Autolysis and the endogenous proteinases characterised in beardless barb (Anematichthys apogon) muscle

Beardless barb is a common fish species used in fermentation of fish paste Ka-pi-plaa. Autolytic profile of beardless barb muscle showed the maximum autolysis was at 50 °C, at both acidic and alkaline pH values. With augmentation concentration of NaCl, autolytic activity slightly decreased. Endogenous proteinases isolated from fish muscle in crude extract forms were also characterised. The acidic proteinases had optimum activity at pH 3.0 and 50°C, and they showed higher proteolytic activity than the alkaline proteinases which were optimally active at pH 9.0 and 50 °C. Proteinases in peak at pH 3.0 were inhibited by pepstatin A, but those in peak at pH 9.0 were highly inhibited by PMSF, TLCK and soybean trypsin inhibitor, suggesting that both aspartic and serine proteinases were existed in beardless barb muscle. The proteinases were stable in pH range of 2.0-5.0 but unstable at the temperatures higher than 40 °C. NaCl suppressed the proteolytic activity, ATP activated the proteinase activity, while CaCl₂, MgCl₂ and CoCl₂ exhibited no influence on the activity. The results implied that cathepsin D is the predominant proteinase responsible for autolysis in beardless barb. The findings were useful to improve the processing and qualities of Ka-pi-plaa product using beardless barb as raw material.     

Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)

Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent K_m and K_cat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S⁻¹, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.     

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii) and elkhorn sculpin (Alcichthys alcicornis): Isolation and characterization

Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii), TR-J, and elkhorn sculpin (Alcichthys alcicornis), TR-E, were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The molecular weights of TR-J and TR-E were estimated to be 24,000 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. TR-J and TR-E revealed optimum temperatures of 60 and 50 °C, respectively, and showed the same optimum pH (pH 8.0) for hydrolysis of N-p-tosyl-l-arginine methyl ester. TR-J and TR-E were unstable at above 50 and 40 °C, respectively, and were more stable at alkaline pH than at acidic pH. Thermal stabilities of TR-J and TR-E were highly calcium dependent. These purified trypsin enzymes were inhibited by serine protease inhibitors, such as TLCK and soybean trypsin inhibitor. The N-terminal amino acid sequences of TR-J and TR-E were also investigated. The N-terminal amino acid sequences of TR-J, IVGGYECKPYSQPHQVSLNS, and TR-E, IVGGYECTPHSQAHQVSLNS, were found, and these sequences showed highly homology to other fish trypsins.     

Optimum extraction and recovery of trypsin inhibitor from yellowfin tuna (Thunnus albacores) roe and its biochemical properties

Effect of lipid removal, extraction medium and extraction time on the isolation and recovery of trypsin inhibitor from yellowfin tuna (Thunnus albacores) roe was investigated. Trypsin inhibitor extracted from defatted tuna roe showed the higher specific inhibitory activity than extracted from origin tuna roe. Optimal extraction medium was attained by shaking the defatted yellowfin tuna roe powder in 10 mm Na phosphate buffer (pH 7.0) containing 0.5 m NaCl (P < 0.05). The extraction time affected the inhibitor recovery significantly (P < 0.05). The extraction time of 30 min was optimum for recovery of trypsin inhibitor from yellowfin tuna roe. The biochemical properties of trypsin inhibitor from yellowfin tuna roes were also determined. The inhibitor was stable to heat treatment up to 60C and over a broad pH range (5‐8). Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the trypsin inhibitory activity. However, the activity decreased when trypsin inhibitor was incubated with metal ions at ambient temperature for 30 min.     

Functional properties and antioxidative activity of protein hydrolysates from toothed ponyfish muscle treated with viscera extract from hybrid catfish

Functional properties and antioxidative activity of a protein hydrolysate prepared from toothed ponyfish (Gazza minuta) muscle, using viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus), with a degree of hydrolysis (DH) of 70%, were investigated. The protein hydrolysate had a good solubility. It was soluble over a wide pH range (3–9), in which more than 77% solubility was obtained. The emulsifying activity index of the protein hydrolysate decreased with increasing concentration (P < 0.05). Conversely, the foaming abilities increased as the hydrolysate concentrations increased (P < 0.05). Protein hydrolysate exhibited the increases in 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) radical scavenging activities, ferric reducing power (FRAP) and metal chelating activity as hydrolysate concentration increased (P < 0.05). ABTS radical scavenging activity of protein hydrolysate was stable when heated at 100 °C for 180 min and subjected to a wide pH range (1–11). Therefore, protein hydrolysate from the muscle of toothed ponyfish produced by viscera extract from hybrid catfish can be used as a promising source of functional peptides with antioxidant properties.     

A heat-stable trypsin inhibitor in adzuki bean (Vigna angularis): effect of extraction media, purification and biochemical characteristics

Trypsin inhibitor from adzuki bean ( Vigna angularis ) seed was isolated and characterised. Extraction of seed with NaCl at the concentration of 0.15  m showed a higher recovery of trypsin inhibitor than other solvents tested ( P  <   0.05). Optimal extraction time for the recovery trypsin inhibitor from adzuki bean seed was 30 min ( P  <   0.05). Purification of inhibitor was achieved by heat-treatment at 90 °C for 10 min, followed by ammonium sulphate precipitation with 30–65% saturation and size exclusion chromatography on Sephacryl S-200, presenting a yield and purification of 53.9% and 10.91-fold, respectively. The apparent molecular weight of trypsin inhibitor was estimated to be 14 kDa based on SDS-PAGE and inhibitor activity of zones separated by electrophoresis. The purified inhibitor was stable over a broad pH range and retained high inhibitory activity toward trypsin after incubation at 90 °C for 60 min. NaCl, at 0–3% concentration, did not affect the inhibitory activity of purified trypsin inhibitor, however, the activity was lost when sample was treated with β-mercaptoethanol prior to electrophoresis.     

Ka-pi-plaa fermented using beardless barb fish: physicochemical,microbiological and antioxidant properties as influenced by production processes

Production of Thai fish paste Ka-pi-plaa fermented using beardless barb, Cyclocheilichthys apogon was monitored. The physicochemical, microbiological and antioxidant properties were compared after each process, that is autolysis, salting, sun-drying and fermentation. Colour parameters L* decreased while a* and b* increased during production (P < 0.05). The Ka-pi-plaa finished product presented an intense brown colour as shown with the increase in browning intensity (A₄₂₀). Contents of formal nitrogen, ammonia nitrogen and amino nitrogen showed continuous increase (P < 0.05) indicating the formation of peptides and free amino acids, which were verified by protein patterns. Populations of total, halophilic, proteolytic, lipolytic and lactic acid bacteria (LAB) generally increased. Halophilic bacteria grew rapidly after salting. LAB counts were correlated with the pH change. It was suggested that a few biochemical reactions occurred during production, including protein hydrolysis by microbial and fish proteases, lipid oxidation as presented by the increasing thiobarbituric acid reactive substances value, and Maillard reaction based on the determined precursors and products. Antioxidant activities generally increased during production particularly fermentation, suggesting Ka-pi-plaa possessed 2,2-diphenyl-l-picrylhydrazyl and 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) scavenging and metal chelating activities, and ferric reducing power. This study provides important information on the relationship among production steps, various properties and chemical reactions for fish fermentation.     

Structural properties of trypsin from cold-adapted fish,arabesque greenling (<Emphasis Type="Italic">Pleurogrammus azonus</Emphasis>)

A cDNA clone encoding trypsin (AG-T) was isolated from the pyloric ceca of cold-adapted fish, arabesque greenling (Pleurogrammus azonus). The cDNA was composed of 892 bp with an open reading frame of 729 bp at nucleotide positions 25–753. Similar to all the known trypsin, the AG-T seemed to be synthesized as preproenzyme that contains a hydrophobic signal peptide, an activation pentapeptide and a mature trypsin of 222 amino acid residues. The AG-T also completely conserved the major structural features common to trypsin such as the catalytic triad (His57, Asp102, and Ser195), the obligatory Asp189 and twelve Cys residues. On the other hand, the AG-T possessed the deletion of Tyr151 and substitution of Pro152 for Gly in the autolysis loop when aligned with the sequence of tropical-zone fish and bovine trypsins. In addition, Val75 concerned in a combination with calcium ion was exchanged for Ala in the AG-T, and the content of positively charged amino acid residues at the calcium-binding site of the AG-T was three times higher than those of tropical-zone fish trypsins. Moreover, the ratio between charged and hydrophobic amino acid residues in the N-terminal region of the AG-T was also higher than those of temperate-zone fish and tropical-zone fish trypsins. Such structural properties of the AG-T would contribute to its low thermostability.     

Laundry detergent-stable lipase from Pacific white shrimp (Litopenaeus vannamei) hepatopancreas: Effect of extraction media and biochemical characterization

Extraction and biochemical properties of a new lipase from the hepatopancreas of Pacific white shrimp were studied. Recovery of the hepatopancreas powder with 50 mM Tris-HCl, pH 7.0 containing 0.2% (v/v) Brij35 gave a higher recovery of lipase activity than other extractants tested (p < 0.05). The optimal pH and temperature for lipase activity were 8.5 and 60°C, respectively, when p-nitrophenyl palmitate was used as a substrate. The enzyme was stable to heat treatment up to 40°C and over a pH range of 7.0–10.0 for 30–120 min. Lipase activities continuously decreased as the sodium deoxycholate (NaDC) concentration increased, but activities increased as NaCl concentration increased up to 3.0 M. Hydrolytic activity was enhanced by NaN₃, but strongly inhibited by Hg²⁺, Cu²⁺, Al³⁺, and phenylmethanesulfonyl fluoride. The lipase was evaluated as highly stable against surfactants (Tween 20, Tween 80, Triton X-100, and gum arabic). However, the enzyme was unstable against sodium dodecyl sulphate. Stability of the lipase with commercial liquid and solid detergents (Attack^®, Bres^®, Omo^®, and Pao^®) was also investigated. The lipase exhibited substantial stability and compatibility with tested commercial liquid and solid laundry detergents for 30–60 min. The overall properties of the lipase from Pacific white shrimp hepatopancreas, thus leading us to propose that it is an excellent candidate for use as biocatalysts for better detergent formulation.