Purification and characterization of heat-stable alkaline proteinase from bigeye snapper (Priacanthus macracanthus) muscle

Heat-stable alkaline proteinase was purified from bigeye snapper (Priacanthus macracanthus) ordinary muscle by heat-treatment and a series of chromatographies including Phenyl-Sepharose 6 Fast Flow, Source 15Q and Superose 12 HR 10/30. It was purified to 5180-fold with a yield of 0.8%. The molecular weight of purified proteinase was estimated to be 72 kDa by gel filtration. The proteinase appeared as two proteinase activity bands with molecular weights of 66 and 13.7 kDa on non-reducing SDS-substrate gel. Accordingly, it was found to consist of two different subunits. The optimum pH and temperature for casein hydrolysis were 8.5 and 60 °C, respectively. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor and partially inhibited by ethylenediaminetetraacetic acid, while pepstatin A and E-64 showed no inhibition. Purified proteinase was able to hydrolyze Boc-Phe-Ser-Arg-MCA, but rarely hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA. In addition, it mainly degraded myosin heavy chain, not actin. These results suggest that purified proteinase was serine proteinase, which is probably involved in gel weakening of bigeye snapper surimi.     

Effect of pH on the properties of protein-based film from bigeye snapper (Priacanthus tayenus) surimi

The effects of the pH and protein content on the properties and compositional changes of protein-based films from bigeye snapper (Priacanthus tayenus) surimi were investigated. Tensile strength of bigeye snapper surimi films prepared at the acidic (pH 3) and alkaline (pH 11) conditions were not significantly different (P > 0.05). Nevertheless, elongation at break of films prepared at pH 3 was higher than that at pH 11 (P < 0.05). The acidic and alkaline conditions had no effect on water vapor permeability of the films obtained. However, the film prepared at acidic condition was more yellowish than that prepared at alkaline pH. Protein content influenced the mechanical properties and color of films.     

Properties and Characteristics of Multi-Layered Films from Tilapia Skin Gelatin and Poly(Lactic Acid)

Multi-layered films based on tilapia skin gelatin and poly(lactic acid) (PLA) were characterized, in comparison with the control gelatin and PLA films. Three different layers of multi-layered films (PLA/Gelatin/PLA) were visualized by scanning electron microscopic (SEM) analysis. The synergetic effect of lamination was evidenced by the increased mechanical properties (P < 0.05). Multi-layered films had higher water vapor barrier property and water resistance, compared to control gelatin film (P < 0.05). Gelatin films showed increased lightness (L*) with coincidental decrease in total color difference (?E*) in the presence of PLA layers (P < 0.05). Transparency and solubility of films decreased with increasing ratio of PLA (P < 0.05). In addition, multi-layered films showed the enhanced hydrophobicity and thermal stability as evidenced by increased water contact angle and degradation temperature, respectively. Thus, PLA/Gelatin/PLA multi-layered film with improved water vapour barrier property could serve as bio-degradable packaging material for wider applications.     

Impact of legume seed extracts on degradation and functional properties of gelatin from unicorn leatherjacket skin

Trypsin inhibitor was extracted from the seed flour of soybean (SB; Glycine max), mung bean (MB; Vigna radiata), cowpea bean (CP; Vigna unguiculata) and adzuki bean (AB; Vigna angularis) using 0.15 M NaCl, followed by heat precipitation at 70 °C. The extract from SB showed the highest specific trypsin inhibitory activity, followed by those from MB, CP and AB, respectively. Based on inhibitory activity staining, molecular weights (MWs) of trypsin inhibitor from SB, MB, CP and AB were 20.1, 14, 10 and 13 kDa, respectively. The SB extract powder (SBEP) containing trypsin inhibitor in the range of 10–100 TIU/g effectively prevented the degradation of γ-, β- and α-chains of collagenolytic proteins of leatherjacket skin subjected to incubation at 50 °C for 30 min. The impact of SBEP on the extraction yield, physical and functional properties of gelatin from leatherjacket skin was investigated. The gelatin extracted in the presence of SBEP contained α₁ and α₂ chains as the predominant components with some degradation peptides. FTIR spectra indicated the significant loss of molecular order of triple helix and higher degradation was found in gelatin extracted in the absence of SBEP. Gelatin extracted in the presence of SBEP had the higher gel strength (232.8–268.5 g) than that extracted in the absence of SBEP (90.4 g). Higher foam stability (FS) but lower emulsion stability index (ESI) was observed in the former. Therefore, the addition of SBEP effectively prevented the degradation of gelatin from the skin of unicorn leatherjacket, thereby yielding the gelatin with improved gel strength and foam stability.     

Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix)

Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 °C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 °C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl₂ partially protected trypsin against activity loss at 40 °C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.     

Properties and microstructure of protein-based film from round scad (Decapterus maruadsi) muscle as affected by palm oil and chitosan incorporation

The properties of protein-based film prepared from round scad (Decapterus maruadsi) muscle in the absence and the presence of palm oil and/or chitosan were investigated. Films added with 25% palm oil (as glycerol substitiution) had the slight decrease in water vapor permeability (WVP) and elongation at break (EAB) (p < 0.05). WVP and tensile strength (TS) of films increased but EAB decreased when 10–40% chitosan (as protein substitution) was incorporated (p < 0.05). Hydrophobic interactions and hydrogen bonds, together with disulfide and non-disulfide covalent bonds, played an important role in stabilizing the film matrix. The a* and b*-values increased with increasing chitosan levels (p < 0.05). Films added with chitosan were less transparent and had the lowered transmission in the visible range. The incorporation of 25% palm oil and 40% chitosan yielded the films with the improved TS but decreased water vapor barrier property. Apart from film strengthening effect, chitosan inconjunction with Tween-20 most likely functioned as the emulsifier/stabilizer in film forming solution containing palm oil.     

Comparative study on the proteases from fish pyloric caeca and the use for production of gelatin hydrolysate with antioxidative activity

Proteases from pyloric caeca extract of three fish species including brownstripe red snapper (Lutjanus vitta), bigeye snapper (Priacanthus tayenus) and threadfin bream (Nemipterus marginatus) were comparatively studied. The extracts from bigeye snapper and threadfin bream exhibited the highest hydrolytic activities toward casein, α-N-benzoyl-dl-arginine-p-nitroanilide and α-N-ρ-tosyl-l-arginine methyl ester at pH 8.0 and 60 °C and pH 8.5 and 55 °C, respectively. The extract of brownstripe red snapper showed the optimal pH and temperature of 8.0 and 60 °C with all substrates used except the optimal temperature was 65 °C when casein was used. All proteases were strongly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-l-lysine chloromethylketone (TLCK) and partially inhibited by N-tosyl-l-phenylalanine chloromethylketone for all substrates tested, suggesting that trypsin-like proteases were the major enzymes. Substrate-gel activity staining of 40–60% ammonium sulfate (AS) fraction revealed that major activity bands were observed with molecular mass of 24, 22 and 20 kDa for brownstripe red snapper, bigeye snapper and threadfin bream, respectively. Those activity bands were partially inhibited by SBTI and TLCK. AS fraction was further used to produce gelatin hydrolysate from the skin of brownstripe red snapper with different degrees of hydrolysis (DH). Hydrolysate with DH of 15% exhibited the highest DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power (p < 0.05). Therefore, the extract from pyloric caeca could be used to produce the gelatin hydrolysates possessing antioxidative activities.     

Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle

A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.     

Effect of Longan Seed Extract and BHT on Physical and Chemical Properties of Gelatin Based Film

Gelatin films developed from fish skin incorporated with longan seeds extract (LS) or butylated hydroxytoluene (BHT) at different concentrations were prepared and characterized. The film thickness was in the range of 35 to 37 μm, and the transparency was 3.24 to 3.36 for the films with and without the addition of LS or BHT (p?<?0.05). Significant increases in redness (a*) and yellowness (b*) values were observed when the concentration of LS increased (p?<?0.05). Water vapour permeability (WVP) slightly decreased when the concentration of LS increased, while no significant change was observed between the control and the BHT films (p?>?0.05). Tensile strength (TS) and elongation at break (EAB) were in the range of 48 to 53 MPa and 16 to 19 %, respectively. The highest (65.7 J/g) and lowest (38.7 J/g) transition enthalpy was found in the control and LS 500 ppm films, respectively. Slight differences in protein pattern were observed under SDS-PAGE between treatments of the film. These differences were also observed in the FTIR spectrum. Films incorporated with LS or BHT showed the preventive effect on lipid oxidation of soybean oil during 30 days of storage. At the level of 500 ppm, LS provided the highest efficacy for lipid oxidation retardation as evidenced by lower peroxide value (PV), and thiobarbituric acid reactive substances (TBARS) values (p?>?0.05). The addition of LS or BHT had an impact on the film properties derived from fish skin gelatin, especially when high levels were added.     

Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization

Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.