Enzymatic Protein Cross-linking Research Articles

Utilization of Reconstituted Whey Powder and Microbial Transglutaminase in Ayran (Drinking Yogurt) Production.

SUMMARYResearch backgroundIn industrial ayran production, milk is diluted to the desired protein content (2% (m/V)) prior to fermentation by yogurt starter cultures by partial replacement of cheese whey in reconstituted form with potable water. This may be an alternative way of protein recovery from cheese by-products as well as reducing the production costs since less milk is used in ayran production. On the other hand, the balance between milk caseins and whey proteins is disturbed when cheese whey is added to milk for ayran production, which likely leads to a time-dependent phase separation during cold storage. Modification of ayran matrix by enzymatic crosslinking of proteins may be a solution to overcome this potential physical instability of ayran. This topic has not been explored to date, and the present study was designed to investigate the possibilities of utilization of reconstituted whey powder (RWP) and microbial transglutaminase (MTG) in ayran production.Experimental approachMilk was diluted to obtain 2% protein level using RWP and potable water. The aim of utilization of RWP was to meet 5, 10 or 15% of the protein content of the final product. RWP solutions were obtained by calculating the amounts of whey powder required to meet the specified ratios and mixing it with the water required for dilution. We prepared eight different ayran samples divided in three groups, namely group A: prepared by partially diluting milk with RWP to obtain 5, 10 or 15% of total protein amount in the product, group AMTG: prepared by adding microbial transglutaminase (0.5 U per g of protein) to group A samples, and control group without RWP and with or without the addition of MTG. The gross composition, physical (phase separation and viscosity, chemical (volatile and peptide profiles and SDS-PAGE electrophoresis patterns) and sensory properties of the samples were evaluated throughout 15 days of storage with weekly intervals.Results and conclusionsSince the amounts of whey powder used to obtain RWP were different, dry matter levels of the samples differed. Using RWP in ayran production increased the phase separation slightly. Incorporation of MTG affected the physical properties of the ayran samples positively and prevented phase separation at a satisfactory level. SDS-PAGE electrophoretograms revealed that cross-linking between proteins triggered by MTG formed intense bonds at high molecular mass regions. The remaining parameters were not affected by MTG. Results revealed that the samples with 10% RWP with and without addition of MTG were determined as the formulations with the highest commercialization potential.Novelty and scientific contributionUtilizing RWP in the production of ayran to reduce the protein content of the final product to the desired level is a new approach. Since complete replacement of RWP with potable water to dilute milk to reach 2% (m/V) protein likely leads to lower sensory scores, we have investigated a possibility of partial replacement of RWP with potable water. A time-dependent phase separation is the major challenge of industrial ayran production. This physical problem was largely eliminated by means of MTG-mediated cross-linking of milk proteins. The proposed novel ayran production method offers dairy industry reduction of production costs and contributes to sustainability in milk production since smaller volume of milk is used to reach desired protein content in the final product.

Influence of Transglutaminase on Glucono-δ-lactone-Induced Soy Protein Gels

Enzymatic cross-linking of proteins can be used to create a variety of foods with appealing textures and facilitate further processing such as slicing or packaging. Transglutaminase (TG), a protein-glutamine-γ-glutamyltransferase, has been used in the production of processed meat and fish products. However, it has a reaction optimum at pH 7 and is therefore typically unsuitable for application in up-and-coming acidic food products such as fermented vegan meat product alternatives. To determine whether a simultaneous addition of slowly acidifying glucono-δ-lactone (GDL) and TG can facilitate protein cross-linking prior to the pH becoming too low, TG and GDL were added separately or in combination to soy protein suspensions composed of 10–15% soy protein concentrates or isolates (SPI). Texture analysis showed that SPI gels at pH 5–6, induced by GDL and TG combined, were harder than only GDL-induced gels and as hard as TG-induced gels at the optimum pH of 7. Decreased tan δ values and confocal laser scanning microscopy images indicated that protein cross-linking had taken place in combined gels. The results suggest an initial network formation induced by TG, which later in the acidification is being supplemented by an agglomeration of proteins due to weakened electrostatic repulsion. Taken together, the combination of slow acidification by GDL and TG addition leads to acidic gels with enhanced textural properties and shelf life.

The non-covalent interaction between two polyphenols and caseinate as affected by two types of enzymatic protein crosslinking

Caseinate was crosslinked by horseradish peroxidase (HRP) or microbial transglutaminase (TGase) and mixed with kaempferol and quercetin at 293–313 K (i.e. 20–40 °C), respectively. Generally, these two polyphenols dose-dependently induced fluorescent quenching in caseinate or its crosslinked products via a static mechanism, while enzymatic crosslinking endowed caseinate with higher affinity for the polyphenols with increased apparent binding constants [(9.94–168.77) × 105versus (4.92–6.53) × 105 L/mol], unchanged binding site number and slightly shortened binding distance. To form protein–polyphenol complexes, hydrophobic force was the main driving force for the HRP-crosslinked caseinate and unreacted caseinate, while hydrogen-bonds and van der Waals force were the main driving forces for the TGase-crosslinked caseinate. Overall, quercetin was more potent than kaempferol to bind to the proteins, while TGase-mediated caseinate crosslinking induced the highest affinity to the polyphenols with the largest ΔG decrease. Thus, two types of crosslinking impacted the driving forces, apparent binding constant and thermodynamic indices of caseinate-polyphenol interaction.

The Impact of Microbial Transglutaminase on the Quality and Antioxidant Activity of Camel-Milk Soft Cheese

This study aimed at investigating the impact of adding microbial transglutaminase (MTGase) after rennet addition on some properties of fresh soft cheese made from camel milk. MTGase was added to milk at concentration of 80, 100 and 120 U/L after 20 and 30 min of renneting. The chemical composition, yield, hardness, antioxidant activity and sensory properties of cheese were estimated. Enzymatic protein crosslinking was analyzed by SDS-PAGE. Results revealed that MTGase-treated cheeses were higher in moisture and lower in protein content compared to control. In addition, the concentration of MTGase and time of addition significantly (P 0.05) impacted these parameters. Among treated cheeses, samples with 80 U of MTGase and addition time of 20 min were the highest in total solids and protein content. Adding MTGase significantly (P 0.05) increased the cheese yield, however, increased MTGase concentration at any time of addition did not improve it. The electrophoretic patterns of MTGase-cheese proteins showed a reduction in the intensity of caseins bands and the appearance of new protein fractions with high molecular weights. However, the changes in the intensity of the whey proteins bands were not sufficiently clear as caseins. The cheese hardness was significantly (P 0.05) affected by adding MTGase. Cheese containing 80 U of MTGase had the highest hardness value compared to control and other treated samples. The antioxidant activity of cheese was negatively influenced by adding the enzyme. The use of MTGase enhanced the mouthfeel, texture and overall acceptability of cheese. However, the effect of MTGase concentration and addition time was not significant (P > 0.05) on the sensory attributes. In conclusion, adding MTGase to milk at concentration of 80 U after 20 min of renneting is recommended to improve the yield, textural and some sensory properties of fresh soft cheese made from camel milk.

Chemical and Enzymatic Protein Cross-Linking To Improve Flocculant Properties

Some proteins promote the flocculation of aqueous suspensions, but proteins are much smaller than modern polymeric flocculants, and they are often not as potent. In this study, two globular protein...

Allergenicity and Oral Tolerance of Enzymatic Cross-Linked Tropomyosin Evaluated Using Cell and Mouse Models.

The enzymatic cross-linking of proteins to form high-molecular-weight compounds may alter their sensitization potential. The IgG-/IgE-binding activity, digestibility, allergenicity, and oral tolerance of cross-linked tropomyosin with tyrosinase (CTC) or horseradish peroxidase (CHP) were investigated. ELISA results demonstrated CTC or CHP reduced its IgE-binding activity by 34.5 ± 1.8 and 63.5 ± 0.6%, respectively. Compared with native tropomyosin or CTC, CHP was more easily digested into small fragments; CHP decreased the degranulation of RBL-2H3 cells and increased endocytosis by dendritic cells. CHP can induce oral tolerance and reduce allergenicity in mice by decreasing IgE and IgG1 levels in serum, the production of T-cell cytokines, and the percentage composition of dendritic cells. These findings demonstrate CHP has more potential of reducing the allergenicity than CTC via influencing the morphology of protein, changing the original method of antigen presentation, modulating the Th1/Th2 immunobalance, and inducing the oral tolerance of the allergen tropomyosin.

Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking.

Considerable interest has arisen in precision fabrication of cell bearing scaffolds and structures by free form fabrication. Gelatin is an ideal material for creating cell entrapping constructs, yet its application in free form fabrication remains challenging. We demonstrate the use of gelatin, crosslinked with microbial transglutaminase (mTgase), as a material to print cell bearing hydrogels for both 2-dimensional (2-D) precision patterns and 3-dimensional (3-D) constructs. The precision patterning was attained with 3 % gelatin and 2 % high molecular weight poly (ethylene oxide) (PEO) whereas 3-D constructs were obtained using a 5 % gelatin solution. These hydrogels, referred to as “bioinks” supported entrapped cell growth, allowing cell spreading and proliferation for both HEK293 cells and Human Umbilical Vein Endothelial Cells (HUVECs). These bioinks were shown to be dispensable by robotic precision, forming patterns and constructs that were insoluble and of suitable stiffness to endure post gelation handling. The two bioinks were further characterized for fabrication parameters and mechanical properties.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-014-9915-8) contains supplementary material, which is available to authorized users.

Enzymatic cross-linking of α-lactalbumin to produce nanoparticles with increased foam stability.

Hard colloidal nanoparticles (e.g. partly hydrophobised silica), are known to make foams with very high foam-stability. Nanoparticles can also be produced from proteins by enzymatic cross-linking. Such protein based particles are more suitable for food applications, but it is not known if they provide Pickering foam stabilisation to the same extent as hard colloidal particles. α-Lactalbumin (α-LA) was cross-linked with either microbial transglutaminase (mTG) or horseradish peroxidase (HRP) to produce α-LA/mTG and α-LA/HRP nanoparticles. With both enzymes a range of nanoparticles were produced with hydrodynamic radii ranging from 20-100 nm. The adsorption of nanoparticles to the air-water interface was probed by increase in surface pressure (Π) with time. In the beginning of the Π versus time curves, there was a lag time of 10-200 s, for nanoparticles with Rh of 30-100 nm, respectively. A faster increase of Π with time was observed by increasing the ionic strength (I = 0-125 mM). The foam-ability of the nanoparticles was also found to increase with increasing ionic strength. At a fixed I, the foam-ability of the nanoparticles decreased with increasing size while their foam-stability increased. Foams produced by low-shear whipping were found to be 2 to 6 times more stable for nanoparticles than for monomeric α-LA (Rh≈ 2 nm). At an ionic strength of 125 mM ionic strength and protein concentration ≥ 10 g L(-1), the foam-stability of α-LA/mTG nanoparticles (Rh = 100 nm, ρapp = 21.6 kg m(-3)) was 2-4 times higher than α-LA/HRP nanoparticles (Rh = 90 nm, ρapp = 10.6 kg m(-3)). This indicated that foam-stablity of nanoparticles is determined not only by size but also by differences in mesoscale structure. So, indeed enzymatic cross-linking of proteins to make nanoparticles is moving a step towards particle like behavior e.g. slower adsorption and higher foam stability. However, the cross-link density should be further increased to obtain hard particle-like rigidity and foam-stability.

Tyrosinase-catalyzed site-specific immobilization of engineered C-phycocyanin to surface.

Enzymatic crosslinking of proteins is often limited by the steric availability of the target residues, as of tyrosyl side chains in the case of tyrosinase. Carrying an N-terminal peptide-tag containing two tyrosine residues, the fluorescent protein C-phycocyanin HisCPC from Synechocystis sp. PCC6803 was crosslinked to fluorescent high-molecular weight forms with tyrosinase. Crosslinking with tyrosinase in the presence of L-tyrosine produced non fluorescent high-molecular weight products. Incubated in the presence of tyrosinase, HisCPC could also be immobilized to amino-modified polystyrene beads thus conferring a blue fluorescence. Crosslinking and immobilization were site-specific as both processes required the presence of the N-terminal peptide in HisCPC.

Nanostructure development during peroxidase catalysed cross-linking of α-lactalbumin

Whereas extensive work has been done on the food functional and chemical aspects of enzymatic protein cross-linking, relatively little is known about the nanostructure and physical-chemical properties of enzymatically cross-linked protein. We investigate how nanostructure develops during enzymatic cross-linking of the 4 tyrosine residues of the globular protein apo α-lactalbumin. Protein cross-linking is catalysed by Horseradish Peroxidase, under the periodic addition of peroxide. We use on-line static and dynamic light scattering, combined with on-line UV-spectroscopy to simultaneously probe the development of nanostructure, the extent of dityrosine formation, and the catalytic state of the enzyme, as a function of the number of peroxide additions. It is found that initially, the rate of dityrosine formation is high, whereas the increase in the solution size of the cross-linked protein is limited. At later stages, the increase in solution size is significant whereas dityrosine formation slows down. Finally, the reaction stops due to enzyme inactivation. Off-line size exclusion chromatography shows that the initial phase corresponds to a fast cross-linking of monomers into small oligomers, followed by a slower joining of oligomers into large protein polymers. Consistent with this, Atomic Force Microscopy shows very heterogeneous polymers, apparently consisting of subunits that we identify with the oligomers formed in the first phase of the reaction. The dependence of the solution size on the molar mass of the cross-linked protein is determined using static and dynamic light scattering on fractionated reaction products. For sizes ranging from 30 nm to 80 nm, the protein polymers consist of 100–1000 α-lactalbumin subunits, and have molar masses of 106–107 g/mol. Apparent internal protein densities of the protein polymers calculated from these numbers are only a few weight percent, indicating a very dilute, open architecture of the cross-linked protein.

Directing enzymatic cross-linking activity to the air–water interface by a fusion protein approach

Enzymatic cross-linking of proteins is of great interest due to an effective and controlled way of modifying the structures of protein networks. Enzyme-aided structural engineering aims at enhanced stabilisation of foams, emulsions and dispersions by enzymatically inducing intra- and intermolecular cross-links between proteins in continuous phase and/or at interfaces. Formation of stronger interfacial structures protects air bubbles or oil droplets against coalescence, and in some applications, may even be preferable to bulk reactions. In this work we studied direction of enzymatic cross-linking reactions to the air–water interface by enhancing the functionality of enzymes as fusion proteins. We used a hydrophobin–laccase fusion protein, HFBI–MaL, where the surface active hydrophobin component facilitates the access for laccase to cross-link β-casein film directly at the air–water interface. As a result, enhanced cross-linking was shown by means of surface dilatational rheology, where increased G moduli indicated formation of a stronger film with the fusion protein compared to native laccase.

Discovery and Structure–Activity Relationship of Potent and Selective Covalent Inhibitors of Transglutaminase 2 for Huntington’s Disease

Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.

Effect of protein structural integrity on cross-linking by tyrosinase evidenced by multidimensional heteronuclear magnetic resonance spectroscopy

Enzymatic cross-linking of proteins can be catalyzed either by transferase-type enzymes, e.g., transglutaminases, or by oxidoreductases, e.g., tyrosinases or laccases. Three-dimensional structure of protein substrate plays a key role in these reactions, that is, the reactivity and end product are strongly modulated by the accessibility of target amino acid residues to the cross-linking enzyme. Typically structural integrity of protein can be distorted by heat, pH, or mechanical action, as well as by varying ionic concentration of the solution. In this study we used partially unfolded protein (wild-type DrkN SH3) and its structurally stabilized mutant (T22G) to investigate the impact of folded/unfolded conformations on cross-linking by Trichoderma reesei tyrosinase. Our results clearly showed formation of intermolecular cross-links solely between unfolded conformations, making them superior substrates to folded proteins when using tyrosinase as a cross-linking enzyme. Multidimensional heteronuclear magnetic resonance experiments in solution state were employed to investigate cross-linked end-products. The results presented in this study form basis for application development in food, medical, cosmetic, textile, packing and other sectors. In addition, the outcome of this study has a high value for the basic understanding of reaction mechanism of tyrosinases on proteins.

Directing the Oligomer Size Distribution of Peroxidase-Mediated Cross-Linked Bovine α-Lactalbumin

Enzymatic protein cross-linking is a powerful tool to change protein functionality. For optimal functionality in gel formation, the size of the cross-linked proteins needs to be controlled, prior to heating. In the current study, we addressed the optimization of the horseradish peroxidase-mediated cross-linking of calcium-depleted bovine alpha-lactalbumin. To characterize the formed products, the molecular weight distribution of the cross-linked protein was determined by size exclusion chromatography. At low ionic strength, more dimers of alpha-lactalbumin are formed than at high ionic strength, while the same conversion of monomers is observed. Similarly, at pH 5.9 more higher oligomers are formed than at pH 6.8. This is proposed to be caused by local changes in apo alpha-lactalbumin conformation as indicated by circular dichroism spectroscopy. A gradual supply of hydrogen peroxide improves the yield of cross-linked products and increases the proportion of higher oligomers. In conclusion, this study shows that the size distribution of peroxidase-mediated cross-linked alpha-lactalbumin can be directed toward the protein oligomers desired.

Effect of crystalline substances in biodegradable films

Films made of sodium caseinate and gelatine were prepared by casting method from a water solution containing glycerol as a plasticizer to obtain environmentally friendly and fully biodegradable materials for agricultural and packaging applications. Additionally, enzymatic protein cross-linking by microbial transglutaminase was applied. Potassium nitrate (KNO 3) was used as additive to investigate the influence of crystallization on the physical properties of protein films. Mechanical properties (tensile strength and elongation at break) of the protein films were determined versus ratio of protein to potassium nitrate in the presence and absence of microbial transglutaminase. Furthermore, optical properties (film formation and consistency, surface texture, crystal size, shape, and distribution of incorporated potassium nitrate) were examined. An increase of 122% and 177% in the elongation of the films was adopted due to the crystallization of KNO 3 in enzymatic-modified sodium caseinate and gelatine films, respectively. Pure sodium caseinate films distinguished ultimate tensile strengths of 4.95 MPa, while MTG-treated gelatine films achieved ultimate tensile strengths of 13.52 MPa. Altogether, the most appropriate overall mechanical performance was obtained for KNO 3/protein ratios of 1:6 in enzymatic-modified films. Furthermore, an increasing content of crystalline KNO 3 results in increasing thickness, rough surfaces, decreased opacity, and whitish coloring of the films.

Yoghurt gel formation by means of enzymatic protein cross-linking during microbial fermentation

A novel yoghurt fermentation process was investigated where the gel formation by isoelectric coagulation is enhanced by enzymatic protein cross-linking by transglutaminase (TG). In contrast to most previous studies the TG was applied during fermentation so that no additional process time is required. The challenge was whether the microbial acidification leaves enough reaction time for the enzyme, which gradually looses its activity upon pH drop and whether the enzyme is completely inactive when the pH has reached values below 4.5. Residual enzyme activities were measured in model experiments as a function of the pH in the range of 3.0–7.0. No residual activity was found below a pH of 4.0 at 40 °C for 120 min exposure time. During yoghurt fermentation, there was no effect of the enzyme on the fermentation time. It was found that 80% of the cross-linking effect is obtained within the pH range of 6.6–5.7. A higher viscosity and degree of protein polymerisation of TG-treated stirred yoghurt was found in comparison to non-cross-linked controls. Over storage time, however, the apparent viscosity of cross-linked yoghurt increased and a visibly coarse gel structure was formed which appears to be related to the high casein content. However, no formation of TG-induced covalently cross-linked protein polymers was measurable while storage. Hence, these structural changes are to be explained by a secondary effect of TG treatment, i. e. by other non-covalent bonds influencing the protein network.

Effects of transglutaminase treatment on functional properties and probiotic culture survivability of goat milk yogurt

The objectives of this study were to investigate the effects of enzymatic cross-linking of milk proteins on the functional properties and probiotic culture survivability of goat milk yogurt. After pre-incubation (50 °C, 1 h) of the milk with microbial transglutaminase [MTGase, 0–4 units (U)/g protein] and subsequent heat inactivation (75 °C, 5 min), yogurt was prepared by inoculating the milk with a commercial probiotic yogurt starter containing probiotic cultures ( Lactobacillus acidophilus, Bifidobacteria, and Lactobacillus subsp. casei) and incubating the inoculated milk at 43 °C for 5 h. Compared to the untreated control trial, pretreatment of the milk with MTGase (2 and 4 U/g protein) increased ( P < 0.05) the viscosity and decreased the syneresis of yogurt samples. The effect of enzyme treatment was more significant in improving the viscosity of yogurt compared to the method of increasing total solids (TS) in the milk. No significant differences in basic nutrient contents between control and enzyme-treated samples were observed. The probiotic cultures were relatively stable in the goat milk yogurt and their populations remained above 10 6 CFU/g during the 8-week storage period at 4 °C. No significant differences in rate of change of population of all the three probiotic cultures were observed between the control and the MTGase treated groups. Microbiological analysis shows that the enzymatic cross-linking of proteins seems to have a positive role in the survivability of the probiotic cultures, which requires further investigation. Scanning electron microscopic studies revealed that the microstructure of the enzyme-treated samples appeared denser than the control, suggesting that pretreatment of milk by MTGase could be used to improve the microstructure of the yogurt gel. Results of this study indicate that enzymatic cross-linking of milk proteins by MTGase appears to be an effective means for improving the functional properties of goat milk yogurt. MTGase treatment may be a useful method in the production of probiotic goat milk yogurt products.

Peptidyl linkers for protein heterodimerization catalyzed by microbial transglutaminase.

Specific peptidyl linkers that result in the heterodimerization of functional proteins, which is catalyzed by microbial transglutaminase from Streptomyces mobaraensis (MTG), were generated based on a ribonuclease S-peptide using site-directed mutagenesis. The peptidyl linkers designated as Lys-tag and Gln-tag were designed to possess sole reactive Lys or Gln residue that was amenable for selective Lys-Gln cross-linkage of different proteins. Green fluorescent protein variants, ECFP and EYFP, were employed as model proteins, and those Lys- and Gln-tags were fused to the N-termini of ECFP and EYFP, respectively. As a result, we succeeded in solely obtaining the ECFP-EYFP heterodimer without forming multiply cross-linked byproducts. It was found that the reactivity of peptidyl linkers varied according to the type of amino acid to be replaced. Peptidyl linkers with a basic amino acid (Arg) exhibited the highest reactivity in the cross-linking reaction, suggesting the cationic residue substrate preference of MTG. Kinetic analysis utilizing fluorescent resonance energy transfer (FRET), that is only observed upon the heterodimeric ECFP-EYFP conjugation, revealed that the amino acid replacement contributed to the acceleration of cross-linking reactions by increasing catalytic turnover (k(cat)), rather than substrate binding affinity (K(m)). Finally, using a ribonuclease S-protein, the manipulation of enzymatic protein cross-linking based on specific S-peptide:S-protein interactions was explored. Since newly designed Lys- and Gln-tags retained binding affinities to the S-protein, the heterodimerization was perfectly restrained by wrapping them with the S-protein. The results suggest the possibility of limited protein conjugation by tuning steric hindrance against the MTG. Tailoring enzymatic posttranslational modifications with either engineering peptidyl substrates or by taking specific peptide-protein interactions into consideration may facilitate the development of a new sequential protein conjugation method for the preparation of multifunctional protein.

Enzymatic cross-linking of proteins with tyrosinase

Tyrosinase (EC 1.14.18.1), usually known as the enzyme responsible for the enzymatic browning of fruits and vegetables, has been demonstrated to induce cross-linking of the whey proteins α-lactalbumin and β-lactoglobulin. The maximum degree of polymerisation for 1.5 mg/ml protein has been achieved at a tyrosinase activity of about 330 U/ml in the presence of a 2 mM concentration of caffeic acid. The pH optimum for the cross-linking reaction was detected at pH 4–5 for α-lactalbumin and β-lactoglobulin, in contrast to pH 7 for lysozyme, a non-whey protein, indicating that for the whey proteins the initial enzymatic oxidation of the phenolic compound is not the rate limiting step for the reaction. In contrast to β-lactoglobulin and lysozyme, for α-lactalbumin a direct cross-linking even without addition of caffeic acid has been proved. In this manner α-lactalbumin polymers with a molecular weight larger than 300 kDa have been produced by increasing the reaction temperature up to 50 °C. The results of the present study represent a basis for the creation of new high molecular weight proteins. Due to a different reaction mechanism and different points of linkage, tyrosinase-induced protein cross-linking may be a promising alternative to the already established use of transglutaminase for food application.

Effect of a microbial calcium-independent transglutaminase on functional properties of a partially purified cowpea (vigna unguiculata) globulin

A partially purified globulin from cowpea seeds was polymerised by calcium-independent microbial transglutaminase. The level of free amino groups in the globulin decreased with increase in enzyme concentration, suggesting that the cross-linking reaction involved participation of the amino residues. At transglutaminase concentrations of 6–18 μg ml−1 emulsifying activity decreased with increasing enzyme concentration and degree of cross-linking, while a similar trend was observed for the foaming property at 6–12 μg ml−1 transglutaminase concentrations. The resultant emulsions and foams formed by the cross-linked proteins were more stable than those formed by the untreated protein. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that of the two major polypeptides, the 55 kDa protein was more susceptible to transglutaminase action than the 50 kDa protein. Scanning electron microscopy revealed an amorphous structure for the control protein gel while a more defined and cross-linked gel structure was observed for the protein treated with transglutaminase. The results suggest that protein products differing in functionalities can be obtained by controlling the degree of enzymatic protein cross-linking. © 1999 Society of Chemical Industry