Fish skin as a biomaterial for halal collagen and gelatin.

Around 40% of the total catch weight of fish is regarded as byproducts, consisting of skin, fins, bones, scales, viscera, etc. The utilization of these byproducts is important to increase their commercial values as well as to prevent environmental pollution. Meanwhile, nowadays, it is getting a global trend to provide foods and other industrial materials which have been accredited as halal products for Moslem communities. As a way of processing fish byproducts to meet the halal criteria, preparation of collagen and gelatin would be useful to fulfill the market demand. As a result of screening studies on fishery byproducts, fish skin has been found to be the good source for halal collagen and gelatin, which show satisfactory quality compared with those from bovine sources which could cause bovine spongiform encephalopathy (BSE).

Expression and characterization of rainbow trout Oncorhynchus mykiss recombinant myoglobin.

Recombinant expression system was established for rainbow trout myoglobin (Mb) considering its unique primary structure of having one unusual deletion and two cysteine residues in contrast to the other fish Mbs. The obtained recombinant Mb without His-tag showed non-cooperative thermal denaturation profile. The presence of free cysteine residue(s) in rainbow trout Mb was demonstrated by reacting with a sulfhydryl agent, 4, 4´-dithiodipyridine, which ultimately resulted in the oxidation of Mb with characteristic changes in visible absorption spectra. Besides, the recombinant Mb displayed steady peroxidase reactivity indicating in vivo roles of Mb as a reactive oxygen species scavenger. The findings of the present study indicate that the solitary rainbow trout Mb, which ultimately manifest typical secondary structure pattern and corroborate characteristic functionality, can be over expressed in recombinant system devoid of fusion tag.

Thermal denaturation and autoxidation profiles of carangid fish myoglobins.

Although myoglobin (Mb) has been considered to be one of the well-characterized proteins, screening of post-genomic era databases revealed the lack of adequate information on teleost Mbs. The present study was aimed to investigate stability and functional features of Mbs from three teleosts of the same family. To unfold how primary structure influences the stability and function of proteins, Mbs were purified from the dark muscles of three carangids, namely, yellowtail, greater amberjack, and silver trevally. Thermostabilities measured by circular dichroism (CD) spectrometry revealed species-specific thermal denaturation pattern, i.e., silver trevally > yellowtail > greater amberjack Mbs. On the other hand, autoxidation rate constants of the ferrous forms of those three carangid Mbs showed positive correlation between the ferrous state of the heme iron and rising temperature. The order of autoxidation rate was in the order of greater amberjack > yellowtail > silver trevally Mbs. The finding of the present study denotes that the thermal stability is not necessarily correlated with the functional stability of carangid Mbs even though their primary structures shared high homology (84-94%).

Characterization and Antioxidant Activity of Collagen, Gelatin, and the Derived Peptides from Yellowfin Tuna (Thunnus albacares) Skin.

Skin waste from tuna processing needs to be utilized, such as extraction of its collagen and gelatin. Their functional properties can be improved by enzymatic hydrolysis for conversion to peptides. Thus, the research objectives were to examine the characteristics and antioxidant activity of collagen, gelatin, and the derived peptide from yellowfin tuna skin. Collagen was extracted using 0.75 M acetic acid at 4 °C, while gelatin was prepared using 0.25% citric acid and extracted at 65 °C. Hydrolysis was carried out with 2% Alcalase, followed by fractionation with a molecular weight cut off sieve for both collagen and gelatin. Collagen yield was 22.6% with pH value of 6.63 and whiteness of 96.7%. Gelatin yield was 20.0% with pH value of 4.94 and whiteness of 51.0%. Hydrolysis for three hours resulted in 52.7% and 45.2% degree of hydrolysis for collagen and gelatin, respectively. The molecular weights of collagen peptides ranged from 2.94 to 11.93 kDa, while those of gelatin peptides ranged from 3.54 to 16,620 kDa. Antioxidant activities of these peptides were higher than those before hydrolysis. The high antioxidant activity (IC50) of collagen peptides were found in <3, 3-10, and 10-30 kDa fractions as well as in the gelatin peptides.

Thermostability of tropomyosins from the fast skeletal muscles of tropical fish species.

In order to investigate the species-specific heat tolerance of tropical fishes, the thermodynamic properties of muscle tropomyosin, a member of myofibrillar proteins, were compared among milkfish, tilapia, grouper, and mudskipper. The purified tropomyosins were subjected to differential scanning calorimetry and circular dichroism spectrometry. To unveil the relationship between the stability and the amino acid sequences, the muscle tropomyosin genes of the four species were also cloned, and their deduced amino acid sequences were compared. Thermodynamic analysis revealed that the milkfish tropomyosin showed lower refolding ability after thermal denaturation, compared with those of the other species. The amino acid sequences of these tropomyosins were similar to each other, with the identity being in the range of 95-96%.

Expression levels of myoglobin in muscle and non-muscle tissues of rainbow trout Oncorhynchus mykiss, a hypoxia intolerant species.

Myoglobin (Mb) is one of the most intensively studied intracellular respiratory muscle proteins. Since the discovery of the fascinating fact that Mb is not confined only to oxidative muscle tissues but also is co-localized in different non-muscle tissues of cyprinids, hypoxia tolerant cyprinids have been established as the model teleost. Mb both at mRNA and protein levels have been reported in this study for the first time from a number of muscle and non-muscle tissues of rainbow trout Oncorhynchus mykiss, a hypoxia intolerant species. Mb transcript levels were high in the heart and slow skeletal muscle, and were comparatively high in the gonad and gill among the non-muscle tissues. Western-blotting by using anti-rainbow trout Mb peptide rabbit antibody detected Mb protein in the muscles and several non-muscle tissues. By both RNA in situ hybridization and immunofluorescence, Mb was localized in the cardiomyocytes and oxidative muscle fibers. On the other hand, Mb both at mRNA and protein levels was restricted to the lamellar epithelial cells of the gill, epithelial layers of hepato-biliary duct, neurons and endothelial cells of brain, ooplasm of gonad, kidney tubules, endothelial cells, and epithelial layer of intestine. Neuroglobin isoform 1 and 2 mRNAs along with Mb mRNA were localized in the granular layer of cerebellum. Considering the previous data reported for cyprinids, the expression sites of Mb in the muscle and non-muscle tissues of teleost could be universal, where Mb concerted with the other globins might play meaningful physiological roles.

A streamlined isolation method and the autoxidation profiles of tuna myoglobin.

Determination of the redox state of myoglobin (Mb) gives useful information for evaluating the quality of tuna meat. To attain this purpose, a fast streamlined method has been established basically based on preparative native gel electrophoresis to isolate Mb from the dark muscle of Pacific bluefin tuna. Crude Mb fraction was prepared from dark muscle by ammonium sulfate saturation fractionation and subsequently Mb was purified by preparative native gel electrophoresis under the isoelectric pH of the Mb, resulting in absorption (or trapping) of all the contaminating proteins in the gel. Purified Mb was converted to oxy form with a trace amount of sodium hydrosulfite, and subsequently dialyzed against 50 mM sodium citrate (pH 5.6) or 50 mM sodium phosphate (pH 6.5). The purified tuna Mb was examined for the temperature and pH dependencies of autoxidation using horse Mb as a reference. Tuna Mb was oxidized 2.5-3 times faster than horse Mb irrespective of the pH conditions examined. The highest autoxidation rates both at 0 and 37 °C were observed at pH 5.6. These data were comparable to those obtained for Mbs isolated by conventional chromatographic methods.

Structural and dynamical characteristics of tropomyosin epitopes as the major allergens in shrimp.

Ingestion of marine invertebrates often causes food allergy, where the major allergens have been reported to be derived from tropomyosin (TM). Intact or the digestive fragments of food allergens generally show resistance to digestion, which is usually attributable to the structural stability (or rigidity). The difference in the structural and dynamical characteristics between the epitope and the non-epitope regions in TM has not yet been well understood. In the present study, molecular dynamics simulation was performed at constant pHs for shrimp TM. By analyzing the main-chain dihedral angle fluctuations and local α-helix contents, we found that the epitope regions are more stable than the non-epitope counterparts, providing a possible physical reason for the resistance to digestion in the epitopes regions. The difference of the structural stability between the epitope and the non-epitope regions was largest at low pHs, even though pH dependence of the structural stability in itself was not significant in both regions. The lower content of the Ala cluster in the epitope region is considered to cause the higher stability of the epitope region.

Molecular characterization of southern bluefin tuna myoglobin (Thunnus maccoyii).

The primary structure of southern bluefin tuna Thunnus maccoyii Mb has been elucidated by molecular cloning techniques. The cDNA of this tuna encoding Mb contained 776 nucleotides, with an open reading frame of 444 nucleotides encoding 147 amino acids. The nucleotide sequence of the coding region was identical to those of other bluefin tunas (T. thynnus and T. orientalis), thus giving the same amino acid sequences. Based on the deduced amino acid sequence, bioinformatic analysis was performed including phylogenic tree, hydropathy plot and homology modeling. In order to investigate the autoxidation profiles, the isolation of Mb was performed from the dark muscle. The water soluble fraction was subjected to ammonium sulfate fractionation (60-90 % saturation) followed by preparative gel electrophoresis. Autoxidation profiles of Mb were delineated at pH 5.6, 6.5 and 7.4 at temperature 37 °C. The autoxidation rate of tuna Mb was slightly higher than that of horse Mb at all pH examined. These results revealed that tuna myoglobin was unstable than that of horse Mb mainly at acidic pH.

Structural characterization of carangid fish myoglobins.

The primary structures of myoglobin (Mb) from the following five carangid species were determined: yellowtail Seriola quinqueradiata, greater amberjack Seriola dumerili, yellowtail kingfish Seriola lalandi, Japanese horse mackerel Trachurus japonicus, and silver trevally Pseudocaranx dentex. The sequences were of varying composition both in the coding and in the noncoding regions, but all contained the open reading frame of 444 nucleotides encoding 147 amino acids. Amino acid sequence identities of carangid Mbs were in the range of 81-99%. The similarity of the heme pocket and associated heme-binding residues of carangid Mbs were evidence of the conservative nature of Mbs. Similar to the other teleost Mbs, carangid Mbs did not contain a D helix and had mostly conserved A and E helices as well as E-F and G-H inter-helical segments. Hydropathy profiles of carangid Mbs showed species-specific variations where silver trevally Mb exhibited generally higher hydrophobicity. Phylogenetic analysis based on the primary structures was in agreement with conventional morphological taxonomy, establishing close proximity of carangid Mbs with those of cichlid and scombroid, the other members of the Perciformes order.

Thermodynamic characterization of muscle tropomyosins from marine invertebrates.

Structural properties of invertebrate muscle tropomyosin (TM) have not been characterized in detail to date. TMs were thus purified from the mantle muscle of Japanese common squid Todarodes pacificus, the foot muscle of tokobushi abalone Haliotis diversicolor and the tail muscle of kuruma prawn Marsupenaeus japonicus, and investigated for their thermodynamic properties by circular dichroism (CD) spectrometry and differential scanning calorimetry (DSC). From the CD spectrometry data, the apparent melting temperature and the apparent free energy of unfolding at 20°C were calculated to be 43.5°C and 14.5 kJ/mol for the squid TM, 43.0°C and 23.9 kJ/mol for the abalone TM, and 47.3°C and 50.2 kJ/mol for the prawn TM, respectively. From the DSC data, the total free energy of unfolding at 20°C was calculated to be 129 kJ/mol, 253 kJ/mol, and 271 kJ/mol for the squid, abalone, and prawn TMs, respectively. These results suggest that the thermal stability was in the order of prawn TM>abalone TM>squid TM.

Thermal denaturation profiles of tuna myoglobin.

Myoglobin (Mb) purified from fast skeletal muscle of bluefin tuna Thunnus thynnus orientalis was subjected to thermal treatment, and the denaturation profiles were examined by thermodynamic analysis. Based on the ellipticity or helical content obtained by circular dichroism (CD) spectrometry, it was found that denaturation of tuna Mb consisted of three steps, and that slight structural changes of Mb started below 20 degrees C. However, major structural changes were observed at around 58 and 72 degrees C. Differential scanning calorimetry (DSC) analysis revealed a similar but somewhat different thermal denaturation profile of Mb. In comparison with the denaturing profiles of whale Mb under the same conditions, the thermal stability of tuna Mb was found to be much lower. In the modeled tertiary structures of these Mbs, they were roughly similar to each other, though minor conformational differences were recognized and the total energy was found to be lower for tuna Mb.

Characterization of two tropomyosin isoforms from the fast skeletal muscle of bluefin tuna Thunnus thynnusorientalis.

Fast skeletal muscle tropomyosin (TM) of tunas is composed of nearly equimolar amount of two isoforms designated alpha-TM and beta-TM expediently based on their migration behavior in SDS-PAGE, whereas corresponding TMs from the other fish species are homogenous (alpha-type). The presence of beta-TM is thus specific to tunas so far. The amino acid sequence of beta-TM from bluefin tuna Thunnus thynnus orientalis, which has not been revealed to date unlike alpha-TM, was successfully obtained in this study by cDNA cloning. The coding region of beta-TM cDNA comprised of an open reading frame of 855bp encoding 284 amino acid residues, like most of the TMs. Unexpectedly, the sequence of beta-TM showed high similarity to those of other vertebrate alpha-type TMs including tuna alpha-TM. Phylogenetic analysis also showed that beta-TM has the closest relationship with alpha-TM of tuna. This fact was quite unlike the relation of mammalian alpha- and beta-TMs. Based on the distribution of amino acid substitutions, it was suggested that tuna TM isoforms are the products of different genes. By thermodynamic analysis of native and reconstituted TMs, it was demonstrated that beta-TM is less thermostable than alpha-TM. Proteolytic digestion also supported the lower stability of the former.

Antioxidative activities of hydrophilic extracts prepared from the fruiting body and spent culture medium of Flammulina velutipes.

Antioxidative properties of hydrophilic extracts prepared from the fruiting body and spent culture medium of Flammulina velutipes were evaluated by monitoring the total reducing power ability (RPA) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity (RSA), together with antioxidative activities against lipid oxidation in homogenates of yellowtail dark muscle and autoxidation of oxymyoglobin (oxyMb) purified from yellowtail dark muscle. Generally, all of the extracts had RPA, RSA and antioxidative activities against lipid oxidation and oxyMb autoxidation. Extracts prepared from the fruiting body of F. velutipes with a higher ergothioneine (ESH) content exhibited a stronger delay of the autoxidation activity of oxyMb, whereas extracts prepared from the spent culture medium of F. velutipes with higher phenolics content showed more efficient antioxidant capacity against lipid oxidation. On the other hand, the amount of ESH was distributed highest in the inedible (base and mycelium) parts of the mushroom. These results suggest that the inedible parts and spent culture medium of F. velutipes could potentially be considered as a potent and readily available source of natural antioxidants.

Thermostability of striated and smooth adductor muscle tropomyosins from Yesso scallop Mizuhopecten yessoensis.

The striated and smooth adductor muscle tropomyosins (TMs) of Yesso scallop Mizuhopecten yessoensis have been known to express different isoforms, but have not been characterized in detail to date. In the present study, TMs from both muscles of Yesso scallop were purified and their stabilities were compared by circular dichroism (CD) spectrometry and differential scanning calorimetry (DSC). From the CD data, the apparent melting temperature and the apparent free energy of folding at 20 degrees C were calculated to be 30.5 degrees C and -13.4 kJ/mol, and 36.0 degrees C and -31.9 kJ/mol for the striated and smooth muscle TMs, respectively. From the DSC data, DeltaH values were calculated to be 1.87 x 10(3) and 2.19 x 10(3) kJ/mol for striated and smooth muscle TMs. These results suggest that smooth muscle TM has higher thermostability than striated muscle TM. The amino acid residues responsible for such stability difference were considered to be the six amino acid substitutions in the middle region of the TM molecules.

Structural and autooxidation profiles of myoglobins from three species and one hybrid of tilapia (Cichlidae, Perciformes).

cDNAs encoding myoglobin were cloned from the slow skeletal muscles of three representative species of tilapia, namely, Nile tilapia Oreochromis niloticus, blue tilapia O. aureus, Mozambique tilapia O. mossambicus and one hybrid O. niloticus female symbol x O. aureus male symbol, and the primary structures were deduced. All cDNAs contained an open reading frame of 444 base pairs, encoding 147 amino acids. The amino acid sequences of Mb were completely conserved among these species, though species variations in the nucleotide sequences were recognized both in coding and non-coding regions. The amino acid sequence identity was around 70-80% compared to other teleostean Mbs. In comparison of each alpha-helical segment (A through H) and the intersegment regions to the counterparts of tuna myoglobin, the alpha-helical segments C and F as well as the intersegment regions F-G and G-H were identical. The identities of alpha-helical segments B and H and the intersegment region F-G were relatively low. Differences were also recognized in the hydropathy plot and the tertiary structures obtained by homology modeling. The autooxidation rates at 25 degrees C of myoglobin fraction from the slow skeletal muscle were essentially the same among the above tilapia species, as expected from the conserved amino acid sequences.

Effects of point mutations on the structural stability of tuna myoglobins.

Structural stabilities of myoglobin (Mb) from several tuna fish species significantly differ from each other, although the amino acid sequence identities are very high (>95%), suggesting that limited number of substitutions greatly affect the stability of Mb. To address this hypothesis, attempts were made to elaborate recombinant tuna Mbs with point mutations on the different residues among fish Mbs. The expression plasmid constructs were based on bigeye tuna Mb cDNA sequence, and the recombinant proteins were expressed as GST-fusion proteins in Escherichia coli. After removal of the GST segment and affinity purification, the stability of five Mb mutants, namely, A49G, T91K, K92Q, V108A, and H112Q, together with the wild type (WT) were measured, taking temperature dependency of alpha-helical content and denaturant (urea and guanidine-HCl) concentration dependency of Soret band absorbance as parameters. As a result, the mutant H112Q showed much higher stability than WT, while the structures of K92Q, T91K and A49G mutants were destabilized. No essential change in helical content was observed for V108A, but the mutant was found to be destabilized easier by the denaturants. These findings suggested that the highly conserved residues among tuna species are responsible for their stability of Mbs, but a few non-conserved residues dramatically give rise to the differences in stability of Mbs among species.

Effect of amino acid replacements on the structural stability of fish myoglobin.

Structural stabilities of myoglobin (Mb) from several fish (scombridae) species differ significantly, although their amino acid sequence identity is very high (>95%), suggesting that only a few substitutions greatly affect the stability of Mb. Accordingly, recombinant Mbs with point mutation(s) derived from bigeye tuna Mb cDNA were expressed as GST-fusion proteins in the soluble fractions of Escherichia coli. After removal of the GST segment, the stability of five mutants, namely, P13A, I21M, V57I, A62G, and I21M/V57I, together with the wild type (WT) were investigated, taking temperature dependency of alpha-helical content and denaturant concentration dependency of Soret band absorbance as parameters. As a result, the stability of P13A against denaturants and its alpha-helical content at 10 degrees C was found to be the highest among the mutants, whereas those of A62G were the lowest. The stabilities of V57I and I21M/V57I were higher than that of WT, though that of I21M was nearly the same as WT. These findings suggest that the structural stability of fish Mb is tuned up only by the substitutions of a few amino acid residues located in the alpha-helical segments forming the hydrophobic heme pocket.

Structural stabilities of recombinant scombridae fish myoglobins.

An expression system of recombinant myoglobins (Mb) of 3 scombridae fish species was constructed. The stability of these Mbs was compared with native Mbs purified from slow skeletal muscle. The addition of hemin during the cultivation of an Escherichia coli strain harboring a pGEX-2T expression vector was found to be necessary to prevent recombinant Mb from degrading and to attain its proper folding. The stabilities of recombinant Mbs were generally lower than those of native Mbs, partly due to the absence of post-translational modification. The alpha-Helical content of bullet tuna recombinant Mb at 10 degrees C was the lowest (29.0%) among the recombinant Mbs examined (the values for bluefin tuna and bigeye tuna Mbs being 34.8 and 35.5%, respectively). On the other hand, the stabilities of recombinant Mbs of bluefin tuna and bigeye tuna against denaturants (urea and guanidine hydrochloride) were found to be similar, whereas bullet tuna recombinant Mb exhibited the lowest stability among these Mbs. The pattern of temperature-dependent decrease in the alpha-helical content supported these results.

Fish fast skeletal muscle tropomyosins show species-specific thermal stability.

Tropomyosin (TM) was isolated from the fast skeletal muscle of six fish species, whose amino acid sequences of this protein have already been revealed. The thermal stability of these TMs was measured by differential scanning calorimetry (DSC) and circular dichroism (CD), while the molecular weights were measured by mass spectrometry. The results showed clear differences in thermostability among these fish TMs, though the identity of amino acid sequences was more than 93.3%. Therefore, only a few amino acid substitutions could affect the overall stability of the TM molecule. Especially, several residues located on the molecular surface were considered to be responsible for such stability difference. In contrast, the molecular weights of these TMs as measured by mass spectrometry were higher than those calculated from amino acid composition, suggesting the presence of post-translational modification(s) which could also affect their thermal stability.