Search Results (100)

As synthetic biology advances, prokaryotic microorganisms have become critical platforms for heterologous biosynthesis in cell factory applications. However, conventional free enzyme systems encounter substantial challenges, including inefficient intermediate transfer, toxic intermediate accumulation, and vulnerability to temperature and pH fluctuations. Enzyme complex catalytic systems offer promising solutions to these limitations. Bacillus licheniformis, a Generally Recognized as Safe (GRAS) host with exceptional protein secretion capacity, represents an ideal chassis for enzyme complex construction. This study developed a peptide-mediated platform in B. licheniformis to enable enzyme complex self-assembly and evaluated its effects on metabolic pathway performance. Five peptide elements were screened through fusion with enhanced orange/green fluorescent proteins (eOFP/eGFP) and transglutaminase (TGase). Effective peptide pairs were identified by measuring fluorescence intensity, visualizing complex formation via laser confocal microscopy, and assessing TGase activity. Subsequently, recombinant strains expressing peptide-fused key metabolic enzymes (gadTt and KdgA) were constructed for whole-cell biotransformation using gluconate as substrate to investigate the impact of peptide-mediated enzyme complexes on pyruvate synthesis. In the fluorescent protein system, P18/D18—amphipathic peptides that drive enzyme self-assembly via intermolecular hydrophobic interactions—increased extracellular fluorescence intensity of eOFP and eGFP by 31.11% and 25.21%, respectively. The D18 peptide significantly elevated TGase activity by enhancing structural stability to over 1.3-fold that of the control. For pyruvate synthesis, the peptide-mediated enzyme complex exhibited remarkable advantages in substrate conversion rate (up to 53.08%) and thermostability, confirming the platform’s ability to enhance substrate channeling despite no optimization for absolute yield. This study established a novel peptide-mediated multienzyme self-assembly platform in B. licheniformis, providing a valuable strategy for artificial metabolic channel design in synthetic biology. Full article

Microvascular leakage is an early hallmark of diabetic retinopathy (DR), but the redox-dependent mechanisms underlying this dysfunction remain unclear. Here, we investigated whether p38α mitogen-activated protein kinase (MAPK) activates transglutaminase 2 (TGase2) through reactive oxygen species (ROS) generation, thereby promoting hyperglycemia-induced vascular permeability in diabetic retinas. In human retinal endothelial cells (HRECs), vascular endothelial growth factor (VEGF), which is elevated under hyperglycemic conditions, activated both p38α MAPK and TGase2. VEGF-induced TGase2 activation was inhibited by the p38 MAPK inhibitor SB203580 or by p38α MAPK siRNA. Similarly, VEGF-stimulated TGase2 activity in non-diabetic mouse retinas was blocked by knockdown of either p38α MAPK or TGase2. In diabetic retinas, hyperglycemia-increased ROS production and TGase2 activity were reduced by SB203580 or p38α MAPK siRNA, but not by the TGase inhibitor cystamine, indicating upstream ROS-dependent regulation. The antioxidant Trolox also suppressed TGase2 activation in VEGF-treated HRECs and diabetic retinas. Functionally, knockdown of p38α MAPK or TGase2 preserved vascular endothelial (VE)-cadherin integrity and attenuated cytoskeletal remodeling in HRECs and diabetic retinas, resulting in reduced microvascular leakage. These findings identify a redox-dependent p38α MAPK–TGase2 axis as a key mediator of retinal vascular permeability in DR and highlight this pathway as a potential therapeutic target for maintaining vascular integrity. Full article

The increasing demand for sustainable and functional plant-based foods has driven interest in 3D food printing technologies, which require bioinks with tailored rheological and structural properties. This study investigated the effects of transglutaminase (TGase) on the structure–function relationships of plant protein bioinks from fava bean, mung bean, pea, and soybean. TNBS assays showed a dose-dependent increase in crosslinking (27.46–64.57%), with soybean and pea proteins exhibiting the highest reactivity (p < 0.05). ¹H-NMR confirmed protein-specific ε-(γ-glutamyl)lysine bond formation, and synchrotron FTIR revealed TGase-induced α-helix reduction and β-sheet enrichment, indicative of network formation across all proteins. SDS-PAGE analysis demonstrated TGase-mediated polymerization with high-molecular-weight aggregates, particularly pronounced in soybean, while SEM images revealed denser, more continuous protein networks compared to untreated samples. Rheological characterization showed enhanced viscoelasticity and shear-thinning behavior in all bioinks, supporting extrusion and post-printing stability. Textural analysis indicated improvements in hardness, springiness, cohesiveness, and chewiness across all proteins, with soybean and fava showing the most pronounced increases. These results demonstrate that TGase is a versatile tool for reinforcing plant protein networks, improving printability, structural integrity, and texture in 3D-printed foods, while highlighting protein-specific differences in response. Full article

In this paper, the effects of TGase reaction times (0, 6, 12, 18, and 24 h) at 4 °C on the solubility, emulsion stability, cooking yield, gel properties and water distribution of reduced-fat and reduced-sodium chicken meat batter were studied. The results showed that the reaction time had a significant effect on the water fluidity and quality characteristics of reduced-fat and reduced-sodium chicken meat batter. The solubility, cooking yield and water-holding capacity of salt-soluble proteins initially increased then decreased with extended reaction time, reaching maximum values of 65.50%, 96.13% and 96.00%, respectively, at 12 h. The emulsifying stability and textural properties initially increased, then decreased with extended reaction time (p < 0.05), achieving optimal levels at 12 h. In contrast, the initial relaxation time of T21 and T22 initially decreased (p < 0.05) and then increased (p < 0.05) with longer reaction times; the minimum values were 12 h, especially the free water decreased from 17.97% to 6.69%, consistent with the finding on water-holding capacity and gel properties. In conclusion, the reaction time of the TGase affected its effect on improving the gel effect of reduced-fat and reduced-sodium chicken meat batter, and the best effect was achieved at 12 h. Full article

In most mosquito species, reproduction requires mating between the female and the male, followed by the female blood-feeding, completing oogenesis, and laying eggs. Warmer environmental temperature and aging both reduce mosquito fecundity and fertility, and warmer temperature accelerates the aging-dependent decline in reproduction such that reproductive impairment manifests earlier in life. To shed light on how this warming-based acceleration of reproductive senescence occurs, we investigated how temperature (27 °C, 30 °C, and 32 °C) and aging interactively shape female and male reproductive tissue size in the African malaria mosquito, Anopheles gambiae. In blood-fed females, we discovered that warmer temperature accelerates the aging-dependent decrease in the size of the ovaries but not the spermatheca. In males, we discovered that warmer temperature lessens and delays the aging-dependent increase in the size of the male accessory glands but not the testes. Next, we measured the expression of reproductive genes in females and males. In female reproductive tissues, warmer temperature accelerates the aging-dependent decrease in the expression of vitellogenin and the aging-dependent increase in the expression of MISO and HPX15. In male reproductive tissues, warmer temperature accelerates an aging-dependent decrease in the expression of Plugin, TGase3, phLP, and CYP315A1. Altogether, these data shed light on how physical and transcriptional changes underpin the warming-based acceleration of an aging-dependent decline in mosquito fecundity and fertility. Full article

Transglutaminase (TGase) improves protein structure by facilitating cross-linking reactions. However, the effects of TGase on the physicochemical properties of whey protein isolate (WPI)–soymilk complexes and their applications in yuba remain unclear. Therefore, the impacts of TGase concentration on the free sulfhydryl content, free amino content, particle size, and structure of WPI–soymilk complexes and their film-forming properties were studied. The results showed that the physicochemical properties of the composite soymilk were changed by the TGase-induced cross-linking reaction of protein. Compared with the composite soymilk without TGase modification, the particle size of the WPI–soymilk complexes increased from 707.99 ± 9.47 nm to 914.41 ± 2.8 nm as the TGase concentration increased, and the complexes remained relatively stable at low TGase concentrations. TGase modification changed the tertiary structure of the WPI–soymilk complexes. The composite yuba with 0.01% and 0.03% levels of TGase had a higher β-sheet content than composite yuba without addition of TGase. The surface hydrophobicity of composite soymilk was decreased by all the addition levels of TGase. Meanwhile, the TGase-modified composite protein with 0.03% TGase had the lowest free sulfhydryl (35.92 μg/g) and amino groups (0.46). Additionally, the tensile strength of the composite yuba with 0.05% TGase addition reached a peak of 1.66 ± 0.02 MPa, which was 7.8% higher than that of the composite yuba without TGase addition. The SEM results revealed that the composite yuba with 0.01–0.03% TGase addition exhibited a dense and non-porous film structure. Moreover, all the composite yuba with TGase addition had a reduced rate of yuba cooking loss. This study contributes to enhancing the yield and mechanical properties of traditional yuba. Full article

The covalent cross-linking of caseins by the enzyme transglutaminase (Tgase) stabilizes the structure of casein micelles. In our study, the effects of a pretreatment of skim milk (SM) by Tgase on milk protein fractionation by microfiltration were tested. Tgase was found to induce amount-dependent modifications of all milk proteins in SM and a reduction in deposit resistance for laboratory dead-end filtrations of up to 20%. This improvement in process performance could partially be confirmed in pilot-scale cross-flow filtrations of Tgase-pretreated SM and micellar casein solutions (MCC). These comparative trials with untreated retentates under a variation of ΔpTM (0.5–2 bar) at 10 and 50° revealed distinct differences in deposit behavior and achieved the reduction in deposit resistance in a range of 0–20%. The possibility of pre-fouling with enzymatically pretreated MCC prior to SM filtration was also investigated. Under different pre-fouling conditions, practical modes of retentate change, and pre-foulant compositions, a switch to untreated SM consistently resulted in an immediate and major increase in deposit resistance by 50–150%. This was partially related to the change in the ionic environment and the protein fraction. Nevertheless, our results underline the potential of Tgase pretreatment and pre-fouling approaches to alter filtration performance for different applications. Full article

In this study, curdlan was investigated as a substitute for egg-white protein, and the effects of different concentrations (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) on lipid oxidation and the physicochemical properties of a novel low-salt surimi gel containing transglutaminase (TGase) and lysine were evaluated. The results indicated that adding appropriate curdlan concentrations (0.2%–0.4%, especially 0.4%) significantly inhibited lipid oxidation in the surimi gel, achieving the highest L* and whiteness values. The fracture strength, WHC, hardness, and chewiness of the gel increased by 23.87%, 6.70%, 32.80%, and 13.49%, respectively, compared to the control gel containing egg-white protein (p < 0.05). At 0.4% curdlan, the gel also enhanced the crosslinking within the surimi, improved its resistance to shear stress, significantly increased the G’ value, and shortened the T₂₁, T₂₂, and T₂₃ relaxation times, inhibiting the conversion of immobilized to free water in the gel and promoting a denser three-dimensional network structure. However, excessive curdlan amounts (0.6%–1.0%) led to a notable deterioration in the gel performance, causing a more irregular microstructure, the formation of larger cluster-like aggregates, and a negative effect on color. In conclusion, the combination of 0.4% curdlan with TGase and Lys is effective for preparing low-salt surimi products. Full article

Soy isolate protein (SPI), as a high-quality plant protein source, is often processed into various soy products. In this study, the physicochemical properties of SPI treated with transglutaminase (TGase) were investigated in correlation with emulsification characteristics and rheological behavior. The polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) and Fourier-transform infrared spectroscopy (FTIR) and endogenous fluorescence spectrum analysis results showed that TGase was able to promote the covalent binding of lysine and glutamine residues in SPI. The moderate pre-crosslinking treatment of TGase (5–7.5 U/g TGase pre-crosslinked for 2 h or 5 U/g TGase pre-crosslinked for 2–3 h) improved the emulsification and gel properties to varying degrees: the nanoparticle and emulsification performance increased by 24.35% and the storage modulus of the gel increased by 288%. Furthermore, the surface charge of SPI increased due to the crosslinking impact of TGase, indicating a considerable rise in the surface electrostatic potential. Simultaneously, the protein surface exhibited a substantial increase in hydrophobicity, while the level of free sulfhydryl groups reduced. These changes indicate that TGase enzymatic crosslinking could significantly improve the structural stability of nanoparticles by enhancing the generation efficiency of covalent bonds between protein molecules. Full article

To increase the added value of peanut meal (PM, protein content of 46.17%) and expand its application in food processing, cold-pressed PM was modified via transglutaminase (TGase)-coupled glycation to enhance its functional properties. The effects of the modification conditions (i.e., PM concentration, PM/glucose mass ratio, temperature, and time) on the functional properties of PM were investigated, and its structural properties were evaluated using water contact angle measurements, fluorescence spectroscopy, and Fourier-transform infrared spectroscopy. It was found that TGase-coupled glycation modification altered the secondary structure of PM and increased both the water contact angle and the surface hydrophobicity, thereby significantly affecting its functional properties. Additionally, superior emulsification, foaming, and oil-absorbing properties were achieved for the modified PM, which were named EPM, FPM, and OPM, respectively (specimens under different modification conditions). Notably, the emulsification activity of the EPM sample was enhanced by 69.8% (i.e., from 18.48 to 31.38 m²/g); the foaming capacity of the FPM specimen was increased by 84.00% (i.e., from 21.00 to 46.00%); and the oil-absorbing capacity of the OPM sample was enhanced by 359.57% (i.e., from 1.41 to 6.48 g/g protein). Full article

Saltwater crocodile (SC; Crocodylus porosus) bone, an underutilized by-product, can be converted into high-value bio-calcium (Biocal), serving as a potential source of calcium and minerals. This study aimed to produce SC bone Biocal as functional gel enhancer for fish bologna development and to increase calcium intake. The resulting bone powder was evaluated for physicochemical, microbiological, and molecular properties. Additionally, the textural, physicochemical, structural, and sensorial properties of the formulated fish bologna incorporating Biocal at varying levels (0–10% w/w) were also evaluated. Biocal, obtained as a fine white powder, had a 16.83% yield. Mineral analysis showed 26.25% calcium and 13.72% phosphorus, with no harmful metals or pathogens detected. X-ray diffraction confirmed hydroxyapatite with 69.92% crystallinity, while calcium bioavailability was measured at 22.30%. Amino acid analysis indicated high levels of glycine, proline, and hydroxyproline, essential for collagen support. The findings confirmed that SC bone Biocal is beneficial and safe for food fortification. Incorporating SC Biocal (2–10% w/w) significantly affected the fish bologna characteristics (p < 0.05). As the Biocal level increased, the gel strength, hardness, and shear force also increased. The addition of 6% (w/w) Biocal significantly improved the textural property, without a detrimental effect on the sensory attributes of the bologna gel (p < 0.05). SDS-PAGE analysis showed TGase-enhanced myosin heavy chain (MHC) cross-linking, particularly in combination with Biocal. Moreover, the enriched Biocal–bologna gel exhibited a finer and denser microstructure. Thus, SC Biocal, particularly at 6% (w/w), can serve as a functional gel enhancer in surimi-based products, without compromising organoleptic quality. Full article

Surimi gel, a type of hydrocolloidal food, is formed through the gelation of fish meat proteins. Myosin heavy chain (MHC), a key myofibrillar protein, plays a crucial role in the formation of the gel network via both transglutaminase (TGase)-catalyzed and non-enzymatic polymerization. Gel disintegration in surimi is primarily attributed to the proteolytic degradation of MHC. This study focused on golden threadfin bream Nemipterus virgatus, a species characterized by low TGase activity and high protease activity at elevated temperatures. We investigated the competition between non-enzymatic polymerization and proteolytic degradation of MHC and their effects on gel mechanical properties using a mathematical model. A mathematical model based on kinetic reactions accurately reflected the changes in MHC observed through SDS-PAGE analysis during N. virgatus gel disintegration. Our results indicate that not only unpolymerized but also polymerized MHC was significantly degraded, which substantially contributed to the reduction in the mechanical properties of the N. virgatus surimi. Mathematically understanding the dynamics of MHC in surimi during heating helps promote the utilization of noncommercial fish species for surimi processing by enabling better control over surimi gel properties. Full article

The addition of Chinese yam powder (CYP) to wheat flour (WF) can compromise the elasticity of noodles due to weakening of the gluten network. To address this, we investigated the effects of TGase, vital wheat gluten (VWG), and egg white powder + sodium alginate (EWP + SA) on the quality of wheat yam composite flour noodles (color, cooking, textural, thermal properties, and in vitro starch digestibility). Our findings demonstrated that VWG, TGase, and EWP + SA exert distinct yet complementary effects on the quality of composite flour noodles. Combining TGase and VWG yielded the densest microstructure and better textural properties, including hardness, adhesiveness, and springiness. TGase and EWP + SA addition significantly increased slow digestible starch (SDS) content (G6: 33.81%) while reducing starch digestibility. These findings demonstrate that synergetic combinations of improvers, particularly TGase with VWG or EWP + SA, improve both the processing characteristics and nutritional quality of yam-based products. Full article

This study investigated the effects of transglutaminase (TGase) content (0%, 0.5%, 1%, 1.5%) and heat treatment (25 °C, 70 °C, 80 °C, 90 °C) on the structure and gel properties of camel casein protein. The results indicate that a TGase concentration of 0.5% combined with a heat treatment of 90 °C in SDS-PAGE facilitates the aggregation and crosslinking of protein molecules to form polymers, with the degree of crosslinking increasing alongside the TGase concentration. In FTIR, the treatment with TGase and heat resulted in a shift of the absorption peak of the amide I band, indicating a transition of the secondary structure from a loose to an ordered configuration. Additionally, surface hydrophobicity and heat enthalpy values were significantly increased, while the thermal transition temperature of casein gradually decreased. Following TGase binding and heat treatment, casein protein molecules formed a network structure characterized by small pore sizes and close crosslinking. Rheological analysis revealed that 0.5% TGase treatment significantly lowered the gel formation point of casein, promoted gelation, and effectively enhanced the mechanical properties and water-holding capacity of the casein gels. These findings provide theoretical reference for the development of camel protein modification and gel products. Full article

The bay scallop (Argopecten irradians) adductor is an attractive raw material for the production of surimi-like products. The gelling properties of raw materials directly affect the quality of surimi-like products. To assess the potential of processing frozen bay scallop adductors into surimi-like products, the effects of short-term freezing treatment on the endogenous transglutaminase (TGase) activity, myofibrillar protein (MP) structure and gelling properties of bay scallop adductors were investigated during 14 days of frozen storage (−18 °C). The results showed that TGase activity in adductor muscles increased significantly during the first 7 days. After 7–14 days, the carbonyl and sulfhydryl contents of the MPs notably changed (increased then decreased). The β-turn content of the MPs increased, indicating stretching and flexibility. Surface hydrophobicity, fluorescence intensity and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated changes in the tertiary structure of the MPs. Compared with gels from fresh samples, gels from scallop adductors frozen for 1 day presented significantly better texture characteristics (breaking force, gel strength, hardness, springiness, cohesiveness, chewiness) and higher water-holding capacity (p < 0.05). However, these properties significantly decreased on the 7th and 14th days (p < 0.05). Microstructural analysis revealed a more compact gel network from 1-day-frozen adductor muscles. These changes in TGase activity and MP structure are key factors influencing the gelling properties of frozen bay scallop adductors. This study provides new insights for improving gel properties during the frozen storage of bay scallop adductors. Full article