Enzyme-catalyzed Crosslinking Research Articles

Enhancing Alkaline Protease Stability through Enzyme-Catalyzed Crosslinking and Its Application in Detergents

Enzymatic additives, particularly alkaline proteases, play a crucial role in enhancing detergent effectiveness against protein-based stains. Despite advancements in enzyme stabilization techniques, there is a need for innovative strategies to further improve protease stability in laundry detergents. However, research exploring the utilization of substrate imprinting technology to achieve this objective remains limited. Therefore, this study aims to enhance the stability of alkaline proteases in laundry detergents by employing casein as an imprinting substrate and utilizing transglutaminase-mediated (TGase) crosslinking to modify proteases 102 and 306. The optimal temperature, pH, and thermal stability of the modified alkaline proteases 102 and 306 showed no significant changes. However, these two modified alkaline proteases exhibited varying degrees of improvement in stability among the 14 detergent additives tested. Under 40 °C incubation for 24 h, the relative enzyme activity of modified alkaline protease 102 increased approximately 1.4–15-fold in AEO-9, BS-12, CMI, APG, FMEE, and SOE, while the relative enzyme activity of modified alkaline protease 306 increased approximately 1.2–3.7-fold across different additives (FMEE, AEO-9, BS-12, SOE, FAA, and AEC-9Na). These modified proteases demonstrated improved stability and wider applicability across commercial detergent formulations available. Integrated into standard laundry detergent at a 1:7 ratio before and after modification, they effectively removed protein stains from the cotton fabric after 24 h of 40 °C incubation. These findings provide insights into more effective stain-removal techniques.

A hyaluronic acid / chitosan composite functionalized hydrogel based on enzyme-catalyzed and Schiff base reaction for promoting wound healing

The healing of full-thickness skin defect has been a clinical challenge. Hydrogels with multiple functions inspired by extracellular matrix are expected to be used as wound dressing. In this paper, dopamine-grafted oxidized hyaluronic acid was blended with quaternary ammonium chitosan to form a composite functionalized hydrogel by enzyme-catalyzed cross-linking and Schiff base reaction. The hydrogel has convenient preparation, good biocompatibility, antibacterial and antioxidant, high adhesion and self-healing properties. The results in vivo show that the hydrogel can effectively close the wound and accelerate the speed of wound healing by up-regulating the expression of angiogenic protein and promoting the distribution of collagen deposition more uniform and regular. It is expected that this composite functionalized hydrogel dressing has great potential in wound regeneration.

Comparison of Silk Hydrogels Prepared via Different Methods.

Silk fibroin (SF) hydrogels have garnered extensive attention in biomedical materials, owing to their superior biological properties. However, the challenges facing the targeted silk fibroin hydrogels involve chemical agents and shortfalls in performance. In this study, the silk fibroin hydrogels were prepared in different ways: sonication induction, chemical crosslinking, photopolymerization, and enzyme-catalyzed crosslinking. The SF hydrogels derived from photopolymerization exhibited higher compressive properties, with 124 Kpa fracture compressive stress and breaks at about 46% compression. The chemical crosslinking and enzyme-catalyzed silk fibroin hydrogels showed superior toughness, yet sonication-induced hydrogels showed brittle performance resulting from an increase in silk II crystals. The chemical-crosslinked hydrogel demonstrated lower thermostability due to the weaker crosslinking degree. In vitro, all silk fibroin hydrogels supported the growth of human umbilical vein endothelial cells, as the cell viability of hydrogels without chemical agents was relatively higher. This study provides insights into the formation process of silk fibroin hydrogels and optimizes their design strategy for biomedical applications.

Injectable hydrogels of enzyme-catalyzed cross-linked tyramine-modified gelatin for drug delivery

Enzymatically catalyzed cross-linking is a hydrogel fabrication method that generally is considered to have lower cytotoxicity than traditional chemical cross-linking methods. In order to optimize the properties of injectable hydrogels and expand their applications, an enzyme-catalyzed cross-linked injectable hydrogel was designed. The tyramine-modified gelatin (G-T) was formed into a stable injectable hydrogel by the combination of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) catalysis. 1H NMR spectroscopy was used to demonstrate the successful modification of gelatin by tyramine. The surface morphology of the prepared hydrogels was characterized jointly by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Rheological tests demonstrated the tunable mechanical strength, formation kinetics, shear thinning and good self-recovery properties of the hydrogels. In addition, the hydrogels can be formed into various shapes by injection. The hydrogel network structure is complex and interlaced, as such it is suitable to encapsulate drugs for controlled release. The drug release from the prepared hydrogels followed the Peppas–Sahlin model and belonged to Fickian diffusion. This study constructed injectable hydrogels through the enzyme-catalyzed cross-linking of modified gelatin and applied the hydrogels for drug release, which is expected to expand the application in biomedical fields.

Structural characteristics, emulsifying and foaming properties of laccase-crosslinked bovine α-lactalbumin mediated by caffeic acid

In this study, laccase (LA) was chosen as a commonly employed enzyme to research the impacts of enzyme-catalyzed crosslinking (polymerization) on the structural, emulsifying and foaming properties of bovine α-lactalbumin (α-LA) in the presence of caffeic acid (CA). High pressure size exclusion chromatography (HPSEC) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated the formation of α-LA aggregates (dimers, trimers, and polymers) by LA-induced crosslinking with CA, consistent with the particle size distribution results. LA-induced treatment exhibited pronounced influences on the conformation, secondary and tertiary structure of α-LA, as confirmed by circular dichroism (CD), and fluorescence spectroscopy. For α-LA-LA-CA, α-helix content was progressively decreased from 33.3% (0 h) to 24.7% (24 h). LA-catalyzed crosslinking resulted in the exposure of interior hydrophobic groups, and the increase of zeta-potential values of α-LA. Apparent viscosity of α-LA was considerably enhanced by LA-induced crosslinking with CA. The emulsifying activity and stability of α-LA can be considerably improved with LA-catalyzed crosslinking with CA except at 24 h incubation time. Compared with controls, LA-catalyzed polymerizations of α-LA molecules with CA led to the increase of foaming activity and stability regardless of incubation time. After 24 h incubation, the foaming stability (FS) of α-LA was 81.0%, whereas the value was 88.0% for α-LA-LA-CA. This research indicates that LA-induced modification possesses great potential in ameliorating the emulsifying and foaming capacities of α-LA. • Laccase led to the significant polymerization of α-lactalbumin with caffeic acid. • Laccase-catalyzed crosslinking led to the changes of the structure of α-lactalbumin. • Laccase-catalyzed crosslinking increased the emulsifying properties of α-lactalbumin. • Laccase-catalyzed crosslinking increased the foaming activity of α-lactalbumin. • Laccase-crosslinked α-lactalbumin aggregates showed potential as functional ingredients.

Injectable enzyme-catalyzed crosslinking hydrogels as BMSCs-laden tunable scaffold for osteogenic differentiation

Bone defects caused by trauma or tumor are a significant challenge in clinical practice. Hydrogel-based tissue engineering has been considered an effective strategy. This study successfully formed a series of injectable hydrogels by enzyme-catalyzed crosslinking hyaluronic acid-tyramine (HA-TA) and sodium alginate-tyramine (ALG-TA) under physiological conditions in the presence of both horseradish peroxidase and hydrogen peroxide. The morphology, mechanical properties, swelling properties, and biodegradation properties of hydrogels were investigated. The results showed that the mechanical properties, swelling properties and biodegradation of HA/ALG hydrogels varied with the precursor solution concentration. Furthermore, the proliferation and osteogenic differentiation of BMSCs within the HA/ALG hydrogels were evaluated in vitro. The results illustrated that the hydrogels could offer an excellent microenvironment for BMSCs growth and promote osteogenic differentiation. Therefore, the injectable hydrogels can be used as an effective 3 D scaffold for bone repair and regeneration.

Application of injectable silk fibroin/graphene oxide hydrogel combined with bone marrow mesenchymal stem cells in bone tissue engineering

The stem cell composite hydrogel scaffold was constructed following its application in bone tissue engineering. For this study, different silk fibroin (SF) hydrogels were prepared by enzyme-catalyzed crosslinking of SF and graphene oxide (GO), and their physicochemical properties and biocompatibility were examined. It has been observed that with the incorporation of GO, the pore size of the scaffold became more regular, and the porosity changed negligibly and also exhibited a stable silk II structure with excellent mechanical properties. Moreover, bone marrow stromal cells (BMSCs) were encapsulated in the hydrogel compared to SF hydrogels either in the absence or in the presence of high concentrations of GO. It has been noted that the cells in the compound gel at a low concentration of GO had shown a better growth capability, proliferation and osteogenic differentiation. Therefore, such injectable BMSCs hydrogel is an excellent biomedical material, possessing potential applications in the field of bone tissue engineering.

Injectable hydrogels from enzyme-catalyzed crosslinking as BMSCs-laden scaffold for bone repair and regeneration

Bone-marrow-derived mesenchymal stem cells possess great potential for tissue engineering and regenerative medicine. In the work, an injectable BMSCs-laden hydrogel system was formed by enzyme-catalyzed crosslinking of hyaluronic acid-tyramine and chondroitin sulfate-tyramine in the presence of hydrogen peroxide and horseradish peroxidase, which was used as a 3D scaffold to explore the behavior of the mesenchymal stem cells. Afterward, the gelation rate, mechanical properties, as well as the degradation process of the scaffold were well characterized and optimized. Furthermore, bone morphogenetic protein-2 was encapsulated in the scaffold, which was used to improve the osteogenic properties. The results illustrated that such a BMSCs-laden hydrogel not only offered a proper microenvironment for the adhesion, proliferation and differentiation of mesenchymal stem cells in vitro, but also promoted bone regeneration in vivo. Therefore, this injectable BMSCs-laden hydrogel may serve as an efficient 3D scaffold for bone repair and regeneration.

Mussel-inspired in situ forming adhesive hydrogels with anti-microbial and hemostatic capacities for wound healing.

All kinds of commercially available wound dressings are clinically used as fleshly obstacles and therapeutic materials in opposition to microbial incursion. Few researches focused on effective-bleeding and anti-bacteria at the same time. In order to better solve this problem, two hydrogels were synthetized in this study. One is phosphate buffer solution-activated dopamine-modified-γ-poly glutamic acid (PBS-PD) hydrogel, the other one is cirsium setosum extracts-activated dopamine-modified-γ-poly glutamic acid (CSE-PD) hydrogel. The two hydrogels are prepared by applying an enzyme-catalyzed crosslinking means in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The chemical structures were characterized through 1H-NMR and FT-IR. In conclusion, both PBS-PD and CSE-PD hydrogels exhibit superior tissue adhesion properties, and remarkable anti-infection quality. In addition, these two hydrogels manifest prominent hemostatic efficiency. The bio adhesion performance can achieve 30 kPa, meanwhile the CSE-PD hydrogels show good germicidal properties, and the antibacterial rate can reach 98%. The hydrogels could reduce blood loss without any obvious side effect, and present a new prospect in the field of hemostasis rapidly.

Injectable Polypeptide Hydrogels with Tunable Microenvironment for 3D Spreading and Chondrogenic Differentiation of Bone-Marrow-Derived Mesenchymal Stem Cells.

Bone-marrow-derived mesenchymal stem cells (BMSCs) possess vast potential for tissue engineering and regenerative medicine. In this study, an injectable hydrogel comprising poly(l-glutamic acid)-graft-tyramine (PLG-g-TA) with tunable microenvironment was developed via enzyme-catalyzed cross-linking and used as an artificial extracellular matrix (ECM) to explore the behaviors of BMSCs during three-dimensional (3D) culture. It was found that the mechanical property, porous structure as well as degradation process of the hydrogels could be tuned by changing the copolymer concentration. The PLG-g-TA hydrogels showed good cytocompatibility in vitro. After being subcutaneously injected into the back of rats, the hydrogels degraded gradually within 8 weeks and exhibited good biocompatibility in vivo. BMSCs were then encapsulated in the polypeptide-based hydrogels with different copolymer concentration to investigate the influence of 3D matrix microenvironment on stem cell behaviors. It is intriguing to note that the BMSCs within the 2% hydrogel showed a well-spread morphology after 24 h and a higher proliferation rate during 7 days of culture, in contrast to a rounded morphology and lower proliferation rate of BMSCs in the 4% hydrogel. Furthermore, the hydrogels with different microenvironment also regulated the matrix biosynthesis and the gene expression of BMSCs. After incubation in the 2% hydrogel for 4 weeks, the BMSCs produced more type II collagen and expressed higher amounts of chondrogenic markers, compared to the cells in the 4% hydrogel. Therefore, the PLG-g-TA hydrogels with tunable microenvironment may serve as an efficient 3D platform for guiding the lineage specification of BMSCs.

Enzyme catalyzed cross-linking of spruce galactoglucomannan improves its applicability in barrier films

Hemicelluloses are one of the main constituents of plant cell walls and thereby one of the most abundant biopolymers on earth. They can be obtained as by-products from different wood based processes, most importantly from the mechanical pulping. Hemicelluloses have interesting properties in e.g. barrier film applications. However, their relatively low molecular weight after isolation and co-extraction with lignin has limited their use. In this work, we present a novel technique for increasing the molecular weight of different wood hemicelluloses from mechanical pulping process waters as well as from pre-hydrolysis extracts. This is achieved by enzyme-catalyzed cross-linking of aromatic moieties bound to the hemicelluloses. The cross-linking treatment resulted in significantly improved mechanical properties in barrier films made with spruce galactoglucomannan. To our knowledge, this is the first time that wood hemicelluloses have been cross-linked by utilizing the bound aromatic moieties and creates new possibilities for utilizing this raw material source.

Enzyme‐Catalyzed Crosslinking in a Partly Frozen State: A New Way to Produce Supermacroporous Protein Structures

In this study a new way to produce supermacroporous protein structures was investigated. Enzyme-mediated crosslinking of gelatin or casein was performed in a partly frozen state, which yielded stable, protein-based cryogels. The reaction kinetics for the formation of cryogels were found to be fairly slow, most likely due to the low temperature (-12 °C) used or due to an increased viscosity owing to the cryo-concentration taking place. The produced cryogels were characterized with regards to their physical properties and in vitro degradation. Furthermore, cryogels produced from gelatin and casein were evaluated as potential scaffolds by fibroblast cultivation to confirm their in vitro biocompatibility. Gelatin- and casein-based scaffolds both supported cell proliferation and migration through the scaffold.

Oxygen-scavenging coatings and films based on lignosulfonates and laccase

Laccase and lignosulfonates were included in coating colors and embedded in latex-based or starch-based films and coatings on foil or board. After 6 days at 23 °C and 100% relative humidity, the oxygen content in airtight chambers decreased from 1.0% (synthetic gas consisting of 99% N(2) and 1% O(2)) to 0.3% in the presence of board coated with lignosulfonate and laccase, while the oxygen content remained unchanged in control experiments without enzyme. The water stability of lignosulfonate-containing latex-based coatings and starch-based films was improved after laccase-catalyzed oxidation of lignosulfonates, which indicates polymerization to products with lower solubility in water. Furthermore, the E' modulus of starch-based films increased with 30%, which indicates laccase-catalyzed polymerization of lignosulfonates resulting in increased stiffness of the film. The results suggest that laccases and lignosulfonates can be used as an oxygen-scavenging system in active packaging and that enzyme-catalyzed polymerization of lignosulfonates contributes to improved water stability and mechanical properties.

Self-attaching and cell-attracting in-situ forming dextran-tyramine conjugates hydrogels for arthroscopic cartilage repair

Small cartilage defects are frequently treated with debridement or left untreated, predisposing to early onset osteoarthritis. We propose to fill these defects with a cell-free injectable hydrogel comprising dextran-tyramine conjugates (Dex-TA) that can be applied during arthroscopic procedures. In this study, we report on the adhesion mechanism between cartilage and Dex-TA hydrogels and enhancement of cell ingrowth by incorporation of Heparin-tyramine (Hep-TA) conjugates. The enzyme-catalyzed crosslinking reaction of Dex-TA and Hep-TA hydrogels is based on covalent bonding of hydroxyphenyl residues. We hypothesized that this reaction results in covalent bonding of the hydroxyphenyl residues in Dex-TA and Hep-TA to tyrosine residues in cartilage matrix proteins. The involvement of TA residues was confirmed by modelling the enzymatic reaction occurring during gelation. The mechanical analysis indicated that higher tyramine content led to stronger binding. Interfacial cartilage-hydrogel morphology and Raman spectroscopy demonstrated collagens’ reorganization and evidenced the coupling of TA to tyrosine residues in collagen. Moreover, the addition of Hep-TA induced cell recruitment. Collectively, in vitro and ex vivo functional studies evidenced the covalent bonding of TA-containing hydrogels to tyrosine residues in cartilaginous matrix proteins. Moreover, the cell-attracting ability of these hydrogels could be explored to guide tissue repair in focal cartilage defects, preventing or delaying the onset osteoarthritis.

Kinetics of Enzyme-Catalyzed Cross-Linking of Feruloylated Arabinan from Sugar Beet

Ferulic acid (FA) groups esterified to the arabinan side chains of pectic polysaccharides can be oxidatively cross-linked in vitro by horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide (H(2)O(2)) to form ferulic acid dehydrodimers (diFAs). The present work investigated whether the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased as a result of the enzymatic oxidation treatment with HRP and H(2)O(2). At equimolar levels of FA (0.0025-0.05 mM) in the arabinan samples, the initial rates of the HRP-catalyzed cross-linking of the longer chain arabinans were slower than those of the shorter chain arabinans. The lower initial rates may be the result of the slower movement of larger molecules coupled with steric phenomena, making the required initial reaction of two FAs on longer chain arabinans slower than on shorter arabinans.

Biofabrication with Biopolymers and Enzymes: Potential for Constructing Scaffolds from Soft Matter

Regenerative medicine will benefit from technologies capable of fabricating soft matter to have appropriate architectures and that provide the necessary physical, chemical and biological cues to recruit cells and guide their development. The goal of this report is to review an emerging set of biofabrication techniques and suggest how these techniques could be applied for the fabrication of scaffolds for tissue engineering. Electrical potentials are applied to submerged electrodes to perform cathodic and anodic reactions that direct stimuli-responsive film-forming polysaccharides to assemble into hydrogel films. Standard methods are used to microfabricate electrode surfaces to allow the electrical signals to be applied with spatial and temporal control. The enzymes mushroom tyrosinase and microbial transglutaminase are used to catalyze macromolecular grafting and crosslinking of proteins. Electrodeposition of the polysaccharides chitosan and alginate allow hydrogel films to be formed in response to localized electrical signals. Co-deposition of various components (e.g., proteins, vesicles and cells), and subsequent electrochemical processing allow the physical, chemical and biological activities of these films to be tailored. Enzymatic processing allows for the generation of stimuli-responsive protein conjugates that can also be directed to assemble in response to imposed electrical signals. Further, enzyme-catalyzed crosslinking of gelatin allows replica molding of soft matter to create hydrogel films with topological structure. Biofabrication with biological materials and mechanisms provides new approaches for soft matter construction. These methods may enable the formation of tissue engineering scaffolds with appropriate architectures, assembled cells, and spatially organized physical, chemical and biological cues.

Formation of fibrinogen-based hydrogels using phototriggerable diplasmalogen liposomes.

We report the triggered release of Ca2+ from liposomal compartments to induce rapid gelation of protein-based hydrogels. Phototriggerable liposomes were designed by entrapping CaCl(2) within liposomes composed of 38:57:5 diplasmenylcholine (DPPlsC):disteroylphosphatidylcholine (DSPC):bacteriochlorophyll (Bchl). These liposomes release >80% of their entrapped Ca2+ within 15 min when irradiated at 800 nm (800 mW/cm2). A precursor solution, containing liposomes suspended in aqueous human fibrinogen and transglutaminase (TGase), remained fluid for several hours in the dark, but gelled rapidly when exposed to 800 nm excitation, as a result of photosensitized Ca2+ release and TG-induced fibrinogen cross-linking. TGase and hrFXIII activities, determined using a fluorimetric dansylcadaverine assay, were found to depend strongly on irradiation time and reaction temperature. SDS-PAGE of the photolyzed reaction mixture revealed that gelation arises from enzyme-catalyzed cross-linking of predominately the alpha and gamma chains of fibrinogen. This approach to the phototriggered formation of protein hydrogels creates new opportunities for biomaterials applications in drug delivery, tissue engineering, and wound healing.

Triggered release of calcium from lipid vesicles: a bioinspired strategy for rapid gelation of polysaccharide and protein hydrogels

The bioinspired strategy of triggered release of Ca 2+ from liposomal compartments was used to induce rapid gelation of polysaccharide and protein-based hydrogels. Thermally triggerable liposomes were designed by entrapping CaCl 2 within liposomes constructed of 90% dipalmitoylphosphatidylcholine and 10% dimyristoylphosphatidylcholine. These liposomes released greater than 90% of entrapped Ca 2+ when heated to 37°C. A precursor fluid containing liposomes suspended in aqueous sodium alginate remained fluid for several days at room temperature but gelled rapidly when heated to 37°C, as a result of Ca 2+ release and formation of crosslinked Ca-alginate. Alternatively, thermally triggered Ca 2+ release from liposomes was used to activate enzyme-catalyzed crosslinking of proteins to form hydrogels. A mixture of Ca-loaded liposomes, fibrinogen, and a Ca 2+-dependent transglutaminase enzyme (either human recombinant FXIII or guinea pig liver transglutaminase) remained fluid indefinitely when stored at room temperature, but gelled rapidly when heated to 37°C. SDS-PAGE of the reaction mixture revealed that gelation was due to enzymatic crosslinking of the α and γ chains of fibrinogen, and oscillating rheometry revealed gel formation within 10 min of heating to 37°C. This new approach may be useful for developing rapidly gelling injectable biomaterials that can be stored at room temperature and injected in a minimally invasive manner into a body tissue or cavity, upon which rapid solidification would occur. This versatile bioinspired strategy could be utilized for the delivery of biomaterials for tissue repair and reconstruction, and local site-directed drug delivery.

The Intermediate Filament Protein, Vimentin, in the Lens Is a Target for Cross-linking by Transglutaminase

Mere addition of Ca2+ to a lens cortical homogenate (bovine) generates a series of products composed of a variety of high molecular weight vimentin species. The Ca2+-induced cross-linking of this cytoskeletal element seems to be mediated by the intrinsic transglutaminase of lens, because the reaction could be blocked at the monomeric state of vimentin by the inclusion of small synthetic substrates of the enzyme dansylcadaverine or dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val. These compounds are known to compete against the Gln or Lys functionalities of proteins that would participate in forming the Nepsilon(gamma-glutamyl)lysine protein-to-protein cross-links. The cytosolic transglutaminase-catalyzed reactions could be reproduced with purified bovine lens vimentin and also with recombinant human vimentin preparations. Employing the latter system, we have titrated the transglutaminase-reactive sites of vimentin and, by sequencing the dansyl-tracer-labeled segments of the protein, we have shown that residues Gln453 and Gln460 served as acceptor functionalities and Lys97, Lys104, Lys294, and Lys439 as electron donor functionalities in vimentin. The transglutaminase-dependent reaction of this intermediate filament protein might influence the shape and plasticity of the fiber cells, and the enzyme-catalyzed cross-linking of vimentin, in conjunction with other lens constituents, may contribute to the process of cataract formation.

Enzyme-catalyzed Cross-linking and Degradation of Myosin Heavy Chain in Walleye Pollack Surimi Paste during Setting

Walleye pollack surimi paste was incubated (set) at 25, 35, 45, and 55°C in the presence of either transglutaminase (TGase) inhibitor or protease inhibitor. In the absence of inhibitors, cross-linking of myosin heavy chains and formation of 150 kDa component occurred at the same time during the incubation at 25°C. Inhibition of endogenous TGase resulted in complete suppression of myosin cross-linking, while it did not suppress the formation of 150 kDa component. The cross-linking was also depressed above 45°C due to the inactivation of TGase. On the other hand, the addition of E-64, a cysteine protease inhibitor, suppressed completelyand partially the formation of 150 kDa component at 25 and 55°C, respectively. Together with these results, the 150 kDa component might be a proteolytic fragment of myofibrillar proteins, possibly myosin. The proteolysis as well as the cross-linking of myosin during the setting affected the textural properties of final cooked gels.