1 Introduction

Asian seabass (Lates calcarifer) is a prominent aquaculture species in Thailand, especially in the area around Songkhla Lake. As per the Department of Fisheries, Thailand, this lake has more than 7000 fish cages, producing about 33,529 tons of sea bass annually. Fish waste is considered a rich source of oil, proteases, lipases, and transglutaminase (TGase) [1]. Asian seabass has been used to produce biocalcium [2, 3], gelatin or its hydrolysate [4, 5], collagen or its hydrolysate [6, 7], etc. However, rare information is available on the extraction of TGase, which is mainly contaminated with other enzymes, especially protease, which has made TGase extraction from fish waste challenging. In general, TGase catalyzes the acyl transfer reaction between a glutamine residue in a peptide chain and the ε-amino group of a lysine residue, resulting in the formation of an ε-(γ-glutamyl) lysine cross-link, which resulted in the cross-linking of proteins. It has been used widely in various sectors including pharmaceuticals/medicine [8], especially in the food sector to provide the desired texture to foods, improve water-holding capacity and elasticity in meat gels, etc. [9,10,11]. Several reports have been available on the use of TGase from different sources to improve the gel quality of surimi obtained from various fish, especially from sardine, Indian mackerel or other pelagic fish, which possessed poor gel quality [12, 13].

Microbial sources like Amycolatopsis sp. are the most widely raw material for TGase production, with different methodologies such as salt precipitation methods, gel chromatography, aqueous two-phase systems (ATPS), etc. [1, 14]. In addition, TGase from new novel sources such as edible insect larvae, has also been explored [15]. TGase from bigeye snapper, oil sardine, tilapia, and common carp showed different purification folds and yields, highlighting the importance of selecting appropriate sources and purification methods [16]. Laksono et al. [17] extracted TGase from daggertooth pike conger fish and enhanced the purity through dialysis, demonstrating another effective method for enzyme purification. So far, no information has been reported on the extraction of TGase from Asian seabass. Considering their consumption and waste generated in Southeast Asian nations, especially Thailand, effective utilization of visceral organs should be employed to enhance the overall income of the farmer and fish industry. Despite these advances, no information has been reported on the extraction of TGase from Asian seabass, a significant gap considering the potential of this species as a valuable source of TGase.

Natural actomyosin has been used widely to determine the role of several additives such as phenolic compounds, protein isolates, protease inhibitors, hydrocolloids, etc. in vitro model system [18,19,20,21]. NAM lies in its critical role in muscle structure and its extensive use in food products such as surimi and processed meat, where improving texture and gelation properties is of high importance [22]. Since NAM is a major component of fish muscle proteins, evaluating the effectiveness of TGase in cross-linking NAM proteins directly reflects the enzyme’s potential application in enhancing the textural qualities of fish-based food products.

Thus, the current study focused on the extraction, purification, and characterization of TGase from Asian seabass liver. In addition, the effect of partially purified TGase (pTGase) on the cross-linking of natural actomyosin (NAM) proteins was also elucidated. This study aims to fill the knowledge gap and provide insights into the potential of Asian seabass as a source of TGase, contributing to both scientific knowledge and practical applications in various fields, particularly in food science.

2 Materials and methods

2.1 Raw material and chemicals

Asian seabass (Lates calcarifer) visceral organs containing liver were purchased from the local fish market (Hat Yai, Songkhla, Thailand). All chemicals used for the extraction, purification, and characterization of TGase were analytical grades.

2.2 Extraction of TGase

Firstly, the liver was separated from the other visceral organs and washed with iced cooled water to remove blood and other impurities, then blended using a blender. The mashed liver was subjected to fat removal twice using chilled acetone at 1:3 (w/v) followed by drying as per the method of Baloch et al. [23]. The dried powder was homogenized with extraction buffer (EB) containing 50 mM tris-hydrochloric acid (pH 7.5), 10 mM sodium chloride (enhance the solubility of proteins), 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol (DTT) and 10 µM tosyl-l-lysine chloromethyl ketone (TLCK) using a homogenizer at 10,000 rpm for 2 min. EDTA and TLCK act as metal chelator and protease inhibitors, respectively, which helps to control the protease activity. DTT, a reducing agent maintains the thiol groups of cysteine residue, which could be oxidized or degraded during the extraction process. Thereafter, undissolved residues were separated via centrifugation at 10,000 rpm for 30 min and the supernatant was obtained via filtration using two layers of cheesecloth. The obtained supernatant was named “crude extract”.

2.3 Purification of TGase

Crude extract was subjected to ammonium sulfate precipitation at various concentrations ranging from 0–20, 20–40, 40–60, 60–80, to 80–100%. The obtained pellet from each fraction was dissolved in 10 mM Tris–HCl at pH 8.0 containing 1 mM CaCl2 (starting buffer; SB). The obtained fractions were determined for TGase and protease activities. The TGase activity was determined via hydroxamate method using N-carbobenzoxy (CBZ)-l-glutaminylglycine as a substrate [24]. The protease activity was determined using the casein as a substrate following the method given by Karnjanapratum and Benjakul [25]. The protein content of each fraction was determined using the Lowry et al. [26] method.

Ammonium sulfate fraction showing the highest activity was further purified DEAE-Sepharose (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden) column. The column was equilibrated with SB and washed with three-bed volumes of the same buffer at a 1 mL/min flow rate. The bound proteins were eluted with a linear gradient of 0–0.75 M NaCl. Fractions of 6 mL were collected and dialyzed (MW cut off: 500 Da) against SB buffer followed by the protein content and TGase activity determination.

2.4 Characterization of purified TGase

2.4.1 Optimal temperature and pH

TGase activity was measured as described previously over a wide range of pHs and temperatures. For optimal pH, different buffers were used to vary the pH, for 4–7, 50 mM acetate buffer, 50 mM Tris–HCl buffer for pH values ranged between 7–9, and 50 mM glycine–NaOH buffer for pH of 9 and 10, with reactions conducted for 10 min at 30 °C. For the temperature profile study, TGase activity was assayed at various temperatures (25, 37, 40, 45, 50, 55, 60, 65, and 70 °C) for 10 min.

2.4.2 Optimal CaCl2 and NaCl concentration

TGase activity affected by the various concentrations of CaCl2 (0, 10, 20, 50, and 100 mM) and NaCl (0, 1, 2, 3, and 5%) were determined following the method described by Hemung and Yongsawatdigul [27]. TGase was dissolved in SB with varying amounts of different salts.

2.4.3 Optimal incubation time

At previously optimized temperature, pH and salt concentrations, TGase activity was determined as described previously for different incubation time (5, 10, 15, 30, and 60 min).

2.4.4 Temperature and pH stability

The effect of pH on enzyme stability was evaluated by measuring the relative activity after incubation of TGase at various pH values (4–10) for 30 min at 30 °C. Different buffers were used as previously mentioned. For thermal stability, the enzyme, dissolved in SB was incubated at various temperatures (25–70 °C) for 15 min in a temperature-controlled water bath. The treated samples were rapidly cooled in ice water before determining TGase activity [27].

2.4.5 Protein pattern and MW determination

The MW of purified TGase was determined using size-exclusion chromatography on a Sephadex G-50 resin and the protein pattern was determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4% stacking gel and 15% running gel following the method of Patil et al. [28] and Laemmli [29], respectively.

2.5 Effect of pTGase on the NAM extracted from threadfin bream surimi

2.5.1 pTGase-catalyzed polymerization

Firstly, NAM from threadfin bream surimi was prepared according to the method of Balange and Benjakul [18] with a slight modification. Then, NAM was adjusted to 5 mg/mL protein content using chilled 2.5% NaCl. Thereafter, pTGase (0, 1, 2, 4, 6, 8, and 10 unit/g protein) was added into NAM and incubated at 40 °C for different times (0, 5, 10, 15, 20, and 30 min), and then the reaction was terminated by heating at 100 °C for 5 min. The supernatant was subjected to analyses.

2.5.2 Analyses

2.5.2.1 Protein solubility

The protein solubility of NAM was determined according to the method described by Riebroy et al. [30]. NAM solution (5 mL) was centrifuged at 5000 × g for 15 min at 4 °C. The supernatant was collected, and protein content was measured by the Biuret method, using bovine serum albumin (BSA) as a standard. Protein solubility was expressed as the percentage decrease:

$$\% {\text{decrease}} = [({\text{P}}_1 - {\text{P}}_2 )/{\text{P}}_1 ] \times 100$$

where P1 indicates protein content of NAM sample added without pTGase during incubation at 40 °C, P2 indicates protein content of NAM sample added with pTGase during incubation at 40 °C at different times.

2.5.2.2 ε-amino group content

The ε-amino group content was measured according to the method of Bubnis and Ofner [31] using trinitrobenzene sulfonic acid (TNBS) with slight modifications. NAM solution (2 mL) containing pTGase at various levels was placed in a 50 mL screw-cap test tube. To this, 1 mL of each 4% (w/v) NaHCO3 (pH 8.8) and 0.5% TNBS were added. The reaction mixture was then incubated at 40 °C for 4 h with gentle shaking. After incubation, 3 mL of 6 N HCl was added, and the mixture was autoclaved at 120 °C for 1 h to hydrolyze and dissolve any insoluble components. The resulting hydrolysate was diluted to 5 mL with water. To remove excess unreacted TNBS, the hydrolysate was extracted with three 20 mL portions of ethyl ether. A 5 mL aliquot of the aqueous phase was then taken, heated in a hot water bath for 15 min to evaporate any residual ether, and diluted with 15 mL of water. The absorbance of the final solution was measured at 346 nm.

2.5.2.3 Transmission electron microscopy (TEM)

NAM samples treated with pTGase at various concentrations followed by heating at 45 °C for 30 min were diluted to 0.2 mg/mL with 50 mM potassium phosphate buffer (pH 7). A drop of sample was fixed for 5 min on carbon-coated grid, negatively stained with 4% uranyl acetate for 5 min and washed with distilled water until the grid was cleaned. The method of Balange and Benjakul (2010) was used to prepare samples for visualization using a JEOL JEM-2010 transmission electron microscope (JEOL Ltd., Tokyo, Japan).

2.6 Statistical analysis

The entire study employed a completely randomized design (CRD). Analysis of variance (ANOVA) was conducted for all data, and Duncan’s multiple range test was used for comparing means. A T-test was used to statistically compare the selected sample and control. Statistical analysis was performed using the Statistical Package for Social Science (SPSS) software (SPSS 21.0 for Windows, SPSS Inc., Chicago, IL, USA) with a set significance level. All experiments were conducted in triplicate.

3 Results and discussion

3.1 Purification of TGase

The TGase activity of different ammonium sulfate fractions are shown in Table 1. The total TGase activity was increased by increasing the concentration of ammonium sulfate to 60–80% (p < 0.05). This increase can be attributed to the optimal precipitation of unwanted proteins at this concentration, which results in a higher concentration of the desired enzyme [32]. However, with further increasing levels to 80–100%, total TGase activity was decreased slightly (p < 0.05), which was more likely due to the co-precipitation of TGase with other proteins or the inhibition of TGase activity by excessive salt concentration. The highest total TGase activity was noticed for AS 60–80% and the lowest was noticed for AS 20–40% (p < 0.05), indicating that lower AS concentrations are less effective at precipitating the desired enzyme and may lead to the loss of TGase in the supernatant. With increasing AS concentrations, protein content was increased to the maximum at 60–80%, however, with further increasing AS concentrations, lower protein content was noticed, which possibly due to the saturation point of protein precipitation. Overall, the purity of TGase was increased by 1.46 times for 80–100% fraction as compared to the crude sample. The yield of TGase for 80–100% fraction was around 73%. For ammonium sulfate fraction 20–40%, TGase of purification fold was 1.39 times, however lower yield (51%) was attained. In addition, 20–40% fraction also showed protease activity, which was also supported by the increasing protein solubility and free amino group content of NAM (data not shown). Therefore, the authors selected 80–100% fraction as partially purified TGase (pTGase). When the 80–100% fraction was subjected to DEAE-Sepharose, purity was increased by 117.19 and 80 folds than the crude and 80-100% fraction, respectively. Among the different concentrations of NaCl used for the elution of TGase protein, the lowest total activity was noticed for 0.25 M NaCl, where no difference was noticed between 0.5 and 0.75 M NaCl (Fig. 1A). However, when specific activity was measured, the highest values were noticed for the 0.75 M NaCl fractions (Fig. 1B). The result was in agreement with lower protein content (0.25 mg/mL) in 0.75 M NaCl fractions as compared to the remaining fractions. Thus, a higher concentration of NaCl leads to the elution of desired proteins. The protein yields were 2.78 and 3.80% when compared to crude and 80–100% fraction. The purification fold is higher than the TGase extracted from (organ not mentioned) bigeye snapper (10.86), oil sardine (14.58), tilapia (17.91), and common carp (18.82) [16]. However, a much lower yield was noticed in the current studyas compared aforementioned fish species (40–59%) [16]. Laksono et al. [17] extracted TGase from daggertooth pike conger fish (Muraenesox cinerus) meat and enhanced the purity by 1.983 times via dialysis of crude extract. In general, the purification of enzymes mainly depends on the source, chemical composition as well as fish species.

Table 1 Total activity, protein content and specific activity of various fractions of transglutaminase extracted from Asian seabass liver
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Fig. 1
figure 1

Total activity (A) and specific activity (B) of different DEAE-Sepharose fractions, protein patterns of various TGase fractions (C) and elution profile purified TGase (D). Where M, C, AS, and P are the molecular weight markers; crude TGase, ammonium sulfate fraction (80–100%) and purified TGase, respectively

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3.2 Protein patterns and molecular weight of purified TGase

The protein pattern and MW weight of purified TGase were determined using SDS-PAGE and Sephadex G-50 as shown in Fig. 1C and D, respectively. Protein patterns of the crude sample (C) showed several protein bands (20–200 kDa). A similar pattern was noticed in 80–100% fraction (AS), nevertheless, the band intensity was lower as compared to the crude sample, especially bands that appeared after 45 kDa. With further purification using DEAE-Sepharose, a single band was achieved for purified TGase (P), suggesting that most of the unwanted proteins were removed during the ion-exchange chromatography. The MW of purified TGase determined using SDS-PAGE was 41.1 kDa, which was near the MW weight determined using Sephadex G-50 resin (43.4 kDa) (Fig. 1D). The protein patterns under reducing conditions suggested the monomeric form TGase from the seabass liver. Binsi and Shamasundar [16] observed the MW TGase in the range of 73–95 kDa, purified from marine (bigeye snapper and Indian oil sardine) and freshwater (tilapia and common carp) fish species. In addition, TGase extracted by Kishi et al. [33] and Nozawa et al. [34] of MW in the range of 90–100 kDa from the ordinary muscle of carps and six different marine species (scallop, botan shrimp, squid, carp, rainbow trout, and atka mackerel), respectively. The lower MW of TGase from Asian seabass liver as compared to the other fish species could be associated with the several factors, including different fish may express TGase isoforms with differing numbers of amino acids or post-translational modifications, in which TGase from seabass liver could be a smaller isoform compared to those found in other species [35]. Furthermore, most of the TGase extracted are from the fish muscle as mentioned earlier. For example, Kumazawa et al. [36] extracted TGase from walleye pollack (Theragra Chalcogramma) liver showed MW of 77 kDa. Thus, the MW weight of TGase varies from fish species as well as the part of the fish, which could be explored further on the basis of amino acid sequences.

3.3 Characterization of purified TGase

3.3.1 Effect of different temperatures and pHs on the TGase activity

The effect of various temperatures and pHs on the TGase activity is shown in Fig. 2A–C and B–D, respectively. The optimum temperature and pH were 45–50 °C and 8, respectively. A similar temperature and pH profile of TGase from threadfin bream liver was noticed [27]. When the temperature was increased higher than 50 °C, a sharp decline in the TGase activity was noticed, which could be associated with the conformational changes in the enzyme [27]. In general, muscle proteins require slightly higher temperatures to unfold, which could expose the substrates for TGase. Thus, the higher temperature stability of TGase aligned with its applications in the muscle food system. With increasing pH higher to 9, no major change was noticed in the TGase activity. The results suggested the wide range of pH optima for TGase from Asian seabass liver. Normally, amino acid residues at the active sites influence the changes in the pH, which might affect the interactions of enzyme and their substrates. Furthermore, a change in pH can alter the enzyme’s three-dimensional structure, either enhancing or inhibiting its ability to bind substrates and catalyze reactions [27]. In another study by Binsi and Shamasundar [16], TGase from bigeye snapper showed maximum specific activity at 37 °C, whereas oil sardine and common carp showed optimal activity at 37 °C. In general, enzyme activity depends on the habitat of the animal. For example, fish from cold environments, like polar regions, produce cold-adapted enzymes with higher catalytic efficiency at low temperatures due to increased active site flexibility, whereas tropical fish enzymes are more thermostable, maintaining activity at higher temperatures [37]. Additionally, habitat salinity influences enzyme activity, with fish from high-salinity environments exhibiting greater salt tolerance [38].

Fig. 2
figure 2

Effect of various temperatures (A, C), pHs (B, D) on the enzyme activity of transglutaminase purified from Asian seabass liver

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For enzyme stability, relative activity was reduced slightly from a temperature of 25–40 °C, however, when the temperature was higher than 50 °C, a sharp reduction in TGase activity was noticed (Fig. 2C). This could be associated with the conformation changes in the enzyme structure, which might disrupt the active site of the TGase. Binsi and Shamasundar [16] found a 100% loss in TGase activity extracted from the different fishes when the temperature was increased to 75 °C. However, at the optimal temperature, TGase from bigeye snapper exhibited peak specific activity at 37 °C, retaining approximately 89% of its activity even at 50 °C. Similarly, TGase from oil sardine and common carp also demonstrated optimal activity at 37 °C, though they were less stable at elevated temperatures. In contrast, the optimum temperature for TGase from tilapia was higher at 50 °C, with around 65% of its specific activity still present at 60 °C. TGase from carp also showed the highest activity at 37 °C; however, its activity sharply declined to 29% of its maximum when the temperature reached 60 °C. Thus, TGase from the Asian seabass liver showed higher stability than bigeye snapper, Indian oil sardine, tilapia, and common carp at the optimum temperature. Kumazawa et al. [36] reported that the TGase activity remained largely stable during incubations at 2 °C and 20 °C for 120 min. However, approximately 80% of the activity was lost after 1 h of incubation at 37 °C, and nearly all activity was rapidly lost when incubated at 50 °C within a few minutes. For pH stability, lower pH treatment also resulted in the reduction of relative TGase activity, data suggested that TGase showed a pH range of stability from 7 to 9 pH (Fig. 2D). The enzymes with a wide range of temperature and pH stability could be employed in a wide range of food products, which undergo various processing conditions.

3.3.2 Effect of different concentrations of NaCl and CaCl2 on the TGase activity

The effect of various concentrations of CaCl2 and NaCl on the TGase activity is shown in Fig. 3 A and B, respectively. For CaCl2, the TGase activity was increased with increasing concentrations to 20 mM, where the highest value was attained (p < 0.05). However, when the concentration was increased further lower activity was noticed (p < 0.05). Binsi and Shamasundar [16] reported that the activity reached almost maxima at 50 mM CaCl2. Whereas, TGase from threadfin bream fish liver showed increasing activity with augmenting CaCl2 to 5 mM, where the highest activity was attained Hemung and Yongsawatdigul [27]. TGases from various sources exhibit a wide range of Ca2+ concentration requirements for full activation, highlighting the diversity in their biochemical properties. In general, Ca2+ plays a crucial role in maintaining the enzyme’s appropriate conformation and enhancing coordination at the active site [39, 40]. It is postulated that the opening of the active site, by the displacement of a Tyr residue covering the catalytic Cys residue, may be mediated by allosteric calcium binding. This process facilitates the formation of an acyl-enzyme intermediate between the acyl donor and the catalytic Cys residue. Ultimately, this allows the acyl acceptor to access the active site, resulting in the formation of an amide bond [16]. In addition, Ca2⁺ to specific sites within the enzyme, particularly in a region near an α-helix in domain 2 of the enzyme, which is the core domain other than the remaining 3 domains [41]. This binding leading to the unfolding of α-helix, thereby disrupting interactions between different domains of the enzyme and opening up the active site for substrate access [41]. This mechanism underscores the necessity of calcium for the TGase activity, as it transitions the enzyme from an inactive to an active state, ready to perform its biological functions. Furthermore, calcium not only activates TGase but also influences its stability, protecting it from denaturation by heat and chemical agents [41]. Overall, the optimum concentration of Ca2+ ions for enzyme activity is species-dependent [16].

Fig. 3
figure 3

Effect of calcium chloride (CaCl2; A), and sodium chloride (NaCl; B) concentrations and incubation time (C) on the enzyme activity of transglutaminase purified from Asian seabass liver

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With increasing concentrations of NaCl, TGase activity was increased and the maximum values were noticed at 3% of NaCl (Fig. 3B). This is more likely due to the stabilization of TGase by enhancing the enzyme’s conformation and substrate binding, crucial for maintaining its catalytic function. Furthermore, enhancing the solubility through a “salting-in” of TGase could be another reason for enhanced activity. Nozawa et al. [34] reported that 0.5 M NaCl had no effect on TGase activity from carp, rainbow trout, and atka mackerel. The effect of NaCl on TGase activity appears to be influenced by the environmental habitat of each species, directly affecting enzyme behavior [27]. Notably, TGase activity from seabass liver exhibits high activity at 3% NaCl, which is commonly used (2–3%) to solubilize muscle protein during the preparation of fish protein gels. This relationship highlights the adaptation of TGase to the specific salt conditions of its environment, demonstrating the enzyme’s evolutionary optimization for its native habitat [27, 42]. Protein solubilization enables the exposure of available reactive amino residues for TGase. It has been reported that TGase is secreted extracellularly in marine invertebrates during wound-healing process. In this context, the enzyme naturally encounters high NaCl concentrations typical of marine environments during its catalytic activity [34]. Thus, TGase is not activated by the NaCl, rather than it retained at high concentration of the saline, which is associated with the habitat of fish [34, 43]. Whereas TGase from tropical tilapia, a freshwater fish, shows a significant inhibition of activity in the presence of NaCl [44]. These results indicated that seabass TGase could be applied to a food product containing NaCl up to 3% without a significant loss in activity.

TGase showed the highest activity at 50 °C, when EB (pH 7) contained 20 mM CaCl2 and 3% NaCl. These conditions were used to determine the optimum incubation time.

3.3.3 Effect different incubation times on TGase activity

The effect of different incubation time on TGase activity is shown in Fig. 3C. The highest enzyme activity was found at 10 min of incubation time (p < 0.05). With further increasing incubation, TGase activist was decreased (p < 0.05). After 30 and 60 min of incubation, the enzyme activity was decreased by 14 and 19%, respectively as compared to the 10 min. The decrease in TGase activity was more likely due to the depletion of substrate or denaturation of enzyme for the longer temperature exposure [45]. In addition, product inhibition of enzyme could be another possible reason for the decreasing activity [46].

3.4 Effect of pTGase on NAM polymerization

3.4.1 Protein solubility

The percentage decrease in protein solubility of NAM added with pTGase at various levels as a function of time are shown in Fig. 4A. Regardless of the time, protein content was decreased with increasing pTGase concentration as indicated by the increasing %decrease in protein solubility. Among all the samples, NAM added with 10 units had the highest decrease (33–48%) in protein solubility regardless of incubation time (p < 0.05). Whereas control had the lowest decrease (3–12%) in protein solubility than the remaining samples, which suggested highest protein content. The decreasing protein solubility was more likely due the cross-linking of NAM proteins, in which TGase catalyzes the acyl transfer reaction between a glutamine residue in a peptide chain and the ε-amino group of a lysine residue, resulting in the formation of an ε-(γ-glutamyl) lysine cross-link. In addition, the cross-linking NAM molecules resulted in the aggregation associated with the setting at 45 °C, which might expose the glutamine and lysin residue for pTGase [47]. In addition, exposure to hydrophobic residue or other amino acids during heating at 45 °C resulted in enhanced interactions between proteins via hydrophobic interactions, Vander Wall force as well as hydrogen bonding, which could also result in lower solubility [21]. Cao et al. [47] also observed a similar phenomenon when myofibrillar proteins were subjected to microbial TGase (MTGase) under different heating methods (water bath and microwave irradiation).

Fig. 4
figure 4

Effect of various concentrations of partially purified transglutaminase (pTGase) from Asian seabass liver on protein solubility (A) and ε-amino group content (B) of natural actomyosin

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3.4.2 Free amino group content

The free amino group content of NAM treated with pTGase at various times is shown in Fig. 4B. The control sample showed a higher ϵ-amino group content as compared to those treated with pTGase. The content was decreased with increasing both incubation time and pTGase concentration. In general, TGase catalyzes protein cross-linking by acyl-transfer reactions between the γ-carboxamide group and different types of the amino group of lysine including, α- and ε-amino groups, which lead to a decrease in ε-amino groups [20]. Thus, with the addition of pTGase, the reduction in the amino group suggested its cross-linking ability. Similarly, Jia et al. [20] observed a reduction in amino groups of black carp NAM affected by MTGase.

3.4.3 Microstructure

Figure 5 displays TEM images of NAM heated at 45 °C without and with pTGase. Generally, fresh NAM exhibited continuous and filamentous structures, which showed interconnections and clustered filaments formed during setting at 45 °C [48]. Typically, 40–50 °C is sufficient to unfold protein strands, allowing these proteins to aggregate or interact [18, 48]. However, when pTGase was added, more connections, cross-linking, and cluster formation were observed in a dose-dependent manner. The highest aggregated NAM proteins were noticed when 10 U pTGase was added. The results suggested the cross-linking ability of pTGase via disulfide bond formation, which increased with higher concentrations. This observation aligned with the decreasing NAM solubility and free amino acid content (Fig. 4A, B). Additionally, gelation is influenced by protein denaturation and aggregation through various covalent and non-covalent bonds [49]. Heat can induce protein denaturation, leading to subsequent interactions and network structure formation. These large aggregates are typically the result of various bonds, including hydrophobic interactions and disulfide bonds. Thus, pTGase could be an alternative source for protein cross-linking, especially in surimi and surimi products. For example, surimi obtained from sardine, Indian mackerel, etc. possessed lower gel qualities, which is more likely die to the lower endogenous TGase [19]. Thus, the application of pTGase into those surimi gel could possibly tackle the problem with lower gel-forming ability. In addition to this, pTGase could be used in plants proteins isolate based salmon fillets, which can provide the desired texture to the fillets.

Fig. 5
figure 5

TEM images of natural actomyosin added without and with partially purified transglutaminase (pTGase) from Asian seabass liver at various concentrations

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4 Conclusion

This study highlights the purification and characterization of TGase from seabass liver, where the highest TGase activity was observed in the 80–100% ammonium sulphate fraction. When 80–100% fraction was subjected to DEAE-Sepharose purification, purity was increased by 117.19 than that of crude extract. However, the lower yield (2.78%) was attained for the purified TGase The purified TGase exhibited optimal activity at 45–50 °C and pH 8 in the presence of 20 mM CaCl2, and its activity was enhanced at 3% NaCl. Partially purified TGase (pTGase) effectively catalyzed protein cross-linking in NAM, reducing protein solubility and amino group content while promoting cluster formation and gelation. These findings suggest that pTGase can be an effective protein cross-linker in surimi and related products, especially those made from fish with lower gel-forming abilities. However, further optimization to enhance the yield of the purified fraction should be explored. In which, specific strategies such as refining the ammonium sulphate precipitation process or aqueous two phase separation, optimizing buffer conditions, or incorporating alternative purification techniques such as chromatography with different resins or columns could be employed.