size distribution, and zeta potential to evaluate component interactions. Hydrogen bonding, hydrophobic interactions, electrostatic complexation, and covalent crosslinking jointly affect the network morphology of composite gels.

6 Application performance evaluation

6.1 Water-holding and storage stability

(1) Water-holding capacity

Water-holding capacity determines gel mouthfeel, appearance, and shelf-life stability. Enzymatic crosslinking can increase network density and restrict free water migration, thereby reducing syneresis rate. Common indicators include centrifugation water loss rate, water-holding capacity, and low-field NMR water distribution.

(2) Freeze-thaw stability

Freeze-thaw cycles can cause ice crystal growth and gel network destruction. Moderate crosslinking can enhance gel resistance to freeze-thaw processes, while polysaccharide compounding helps improve water distribution and reduce post-freeze-thaw syneresis.

6.2 Thermal stability

(1) Thermal transition behavior

Differential scanning calorimetry can be used to observe the thermal transition temperature and enthalpy changes of protein gels. After enhancement by enzymatic crosslinking, the gel network usually has improved resistance to thermal disturbance.

(2) Thermogravimetric behavior

Thermogravimetric analysis can be used to evaluate water loss, thermal degradation stages, and residual mass changes in gels. For high-water-content hydrogels, early-stage weight loss can reflect the water-binding state.

(3) Structural retention after heat treatment

Texture retention rate after heating, heat-induced syneresis, and volume shrinkage can be used to determine the processing compatibility of gels under steaming, sterilization, baking, or reheating conditions.

6.3 Digestion and nutritional release

(1) Protein digestibility

Enzymatic crosslinking affects the recognition and hydrolysis of protein substrates by digestive enzymes. Moderate crosslinking can delay protein digestion and form sustained-release characteristics, whereas excessive crosslinking may reduce hydrolysis degree, amino acid release efficiency, and nutritional accessibility.

(2) Active substance release

When used for delivery systems, encapsulation efficiency, release curves, gastrointestinal stability, and active retention rate need to be evaluated. The pore size, crosslinking density, and degradation rate of the protein network are key factors affecting release behavior.

6.4 Biocompatibility

(1) Cell behavior

Medical hydrogels, tissue repair materials, and cell delivery systems need to be evaluated for cell viability, cell adhesion, proliferation, and migration. Gel hardness, pore structure, and degradation rate affect the growth state of cells in the material.

(2) Residual risk

Peroxidase systems need to pay attention to hydrogen peroxide residues. Crosslinking efficiency, residual oxidants, and cytocompatibility should be optimized simultaneously to avoid oxidative stress risk caused by rapid gelation.

7 Related products and experimental selection

Product category	Product name	CAS No.	Role in the system	Key points for selection
Crosslinking enzyme	Transglutaminase	80146-85-6	Catalyzes the formation of isopeptide bonds between glutamine and lysine residues	Preferred for enhancement of food protein gels
Oxidase	Laccase	80498-15-3	Catalyzes oxidation of phenolic groups and induces coupling crosslinking	Suitable for protein–polyphenol gels and antioxidant systems
Oxidase	Tyrosinase	9002-10-2	Catalyzes tyrosine oxidation to form quinone intermediates	Suitable for wet-adhesive and oxidatively crosslinked hydrogels
Peroxidase	Horseradish peroxidase	9003-99-0	Catalyzes phenolic crosslinking in the presence of hydrogen peroxide	Suitable for rapid in situ gelation and injectable hydrogels
Oxidation-regulating enzyme	Glucose oxidase	9001-37-0	Generates hydrogen peroxide and participates in oxidative crosslinking regulation	Can be used in mild in situ oxidant-generating systems
Oxidation-regulating enzyme	Catalase	9001-05-2	Decomposes hydrogen peroxide and reduces oxidative residues	Suitable for controlling hydrogen peroxide residue risk
Protein substrate	Gelatin	9000-70-8	Provides a flexible protein skeleton and biocompatible network	Suitable for hydrogels, film materials, and cell carriers
Protein substrate	Casein	9000-71-9	Provides milk protein crosslinking sites and gel structural units	Suitable for milk protein gels and composite food systems
Protein substrate	Sodium caseinate	9005-46-3	Provides emulsification stability and crosslinkable protein components	Suitable for emulsion gels and composite gels
Protein substrate	Bovine serum albumin	9048-46-8	Provides a model protein skeleton and reaction sites	Suitable for mechanistic studies and model gel construction
Protein substrate	Collagen	9007-34-5	Provides cytocompatibility and tissue scaffold structure	Suitable for tissue engineering and wound repair gels
Protein substrate	Soy protein	9010-10-0	Provides a plant protein gel skeleton	Suitable for plant-based gels and composite protein systems
Protein substrate	Whey protein	84082-51-9	Provides a milk protein gel skeleton and nutritional protein components	Suitable for high-protein foods and nutritional delivery gels
Polysaccharide excipient	Sodium alginate	9005-38-3	Regulates water retention, viscosity, and network support	Suitable for protein–polysaccharide composite gels
Polysaccharide excipient	Chitosan	9012-76-4	Provides amino reaction sites and film-forming properties	Suitable for antibacterial gels, composite films, and adhesive materials
Polysaccharide excipient	Pectin	9000-69-5	Regulates gel water-holding capacity and stability in acidic systems	Suitable for food gels and composite network regulation
Polysaccharide excipient	Sodium carboxymethyl cellulose	9004-32-4	Increases system viscosity and improves water distribution	Suitable for optimization of water retention and freeze-thaw stability
Polysaccharide excipient	Sodium hyaluronate	9067-32-7	Provides water-holding capacity and biocompatibility	Suitable for medical hydrogels and cell delivery systems
Polysaccharide excipient	Dextran	9004-54-0	Provides a hydrophilic skeleton and regulates swelling performance	Suitable for hydrogel modification and delivery systems
Polysaccharide excipient	Agarose	9012-36-6	Forms a thermoreversible physical gel network	Suitable for model gels and composite network support
Polyphenol component	Tannic acid	1401-55-4	Participates in oxidative coupling and enhances antioxidant performance	Suitable for protein–polyphenol composite gels
Polyphenol component	Gallic acid	149-91-7	Provides phenolic hydroxyl reaction sites and antioxidant activity	Suitable for functional gels and active delivery systems
Polyphenol component	Caffeic acid	331-39-5	Provides phenolic hydroxyl structure and participates in oxidative coupling	Suitable for antioxidant gels and interfacial functional materials
Polyphenol component	Catechin	154-23-4	Provides polyphenol structure and antioxidant activity	Suitable for protein–polyphenol interaction studies
Amino acid substrate	L-Tyrosine	60-18-4	Provides a tyrosine reaction model and oxidative crosslinking sites	Suitable for mechanistic studies of tyrosinase systems
Amino acid substrate	L-Lysine	56-87-1	Provides an amino donor model	Suitable for validation of transglutaminase reactions
Amino acid substrate	L-Glutamine	56-85-9	Provides an acyl donor model	Suitable for mechanistic studies of isopeptide bond formation
Functional modifier	Dopamine hydrochloride	62-31-7	Introduces catechol structure and improves adhesion and crosslinking ability	Suitable for wet-adhesive hydrogels and surface functionalization

7.2 Application system matching table

Application target	Recommended crosslinking system	Key material combination	Core evaluation indicators
Food gel enhancement	Transglutaminase system	Whey protein, sodium caseinate, soy protein	Hardness, elasticity, water-holding capacity, syneresis rate
Construction of antioxidant gels	Laccase or tyrosinase system	Protein, polyphenols, chitosan	Antioxidant activity, gel strength, color stability
Preparation of injectable hydrogels	Horseradish peroxidase system	Gelatin, phenol-modified polysaccharides, hydrogen peroxide	Gelation time, rheological properties, cytocompatibility
Construction of wet-adhesive materials	Tyrosinase or peroxidase system	Gelatin, dopamine hydrochloride, polyphenols	Adhesive strength, swelling rate, degradation behavior
Active substance delivery	Transglutaminase or oxidase system	Protein, polysaccharides, active ingredients	Encapsulation efficiency, release curve, digestive stability
Improvement of freeze-thaw stability	Transglutaminase–polysaccharide synergistic system	Protein, sodium alginate, pectin	Freeze-thaw syneresis rate, texture retention rate, water distribution
Optimization of plant-based matrix structure	Transglutaminase composite system	Soy protein, pea protein, milk protein or polysaccharides	Elasticity, cuttability, chewiness, network uniformity
Construction of double-network hydrogels	Enzymatic crosslinking–ionic crosslinking synergistic system	Gelatin, sodium alginate, calcium chloride	Compressive modulus, swelling rate, network stability
Control of oxidative residues	Peroxidase–catalase regulation system	Horseradish peroxidase, hydrogen peroxide, catalase	Gelation rate, residual oxidants, cell viability
Stability of emulsion gels	Transglutaminase–milk protein system	Sodium caseinate, whey protein, oil phase	Emulsion stability, gel strength, oil separation rate
Wound repair gels	Oxidase or peroxidase system	Gelatin, collagen, sodium hyaluronate	Adhesiveness, moisture retention, cell migration
Model mechanistic studies	Single-enzyme–model protein system	Bovine serum albumin, L-lysine, L-glutamine	Degree of crosslinking, molecular weight change, reaction kinetics

The key points in the application of enzymatic protein crosslinking systems lie in enzyme system selection, substrate adaptation, and matching of evaluation indicators. By reasonably controlling the degree of crosslinking and the structure of the composite network, the application value of protein gels in food processing, active delivery, and biomaterials can be further improved.