Isopeptide Research Articles

Functional decoration of elastin-like polypeptides-based nanoparticles with a modular assembly via isopeptide bond formation.

Temperature-responsive elastin-like polypeptides (ELPs) exhibit a low critical solution temperature-type phase transition and offer potential as useful materials for the construction of nanoparticles. Herein, we developed a novel decoration method for ELP-based nanoparticles via isopeptide bond formation with the SnoopTag/SnoopCatcher system that is not affected by the heating process required for particle formation. A mixture of a fusion protein of ELP and poly(aspartic acid) (poly(D)), known as ELP-poly(D), and ELP-poly(D) fused with SnoopCatcher (ELP-poly(D)-SnC) formed protein nanoparticles as a result of the temperature responsiveness of ELP, with the resultant nanoparticles displaying the SnoopCatcher binding domain on their surfaces. In the present study, two model proteins fused to SnoopTag were displayed on the surfaces of protein nanoparticles constructed from ELP-poly(D)-SnC and ELP-poly(D). The model proteins are enhanced green fluorescent protein (EGFP) and Renilla luciferace (Rluc), which exhibits luminescent capability and weak thermostability, respectively. EGFP on the particle surface was found to retain 48.7% activity, while Rluc exhibited almost full activity, as calculated from the binding efficiency and nanoparticle activities recovered after purification. ELP-based nanoparticles containing the SnoopTag/SnoopCatcher system offer the opportunity for particle decoration with a wide range of functional proteins via isopeptide bond formation.

Type IV pili-associated secretion of a biofilm matrix protein from Clostridium perfringens that forms intermolecular isopeptide bonds.

For bacteria to secrete folded proteins to the environment, they have to overcome the physical barriers of an outer membrane in Gram-negative bacteria and the thick peptidoglycan layer in Gram-positive bacteria. One mechanism to do this is the use of a Type II secretion system in Gram-negative bacteria, which has a similar structure as type IV pili and is modeled to act as a piston that pumps folded proteins through the outer membrane to the environment. Clostridium perfringens , like all or most all of the clostridia, has type IV pili and, in fact, has two sets of pilus-associated genes. Here we present evidence that C. perfringens uses one set of pilus genes to secrete a biofilm associated protein and may be responsible for secreting the main biofilm protein BsaA. We show that BsaA monomers are, unlike most other biofilm matrix proteins, linked by intermolecular isopeptide bonds, enhancing the physical strength of BsaA fibers.

SUMO: A new perspective to decipher fibrosis.

Fibrosis is characterized by excessive extracellular matrix (ECM) deposition resulting from dysregulated wound healing and connective tissue repair mechanisms. Excessive accumulation of ECM leads to fibrous tissue formation, impairing organ function and driving the progression of various fibrotic diseases. Recently, the role of small ubiquitin-like modifiers (SUMO) in fibrotic diseases has attracted significant attention. SUMO-mediated SUMOylation, a highly conserved posttranslational modification, participates in a variety of biological processes, including nuclear-cytosolic transport, cell cycle progression, DNA damage repair, and cellular metabolism. Conversely, SUMO-specific proteases cleave the isopeptide bond of SUMO conjugates, thereby regulating the deSUMOylation process. Mounting evidence indicates that SUMOylation and deSUMOylation regulate the functions of several proteins, such as Smad3, NF-κB, and promyelocytic leukemia protein, which are implicated in fibrotic diseases like liver fibrosis, myocardial fibrosis, and pulmonary fibrosis. This review summarizes the role of SUMO in fibrosis-related pathways and explores its pathological relevance in various fibrotic diseases. All evidence suggest that the SUMO pathway is important targets for the development of treatments for fibrotic diseases.

Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units

During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the “mispairing problem.” Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce “bsAb by external pairing and excision” (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells.

PH-Controlled Chemoselective Rapid Azo-Coupling Reaction (CRACR) Enables Global Profiling of Serotonylation Proteome in Cancer Cells.

Serotonylation has been identified as a novel protein posttranslational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main "writer" enzyme for this PTM, and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction. Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Effect of transglutaminase on the structure, properties and oil absorption of wheat flour

The structure, properties, as well as the oil absorption characteristics of wheat flour (WF) treated with varying concentrations of transglutaminase (TG) (0 U/g ∼ 50 U/g) were characterized. The content of free amino groups in WF modified by TG (TG-WF) decreased and protein aggregated. The isopeptide bonds and disulfide bonds played important roles in protein crosslinking. The thermal stability, the peak viscosity after gelatinization and protein secondary structure stability of TG-WF were improved. In addition, the oil absorption and surface oil content of TG-WF after frying were reduced. TG enhanced the protein-protein interactions in WF, so that protein played barrier roles in the process of high-temperature frying, protecting the starch particles covered by them from the infiltration of oil, thus reducing the oil absorption of TG-WF during frying. Among them, the oil content of TG-WF-30 U/g after frying was the lowest, which decreased by 10.73 % compared with the control group.

A SARS-CoV-2 peptide antigen purified from bacteria and displayed in a high-density repetitive manner on a virus-like particle could generate anti-SARS-CoV-2 neutralizing antibodies unlike free peptide

SARS-CoV-2 is an enveloped virus. Proteins of the lipid based envelope are not rigidly arranged as compared to non-enveloped viruses lacking lipid based outer layer. Rigidly arranged multivalent display has been reported to be important for potent immune stimulation. We assembled himeric virus-like-particle (VLP) where the core of the particle is composed of the coat protein of Acinetobacter phage. Peptide-based antigen from receptor binding domain (RBD) of SARS-CoV-2 spike protein has been displayed on the coat based VLP matrix using spy-tag/spy-catcher split inteine conjugation system that allows spontaneous, irreversible isopeptide bond formation. Both the matrix as well as peptide have been purified from bacteria which is an easy platform for protein purification. We used the closed state of spike trimer for epitope mapping as this is the conformation of the spike that the immune system visualizes unlike the open state that appears when the virus tries to attach to receptor. Mice based immunization studies were used to test immunogenicity of the antigens. The work demonstrates that peptide-based antigens displayed in high densities can induce neutralizing antibody production unlike free peptide. Careful choice of peptides can deliver better candidates. Also, small size allows easy improvisation. Production in bacteria offers cheaper and robust purification option.

The impacts of different modification techniques on the gel properties and structural characteristics of fish gelatin

Fish gelatin hydrogels are typically characterized by weak texture and poor thermal stability. To address this issue, fish gelatin (FG) was modified using κ-carrageenan (κC) and transglutaminase (TG). FG-κC, FG-TG, and FG-κC-TG gels were prepared. The results demonstrated that the particle size and textural properties of FG gels were significantly enhanced by κC and κC-TG composite modifications, and their zeta potential was reduced (P < 0.05). As opposed to that, the improvement effects of TG modification were weaker. The highest particle size and hardness, along with the lowest zeta potential, were exhibited by the FG-κC-TG0.06% gel. Additionally, the FG-κC-TG gel system possessed higher apparent viscosity and elastic storage modulus. Fluorescence spectroscopy and UV absorption analysis indicated that fluorescence intensity was reduced by all three modification methods, and the conformational structure of FG was altered to varying degrees. Microstructure, FTIR, and XRD results revealed that the number of hydrogen bonds and isopeptide bonds in the composite gel systems was increased by κC and κC-TG modifications, enhancing the stability and orderliness of the gel's helical structure, and forming larger aggregates and denser network structures. Furthermore, due to the presence of covalent cross-linking, improved thermal stability was exhibited by the modified gelatin hydrogels. This study not only provides theoretical support for enhancing the performance of fish gelatin but also facilitates the application of modified fish gelatin in improving the sensory quality and flavor characteristics of food, as well as in serving as a matrix material in the biomedical field.

Structural Analyses of a GABARAP~ATG3 Conjugate Uncover a Novel Non-covalent Ubl-E2 Backside Interaction.

Members of the ATG8 family of ubiquitin-like proteins (Ubls) are conjugated to phosphatidylethanolamine (PE) in the autophagosomal membrane, where they recruit degradation substrates and facilitate membrane biogenesis. Despite this well-characterized function, the mechanisms underlying the lipidation process, including the action of the E2 enzyme ATG3, remain incompletely understood. Here, we report the crystal structure of human ATG3 conjugated to the mammalian ATG8 protein GABARAP via an isopeptide bond, mimicking the Ubl~E2 thioester intermediate. In this structure, the GABARAP~ATG3 conjugate adopts an open configuration with minimal contacts between the two proteins. Notably, the crystal lattice reveals non-covalent contacts between GABARAP and the backside of ATG3's E2 catalytic center, resulting in the formation of a helical filament of the GABARAP~ATG3 conjugate. While similar filament formations have been observed with canonical Ub~E2 conjugates, the E2 backside-binding interface of GABARAP is distinct from those of Ub/Ubl proteins and overlaps with the binding site for LC3 interacting region (LIR) peptides. NMR analysis confirms the presence of this non-covalent interaction in solution, and mutagenesis experiments demonstrate the involvement of the E2 backside in PE conjugation. These findings highlight the critical role of the E2 backside in the lipidation process and suggest evolutionary adaptations in the unique E2 enzyme ATG3.

A dual crosslinking strategy to tailor rheological properties of gelatin methacryloyl

3D bioprinting is an emerging technology that enables the fabrication of three-dimensional organised cellular constructs. One of the major challenges in 3D bioprinting is to develop a material to meet the harsh requirements (cell-compatibility, printability, structural stability post-printing and bio-functionality to regulate cell behaviours) suitable for printing. Gelatin methacryloyl (GelMA) has recently emerged as an attractive biomaterial in tissue engineering because it satisfies the requirements of bio-functionality and mechanical tunability. However, poor rheological property such as low viscosity at body temperature inhibits its application in 3D bioprinting. In this work, an enzymatic crosslinking method triggered by Ca2+-independent microbial transglutaminase (MTGase) was introduced to catalyse isopeptide bonds formation between chains of GelMA, which could improve its rheological behaviours, specifically its viscosity. By combining enzymatic crosslinking and photo crosslinking, it is possible to tune the solution viscosity and quickly stabilize the gelatin macromolecules at the same time. The results showed that the enzymatic crosslinking can increase the solution viscosity. Subsequent photo crosslinking could aid in fast stabilization of the structure and make handling easy.

Ultrasound-responsive theranostic platform for the timely monitoring and efficient thrombolysis in thrombi of tPA resistance

There is no effective and noninvasive solution for thrombolysis because the mechanism by which certain thrombi become tissue plasminogen activator (tPA)-resistant remains obscure. Endovascular thrombectomy is the last option for these tPA-resistant thrombi, thus a new noninvasive strategy is urgently needed. Through an examination of thrombi retrieved from stroke patients, we found that neutrophil extracellular traps (NETs), ε-(γ-glutamyl) lysine isopeptide bonds and fibrin scaffolds jointly comprise the key chain in tPA resistance. A theranostic platform is designed to combine sonodynamic and mechanical thrombolysis under the guidance of ultrasonic imaging. Breakdown of the key chain leads to a recanalization rate of more than 90% in male rat tPA-resistant occlusion model. Vascular reconstruction is observed one month after recanalization, during which there was no thrombosis recurrence. The system also demonstrates noninvasive theranostic capabilities in managing pigs’ long thrombi (>8 mm) and in revascularizing thrombosis-susceptible tissue-engineered vascular grafts, indicating its potential for clinical application. Overall, this noninvasive theranostic platform provides a new strategy for treating tPA-resistant thrombi.

PH-Controlled chemoselective rapid azo-coupling reaction (CRACR) enables global profiling of serotonylation proteome in cancer cells.

Serotonylation has been identified as a novel protein post-translational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

An N-terminal heptad repeat trimer-based peptide fusion inhibitor exhibits potent anti-H1N1 activity

Influenza viruses are susceptible to seasonal influenza, which has repeatedly caused global pandemics and jeopardized human health. Vaccines are only used as preventive medicine due to the extreme mutability of influenza viruses, and antiviral medication is the most significant clinical treatment to reduce influenza morbidity and mortality. Nevertheless, the clinical application of anti-influenza virus agents is characterized by the narrow therapeutic time window, the susceptibility to drug resistance, and relatively limited effect on severe influenza. Therefore, it is of great significance to develop novel anti-influenza virus drugs to fulfill the urgent clinical needs. Influenza viruses enter host cells through the hemagglutinin (HA) mediated membrane fusion process, and fusion inhibitors function antivirally by blocking hemagglutinin deformation, promising better therapeutic efficacy and resolving drug resistance, with targets different from marketed medicines. Previous studies have shown that unnatural peptides derived from Human Immunodeficiency Virus Type 1 (HIV-1) membrane fusion proteins exhibit anti-HIV-1 activity. Based on the similarity of the membrane fusion protein deformation process between HIV-1 and H1N1, we selected sequences derived from the gp41 subunit in the HIV-1 fusion protein, and then constructed N-trimer spatial structure through inter-helical isopeptide bond modification, to design the novel anti-H1N1 fusion inhibitors. The results showed that the novel peptides could block 6-HB formation during H1N1 membrane fusion procedure, and thus possessed significant anti-H1N1 activity, comparable to the positive control oseltamivir. Our study demonstrates the design viability of peptide fusion inhibitors based on similar membrane fusion processes among viruses, and furthermore provides an important idea for the novel anti-H1N1 inhibitors development.

Fabrication of resveratrol-loaded soy peptide nanogels with transglutaminase and in vitro gastrointestinal delivery and cholesterol-lowering effect

To improve the intestinal delivery of resveratrol, soy protein peptides (SPP) and transglutaminase (TGase) were used to prepare nanogels (NGs). The results demonstrated that, compared with native soy protein isolate (SPI) NG, SPP-NG showed a lower encapsulation efficiency (EE, 70.26%), a higher release efficiency (RE, 95.83%), and a smaller particle size (194.43 nm). However, the addition of TGase increased the EE and particle size to 74.37% and 217.73 nm, respectively. The RE of SPP-TGNG was also maintained at as high as 93.28%. Interestingly, SPP-TGNG exhibited optimal bile acid–binding capacity and cholesterol-lowering digests. The differences in delivery effects and morphology were attributed to structural changes in NGs. The unfolding and exposure of hydrophobic residues and weakened hydrogen bonds by hydrolysis resulted in a disordered and loose network structure, but it was beneficial to the production of cholesterol-lowering peptides and amino acids after digestion. The presence of TGase strengthened the crosslinking network by increasing β-sheet contents, hydrogen bonds, hydrophobic interactions, and isopeptide bonds. The alteration in multilevel structure would further affect the digestive products. The exposure of more hydrophobic residues and isopeptide crosslinking may be the intrinsic reason for the production of more hypocholesterolemic peptides and amino acids. This study indicated that the combination of hydrolysis and TGase treatment enhanced the delivery efficiency and bioactive properties of peptide-based NGs to resveratrol and revealed the internal mechanism between the structural changes and delivery efficiency. This work offers a theoretical foundation and technical suggestions for the production of peptide-based cargo systems and hydrophobic active substances.

An i-type lysozyme from a crustacean, Pacifastacus leniusculus, functions as a clot-destabilising enzyme

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots.We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the β-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

The Frequently Used Industrial Food Process Additive, Microbial Transglutaminase: Boon or Bane.

Microbial transglutaminase (mTG) is a frequently consumed processed food additive, and use of its cross-linked complexes is expanding rapidly. It was designated as a processing aid and was granted the generally recognized as safe (GRAS) classification decades ago, thus avoiding thorough assessment according to current criteria of toxicity and public health safety. In contrast to the manufacturer's declarations and claims, mTG and/or its transamidated complexes are proinflammatory, immunogenic, allergenic, pathogenic, and potentially toxic, hence raising concerns for public health. Being a member of the transglutaminase family and functionally imitating the tissue transglutaminase, mTG was recently identified as a potential inducer of celiac disease. Microbial transglutaminase and its docked complexes have numerous detrimental effects. Those harmful aspects are denied by the manufacturers, who claim the enzyme is deactivated when heated or by gastric acidity, and that its covalently linked isopeptide bonds are safe. The present narrative review describes the potential side effects of mTG, highlighting its thermostability and activity over a broad pH range, thus, challenging the manufacturers' and distributers' safety claims. The national food regulatory authorities and the scientific community are urged to reevaluate mTG's GRAS status, prioritizing public health protection against the possible risks associated with this enzyme and its health-damaging consequences.

Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

AbstractUbiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i. e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2 K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein.

Effect of transglutaminase and laccase on pea protein gel properties compared to that of soybean

Enzyme cross-linking is a pivotal method for enhancing the functionality of plant proteins, offering improvements in texture, stability, and nutritional value, which are essential for food applications. Pea protein is increasingly used as it is not identified as allergenic and has fewer other negative health concerns. However, the lower gelling capacity compared with soybean protein has limited its application in plant-based meat or protein-based products. Thus, this study focused on investigating the effects of transglutaminase (TG) and laccase (LAC) and their concentrations on pea protein isolate (PPI) gelation, towards comparison with that of soybean protein isolate (SPI). Results showed that TG and LAC both contribute to the formation of PPI gels while exhibiting different action ways. As the TG content increased, the particle size of PPI significantly increased from 97.27 ± 4.61 μm to 146.67 ± 9.87 μm at 0.8% TG concentration, whereas the particle size remained constant with increasing LAC content. Both TG and LAC significantly enhanced the PPI gel properties, with 0.4% TG addition resulting in gel strength comparable to SPI and 0.8% TG content increasing PPI gel strength by approximately 4.2 times compared to the control, surpassing SPI. Moreover, PPI with TG treated demonstrated superior effects compared to LAC-treated gels at the same concentration. Water retention of gels increased from 89.37 ± 1.97% to 94.90 ± 3.86% with TG concentration increasing, while it decreased with LAC increasing. SDS-PAGE results revealed that TG promoted the formation of larger aggregates in PPI, indicating that legumin and vicilin were the main target sites for TG, while LAC was inapparent on molecular weight. The viscosity of PPI increased from 36.91 ± 2.32 Pa·s to 70.57 ± 1.00 Pa·s with 0.8% TG, while 0.8% LAC increased viscosity to 61.79 ± 1.32 Pa·s. Hydrogen bonds and disulfide bonds play a crucial role in the gel formation process facilitated by TG and LAC. Besides, TG is also expected to contribute through the formation of isopeptide bonds. These findings validated the potential of TG and LAC in improving the quality of PPI gels and provided insights for the application and development of cross-linking enzymes in the protein processing industry.

Lactoferrin-Chitosan Composite Hydrogels Induced by Microbial Transglutaminase: Potential Delivery Systems for Thermosensitive Bioactive Substances.

In this work, lactoferrin (LF)-chitosan (CS) composite hydrogels with good loading capacity of thermosensitive bioactive substances were successfully obtained by microbial transglutaminase (MTG)-induced cross-linking. We evaluated the rheological, textural, and microstructural characteristics of the composite hydrogels under different conditions. The results demonstrated that the concentrations of LF and CS as well as the amount of MTG could regulate the textural properties, rheological properties, and water holding capability. The results of FTIR and fluorescence spectroscopy indicated that the main interactions within the composite gel were hydrogen and isopeptide bonds. Additionally, in vitro digestion simulation results verified that riboflavin kept stable in stomach due to the protection of LF-CS composite hydrogels and was released in small intestine. These results suggested that thermosensitive bioactive substance could be encapsulated and delivered by the LF-CS composite hydrogel, which could be applied in lots of potential applications in functional food as a new material.