Cross-linked Myosin Heavy Chain Research Articles

Comparison of Atlantic menhaden gels from surimi processed by acid or alkaline solubilization

Heat-induced gelling abilities of surimis prepared by pH shifting (isoelectric precipitation following acid (AC) or alkaline (AL) solubilization) were compared to that of conventionally washed (CW) surimi. Greater endogenous transglutaminase activity (evidenced as enhanced strength of cooked gels subjected to 30–40 °C preincubation) was measured for CW and AL surimi than for AC surimi (all at pH 7). Upon addition of microbial transglutaminase (MTGase), increased crosslinking of myosin heavy chain and gel strengthening during 30–40 °C preincubation were apparent for all three types of surimi, most markedly in CW and AL surimi. Salt addition improved CW gels most, but seemed to adversely affect MTGase activity in AC and AL surimi. AC and AL surimi gels were of lower whiteness than were CW surimi gels.

Effects of microbial transglutaminase and starch on the thermal gelation of salted squid muscle paste

The addition of microbial transglutaminase (MTGase) to salted squid muscle paste greatly strengthened the elasticity of the thermal gel, which was produced by the preferential crosslinking of myosin heavy chains through a two-step heating process that consisted of setting at 40°C and subsequent heating to 80°C or 90°C. Starch increased the breaking strength of thermal squid gels, but decreased the deformation. Thus, the starch-added gels became harder and less elastic. Although, when both MTGase and starch were added to squid muscle paste, the changes in viscoelasticity and myosin cross-linking reaction were similar to those with MTGase alone during setting, the storage and loss moduli of the paste sharply increased with an increase in starch content above 70°C. Subsequently, the thermal gel texture became more brittle or breakable than gels prepared by setting with MTGase only.

Combination effects of chicken plasma protein and setting phenomenon on gel properties and cross-linking of bigeye snapper muscle proteins

Effects of chicken plasma protein (CPP) in combination with setting on surimi gel properties and protein cross-linking were investigated. Addition of CPP (0.5g/100g), CaCl2 (10mmole/kg) and 200 units of thrombin/g CPP in combination with setting at 40°C for 30min prior to heating at 90°C for 20min resulted in the highest breaking force and deformation (P<0.05). Regardless of CPP addition, myosin heavy chain (MHC) in surimi proteins and natural actomyosin (NAM) underwent polymerization to some extent in the presence of CaCl2 and thrombin. The cross-linking of MHC in surimi proteins were markedly suppressed by the addition of EGTA and NH4Cl, transglutaminase (TGase) inhibitors. No cross-linking of myosin was observed when CPP, CaCl2 and thrombin were added, even with the prolonged incubation time. The result revealed that CPP showed no cross-linking activity and gel strengthening effect of CPP was attributed to its filler effect and proteolytic inhibitory activity.

Biochemical susceptibility of myosin in chicken myofibrils subjected to hydroxyl radical oxidizing systems.

Biochemical changes of myosin in chicken myofibrils exposed to nonenzymatic, hydroxyl radical generation systems (HRGS) were investigated by means of cross-linking reaction, ATPase activity, salt solubility, and 40% saturated ammonium sulfate (AS) extractability. HRGS treatment of myofibrils caused cross-linking of myosin heavy chains (MHC) via disulfide bonding, an increase in Ca-ATPase activity, and a decrease in K-ATPase activity, suggesting that thiol groups of myosin including those at the active site were modified. The specific changes depended on the concentrations of H(2)O(2) in HRGS as well as the weight ratio of H(2)O(2) to myofibrils. On the other hand, the decrease in salt solubility or AS extractability of myosin in HRGS-treated samples proceeded slowly when compared with the cross-linking reaction of MHC, indicating that considerable amounts of myosin biopolymers remained hydrophilic in the ionic solutions. The results demonstrated that initial cross-linking of MHC occurred inside the myosin molecule, and this was followed by progressive aggregation of myosin molecules through intermolecular cross-linking. Oxidation under the current experimental condition decreased the gel-forming ability of myofibrillar proteins, which coincided with the progress of the intra- and intermolecular cross-linking reactions as well as with ATPase activity changes.

Influence of Endogenous Proteases and Transglutaminase on Thermal Gelation of Salted Squid Muscle Paste

ABSTRACT: Thermal gelation of salted squid mantle muscle paste was studied in relation to endogenous proteases and transglutaminase. Myosin in the paste was preferentially degraded into 130‐kDa and 90‐kDa fragments at an optimum temperature of 30 °C. Degradation was inhibited with EDTA or 1,10 phenanthroline, suggesting the presence of metalloproteases. Myosin degradation was markedly reduced above 40 °C. Although 10 mM Ca2+ increased cross‐linking of myosin heavy chains by activating the endogenous transglutaminase, setting effect on thermal gelation of the paste was offset by degradation induced by simultaneously activated calpains. Ca2+ and the alpain inhibitor, E64, significantly improved the breaking strength and strain of thermal gels preincubated at 40 °C.

Thermal gelation of salted paste from scallop striated adductor muscle

: Salted muscle paste containing 60–100 mg protein/g paste and 0.5 M NaCl at pH 7.0 was prepared from fresh scallop striated adductor muscle and its heat-induced gelling characteristics were examined in order to produce invertebrate kamaboko-like foods. The heating of the paste to 90°C formed a weak gel. The setting effect on the gelation of the paste was introduced by incubation at 25°C for up to 4 h and was greatly strengthened by the addition of 10 mM Ca2+ due to the activation of an endogenous transglutaminase that preferentially cross-linked myosin heavy chains, but not actin and paramyosin. As a result, the texture of the gel produced by a two-step heating method was almost the same as that of a commercial fish kamaboko. It was also observed that the thermal gelation profile of scallop muscle paste was similar to that of fish myosin, rather than that of actomyosin, by means of a dynamic rheological measurement. Paramyosin also contributed to a characteristic increase in the G′ (storage modulus) value over the thermal gelation process and raised the G′′ (loss modulus) value around 65°C. The G′′ increase resulted in a slightly rigid texture of heated scallop gel. Isolated paramyosin demonstrated a characteristic thermal gelation profile showing a two-step increase in G′ and G′′ values, independent of Ca2+ concentration. The higher NaCl and protein concentrations were required to produce stronger and more elastic gels.

Cross-linking activity of sarcoplasmic fraction from bigeye snapper ( Priacanthus tayenus) muscle

Addition of sarcoplasmic fraction from bigeye snapper ( Priacanthus tayenus) into natural actomyosin in combination with setting at 40°C resulted in the cross-linking of myosin heavy chain (MHC). Higher amount of sarcoplasmic fraction and extended setting time resulted in a higher cross-linking, indicating the presence of endogenous transglutaminase (TGase) in bigeye snapper muscle. TGase activity was activated by calcium ion and reducing agents ( β-mercaptoethanol and dithiotreitol), but was inhibited by N-ethylmaleimide (NEM), NH 4Cl and EDTA. TGase in the sarcoplasmic fraction was not stable when heated at temperature above 40°C, particularly with an increasing heating time. TGase was stable at pH ranging from 5.0 to 7.0, in which more than 70% activity was retained. Therefore, sarcoplasmic fraction possessed a cross-linking activity caused by TGase and its recovery for further uses should be considered.

Effect of Endogenous Transglutaminase on Threadfin Bream Surimi Gelation

ABSTRACT: Transglutaminase(TGase) activity of threadfin bream mince was 99.6 units/g of dry weight. After washing and screw‐pressed dewatering, 44% residual activity was retained. Covalent cross‐linking of myosin heavy chain (MHC) was observed at both 25 and 40°C and supported by increased gel strength. When pre‐incubation at 40°C was prolonged to 4 h, breaking force and MHC decreased due to endogenous proteinase(s). TGase activity towards MHC and synthetic substrates was effectively inhibited by iodoacetic acid (IAA). Autolytic activity and degradation of MHC was inhibited by phenylmethanesulfonyl fluoride (PMSF). Addition of 0.2% Ca2+ significantly improved breaking force and increased MHC cross‐linking of surimi gels pre‐incubated at 40°C for 2 h. Keywords: transglutaminase, myosin heavy chain, cross‐linking, threadfin bream

Microbial Transglutaminase and Recombinant Cystatin Effects on Improving the Quality of Mackerel Surimi

ABSTRACT: Effects of microbial transglutaminase (MTGase), recombinant cystatin, or their combination on the gel properties of mackerel surimi were investigated. The addition of MTGase caused the cross‐linking of myosin heavy chain (MHC) and substantially increased the gel strength (from 536.6 to 2012.4 g × cm), while the recombinant cystatin could effectively prevent the MHC degradation and gel softening during the production of mackerel surimi‐based products. Combined use of MTGase and recombinant cystatin revealed synergistic effectiveness on improving the quality of mackerel surimi (increased from 435 to 2438 g × cm, set at 45 °C for 20 min).

Beta-Actinin, Cap Z, connectin and titin: what's in a name?

beta-Actinin, Cap Z, connectin and titin: what's in a name?

Effect of pH on the gelation of walleye pollack surimi and carp actomyosin pastes

: The effect of pH on thermal gelation and transglutaminase (TGase; EC2.3.2.13)-induced suwari (setting) of surimi and actomyosin pastes was investigated. A strong and elastic gel was produced from walleye pollack surimi paste at pH 7.0 in the presence of Ca2+ using a two-step heating method. In contrast, walleye pollack actomyosin paste formed a weak gel under the same conditions as a result of the low concentration of endogenous TGase. In the presence of EGTA [ethyleneglycol bis(2-aminoethylether) tetraacetic acid], weak gels were formed at pH values of 7.0 and 6.0. Non-proteolytic modori (gel weakening) occurred extensively in the course of actomyosin gelation, but not in surimi gelation. Maximum TGase-induced myosin heavy chain cross-linking was observed at a slightly higher pH of 7.5 than at the optimal pH of endogenous TGase activity; the difference being derived from different substrates. Gelation of carp actomyosin paste at pH values of 5.5, 6.0, 6.5 and 7.0 was monitored by measuring storage modulus (G′) and loss modulus (G″). A weak gel was formed at all pH values, but a slightly rigid and less elastic gel was obtained at lower pH values. The addition of microbial TGase (MTGase) formed strong elastic gels at pH 7.0 and 6.5. MTGase cross-linked myosin heavy chains even at pH 5.5, but contributed neither to suwari response nor strong gel formation. Overall, results suggest that the optimal pH for the gelation of surimi paste from easy-setting fish species is a compromise between the pH-optima of TGase activity and of preferable actomyosin conformation for myosin cross-linking.

DIFFERENT ARRANGEMENT OF ?-(?-GLUTAMYL)LYSINE CROSS-LINKING IN ALASKA POLLOCK (THERAGRA CHALCOGRAMMA) SURIMI PROTEINS BY STREPTOVERTICILLIUM AND ENDOGENOUS TRANSGLUTAMINASES DURING SUWARI PROCESS

The objective of the present study is to compare the protein cross-linking reaction in Alaska pollock surimi that is catalyzed by a commercially available microbial transglutaminase and by endogenous Alaska pollock transglutaminase The endogenous transglutaminase was inhibited by EGTA and activated by CaCl 2 . The microbial transglutaminase was added to the salted surimi with and without EGTA and CaCl 2 . These surimi pastes were incubated at 25C up to 24 h followed by cooking at 90C. The resultant gels were fractionated into soluble and insoluble (aggregate) fractions by SDS-urea extraction. Compositional analysis revealed that the aggregate consisted predominantly of cross-linked myosin heavy chain. The distribution of e-(γ-glutamyl)lysine isopeptide in the soluble and aggregate fractions andpeptide mapping analyses ofthe aggregate fraction demonstrate thatthe formation of isopeptide cross-links in Alaska pollock surimi proteins during suwari process differs when catalyzed by the microbial transglutaminase and endogenous transglutaminase. La pâte de surimi peut gelifier a faible temperature (20-40°C) sans procede de cuisson. L'objectif de cette etude est de comparer la reticulation des proteines, pendant le procede de gelification dans du surimi de Theragra chalcogramma, par une transglutaminase commerciale microbienne (Streptoverticillium) et par une transglutaminase endogene. Les pâtes de surimi sont incubees a 25°C pendant 24h puis cuites a 90°C. Les gels produits sont separes en fractions solubles et insolubles et analyses.

PORCINE PLASMA PROTEINS AS GEL ENHANCER IN BIGEYE SNAPPER (PRIACANTHUS TAYENUS) SURIMI

Cohn 's fraction I-Sfiom porcine plasma showed the highest transglutaminase activity, compared to fractions I, II+III, IV, IV-I. The optimum temperature for incorporating monodancylcadaverine into dimethylated casein was 45C. Plasma transgfutaminase in fraction I-S was activated by calcium chloride but was inhibited by N-ethylmaleimide, ethylenediaminetetraacetic acid, and ammonium chloride. The addition of fraction I-S into bigeye snapper surimi resulted in a substantial increase in gel breaking force and deformation, particularly in the presence of calcium chloride and thrombin. No changes in whiteness and water holding capacity were observed in surimi gel with the addition of 0-0.5% of fraction IS. Fraction I-S was found to catalyze nondisulJide covalent cross-linking of myosin heavy chain. The combination of endogenous and plasma transglutaminase enhanced surimi gelation.

Cross-Linking of Mackerel Surimi Actomyosin by Microbial Transglutaminase and Ultraviolet Irradiation

The effect of combining microbial transglutaminase (MTGase) from Streptoverticillium ladakanum with ultraviolet (UV) irradiation on the gelation of minced mackerel was investigated. The gel strength of minced mackerel with MTGase alone at a concentration of 0.47 unit/g reached 1789 g·cm, which was 3 times greater than that of control. When MTGase-supplemented minced mackerel was exposed to UV light for the optimal irradiation time of 20 min, the gel strength could be further increased by 25%. SDS−PAGE analysis suggested that UV irradiation accelerated the MTGase to catalyze the cross-linking of myosin heavy chains in mackerel actomyosin. Keywords: Microbial transglutaminase; mackerel surimi; actomyosin; Streptoverticillium ladakanum; UV irradiation

Proteolysis and gelation of fish proteins under ohmic heating.

Pacific whiting surimi gels heated slowly in a water bath exhibited poor gel quality, while the ohmically heated gels without holding at 55 degrees C showed more than a twofold increase in shear stress and shear strain over conventionally heated gels. Degradation of myosin and actin was minimized by ohmic heating, resulting in a continuous network structure. Ohmic heating with a rapid heating rate was an effective method for maximizing gel functionality of Pacific whiting surimi without enzyme inhibitors. In linear heating, slow heating rates increased proteolysis in Pacific whiting surimi as shown by degradation of myosin heavy chain and low shear stress and shear strain. Proteolysis of whiting surimi was decreased by the presence of beef plasma protein (BPP) to a greater extent at rapid heating rates (20 and 30 degrees C/min) than at slow heating rates (1 and 5 degrees C/min). Shear stress of Alaska pollock surimi gels with or without BPP increased as heating rate decreased, but shear strain was unaffected. An increase in shear stress was accompanied by an increase of cross-linked myosin heavy chain.

加熱に伴うスケトウダラ肉糊中のミオシン重鎖の交差結合と牛血しょう粉末の影響

Kamaboko gels were prepared with 0 ?? 5.0% bovine plasma powder, a food additive, by an ordi-nary manner through preheating at 25°C and 40°C of salt-ground meat from walleye pollack frozen surimi. The quality of kamaboko gel was assessed by measuring breaking strength, breaking strain and the progress of cross-linking of myosin heavy chains.At 25°C, the gelling property was strengthened with plasma powder, that is, the increase in breaking strength and breaking strain were at a maximum when the addition of plasma powder was 5.0% and 1.0%. Simultaneously, the plasma powder in the salt-ground meat suppressed the decrease in myosin heavy chain and the increase in unidentified components, X1, while the cross-linked myosin heavy chains were accumulated with increasing amounts of plasma powder.At 40°C, the plasma powder suppressed one-sided deterioration of the gelling property of the salt-ground meat. The breaking strength and breaking strain of kamaboko gel with plasma powder maintained the same level as those prepared without plasma powder and preheating. The plasma powder suppressed the rates of decrease in myosin heavy chain and increase in the X1 components. However, the cross-linked myosin heavy chains were hardly formed regardless of the presence and absence of plasma powder.These results suggested that the enhancing effect of bovine plasma on the gelling property of salt-ground meat was attributable to strengthening of the interactions among myofibrillar proteins and plasma proteins through noncovalent type bonds.

サケ落し身から形成した加圧ゲルの品質に対する加熱の影響

Mince of chum salmon mixed with 4% sorbitol, 4% sucrose, and 0.3% polyphosphate salt was stored at -45°C. The frozen mince was thawed and ground with 2.5% NaCl and 150ppm chlorumphenicol. The salted mince was texturized under high pressure of 200-1, 000MPa at 0°C. The texturized gel under pressure (termed the pressure-induced gel) was subsequently kept at 5°C or heated at 30-90°C. The salted mince was also texturized under the pressure of 300 or 500MPa at 20-80°C. The quality of 4 types of these pressure-induced gels was assessed by measuring the breaking strength, breaking strain and the change in myosin heavy chains in the same gels. The results were as follows: (1) The breaking strength of the pressure-induced gel was only slightly reinforced with the following aging period at 5°C, although the breaking strength of the salted mince enhanced with increasing pressure. (2) When the salted mince was texturized with 500MPa at 0°C for 10 min and subsequently heated at 80°C for 30 min, the breaking strength of the gel largely increased and reached a higher value (ca. 650g), but the breaking strain remained at a low level (ca. 9.0mm). (3) The cross-linking of myosin heavy chains hardly occurred in all types of the pressure-induced gels.

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.

スケトウダラ坐り‐加熱ゲルのゲル物性に及ぼすピロリン酸塩の効果

スケトウダラ坐り‐加熱ゲルのゲル物性に及ぼすピロリン酸塩の効果

Tissue-Type Transglutaminase from Red Sea Bream (Pagrus major). Sequence Analysis of the cDNA and Functional Expression in Escherichia coli

A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from a cDNA library prepared from the liver of red sea bream (Pagrus major). The cDNA sequence had an open reading frame coding for a protein of 695 amino acids and showed 43 % identity to the sequence of guinea pig liver TGase, revealing a relatively low overall similarity. However, the 25-amino-acid sequence containing the putative active site (Cys272) of the enzyme was completely conserved between the two species, and was also identical to the corresponding regions of human and bovine endothelial cell TGases. In addition, the critical residues (His332 and Asp355) thought to form the catalytic-center triad together with Cys272, were found in the highly conserved region. The red sea bream TGase had an extension of 11 amino acids in the C-terminal region and some differences in the N-terminal region when compared with guinea pig TGase. From the cloned cDNA, a semi-synthetic TGase gene suitable for overexpression in Escherichia coli was constructed (pTTG2-22). At a reduced temperature (28°C), E. coli cells transformed with pTTG2–22 could produce soluble TGase which exhibited catalytic activity in the presence of calcium. E. coli extracts containing the recombinant red sea bream TGase induced gelation of actomyosin solutions, accompanied by a significant increase of ɛ-(γ-glutamyl)lysine bonds, which are predominantly derived from the cross-linking of myosin heavy chains. These results indicate that this fish TGase should be useful for further analysis of TGase structure/function relationships and that it could also be employed to enhance the viscoelastic properties of proteinaceous materials.