Isolation,Purification, and Biochemical Characterization of Trypsin from Indian Mackerel (Rastralliger kanagurta)

Trypsin from viscera of Indian mackerel (Rastralliger kanagurta) was purified by ammonium sulphate precipitation and chromatographic techniques such as size exclusion, ion exchange, and affinity chromatography, with a 14.4-fold increase in specific activity and 18.7% recovery. The molecular weight of the trypsin was estimated to be approximately 26 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified trypsin showed amidase-specific activity which was determined using benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for isolated trypsin activity were 9.0 and 50°C, respectively. The purified trypsin was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK). Purified trypsin showed almost 40% recovery at high NaCl concentration (30%). The N-terminal amino acid sequence of the first 10 amino acids of purified trypsin was IVGGYESQPH. The Michaelis-Menten constant (K_m) and catalytic constant (K_cat) of purified trypsin were 0.430 mM and 0.77 s^?1, respectively, determined using BAPNA as a substrate. Purified trypsin showed digestion of casein similar to bovine trypsin by the fluorometric method.     

Autolysis of krill protein from North Pacific krill Euphausia pacifica during protein recovery via isoelectric solubilization/precipitation

Autolysis of North Pacific krill protein in acidic and alkaline solubilization during protein recovery via isoelectric solubilization/precipitation was investigated. Pronounced auto-degradation of myosin heavy chain and actin from krill protein was observed at alkaline pH values (pH 9.0–12.0), with maximum autolytic activity at about pH 12.0. Meanwhile, proteolytic activity in krill protein at pH 12.0 was observed at low temperatures, suggesting a possible cause for the autolysis of krill protein during the protein recovery process. Three major proteinases were detected by zymographic analysis of myofibrillar protein, with approximate molecular weights of 26 kDa (KP-1), 18 kDa (KP-2) and 17 kDa (KP-3). KP-3 was active over a pH range from pH 5.0–12.0, suggesting that it may be responsible for the autolysis of krill protein during alkaline-induced protein recovery. Further study on substrate specificity and inhibitory specificity of KP-3 showed that KP-3 is serine type proteinase.     

Correlation of Kudoa Spore Counts with Proteolytic Activity and Texture of Fish Mince from Pacific Hake (Merluccius productus)

ABSTRACT

Sixteen batches of fish mince prepared using fillets from Pacific hake (Merluccius productus) harvested near Vancouver Island, Canada were studied for possible correlations of Kudoa spore counts with proteolytic activity and cooked texture. The fish were mainly infected by K. paniformis, and Kudoa spores were distributed throughout 7 sampling sites on 13 individual hake fish tested. Proteolytic activity of fish homogenate was optimum within the range of pH 5.25–5.50 and 52–55°C. Significant (p < 0.05) correlations were observed among fish mince K. paniformis spore counts, endogenous proteolytic activity, cooked texture measured as maximum compression force (g), and the change in free amino groups during autolysis.     

Gel Forming Ability and Other Properties of Eleven Underutilized Tropical Marine Fish Species

Abstract

The gel forming ability and other characteristics of the mince of 11 underutilized marine fish were studied. They were Bombay duck, silverbelly, sea catfish, silver jewfish, jewelled shad, queenfish, Spanish mackerel, hardtail, Indian tuna, tripletail and false conger eel. Mince was prepared from fillet and a portion of the mince was washed two times with cold water (5°C) containing 0.1% NaCl. Both washed and unwashed mince were ground with 3% NaCl. Ground paste was then stuffed into plastic tube and heated for one- and two-step heating. In the one-step heating, the tubes were subjected to 25°, 30°, 35°, 40°, 50°, 60°, 70° and 80°C for 60, 120 and 180 min. In the two-step heating, the tubes were pre-heated at 25°, 30°, 35°, 40°, 50°, 60°, 70° and 80°C for 60, 120 and 180 min. After the pre-heating, the tubes were immediately subjected to 85°C for 30 min. The gel was subjected to puncture, folding, expressible moisture and sensory tests.

Two-step heating distinctly improved the gel strength compared to the one-step heating. The improvement due to two-step heating was more at low preheating temperatures from 25-35°C. Washing improved the texture and color of all of the gels except Bombay duck and decreased the extent of gel-disintegration in silverbelly, queenfish, sea catfish and hardtail. The gels were set optimally at 35°-40°C for most species. Species variation in the disintegration of the gels was observed. Bombay duck mince produced very weak gel. Neither two-step heating nor washing could improve the gel quality of Bombay duck mince. Our data suggested that jewelled shad, queenfish, silver jewfish, sea catfish, tripletail and false conger eel could be suitable as the material for surimi.     

Gelation Characteristics of Mince and Washed Mince From Small-Scale Mud Carp and Common Carp

Small-scale mud carp (SC, Cirrhina microlepis) and common carp (CC, Cyprinus carpio) are underutilized freshwater fish. The objective of this study was to elucidate the gel-forming ability of mince and washed mince from these species in relation to washing cycles and CaCl₂ addition. Protein loss of up to 67% was observed after three-washing cycles of both species, rendering relatively low yield. About 85–90% of total fat was removed from SC flesh after three-washing cycles, but only two-washing cycles were sufficient for fat removal in CC mince. Mince and washed mince of both species did not exhibit severe proteolysis with low autolytic activity at 65°C. SC exhibited superior gel-forming ability to CC. Two- and three-washing cycles in conjunction with a 40°C-preincubation resulted in the highest textural properties of SC and CC, respectively. CaCl₂ increased the breaking force of CC and SC mince gels up to 65 and 95% at 0.3 and 0.5% CaCl₂, respectively, as compared to without CaCl₂. However, it showed negative effects on surimi gels of both species. Both SC and CC are potential freshwater fish resources for mince and washed mince production with reasonable good gel-forming ability.     

Protease Activity and Partial Characterization of the Trypsin-Like Enzyme in the Digestive Tract of the Tropical Sierra Scomberomorus concolor

Abstract

Protease and trypsin-like activities of the digestive system of the tropical sierra Scomberomorus concolorwere evaluated. Sierra digestive tract extracts hydrolyzed specific substrates for trypsin, chymotiypsin, and leucine aminopeptidase At least eight bands of activity were observed by using SDS-PAGE. In PAGE and serineinhibition assays, one fraction resembled bovine trypsin. An ammonium sulfate (40-60%) trypsin-like fraction displayed different inhibitions with tosyllyschloromethyl ketone (96%) and soybean trypsin inhibitor (100%). Maximum activity was found using benzoyl-L-arginine-p-nitroanilide as substrate at pH 9.0 at 25°C and around 50°C at pH 7.8. The capacity of the trypsin-like fraction to work at different pHs (4, 7, and 9) at 25°C and different temperatures (0 and 40°C) at pH 7.8 were detected.     

Antioxidative Activitiy of Protein Hydrolysate from the Muscle of Common Kilka (Clupeonella cultriventris caspia) Prepared Using the Purified Trypsin from Common Kilka Intestine

Trypsin from the intestine of common kilka (Clupeonella cultriventris caspia) was purified using ammonium sulfate precipitation (30–50% saturation), Sephadex G-75, and DEAE-cellulose chromatography with the purity of 30-fold and the yield of 12%. The molecular weight of trypsin was estimated to be 23.2 kDa based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The trypsin had optimal activity at pH 8.0 and 60°C using N-α-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) as a substrate and showed high stability in the pH range of 7.0–10.0. It was stable up to 50°C. Soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) significantly inhibited trypsin activity (p < 0.05). Protein hydrolysate from common kilka muscle with different degrees of hydrolysis (DHs; 20, 30, and 40%) was prepared using the purified trypsin, and antioxidative activities were determined. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power, and ferrous chelating activity of hydrolysate increased with increasing DH up to 40% (p < 0.05). Therefore, trypsin from intestine of common kilka could be used as a processing aid for production of fish protein hydrolysate with antioxidative activity.     

Purification and characterization of three trypsin isoforms from viscera of sardinelle (<Emphasis Type="Italic">Sardinella aurita</Emphasis>)

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20–70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0–11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K _m and k _cat were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s⁻¹, respectively. The N-terminal sequences of the first 10 amino acids were “I V G G Y E C Q K Y” for trypsin A and “I V G G Y E A Q S Y” for trypsins B and C. These sequences showed highly homology to other fish trypsins.     

Organic Solvent and Laundry Detergent Stable Crude Protease from Nile Tilapia (Oreochromis niloticus) Viscera

The objective of this study was to explore the characteristic activity of crude protease extracted from Nile tilapia viscera. The optimal temperature and pH of the crude protease from Nile tilapia viscera were 60°C and 8.0, respectively. The enzyme was stable to heat treatment up to 45°C and over a pH range of 7–11 for 30–120 min. The protease was effectively inhibited by phenylmethylsulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI). It was activated by Ca²⁺ and Fe²⁺, while inhibited in order by Hg²⁺ > Fe³⁺ > Cu²⁺ > Mn²⁺. The protease showed great stability toward Tween 20, Tween 80, and Triton X-100 and moderate stability toward sodium dodecyl sulfate (SDS) and hydrogen peroxide (H₂O₂)_. Nevertheless, it showed excellent stability and compatibility with various solid and liquid laundry detergents at temperatures from 30 to 50°C. The protease showed not only an improved activity but also a satisfied stability in 25% (v/v) organic solvents for 7 days at 37°C. At higher concentrations (50–75%), the protease activity was decreased by hydrophilic solvents, except dimethyl sulfoxide (DMSO), whereas it was enhanced by hydrophobic solvents. Thus, the crude protease is an excellent candidate as a biocatalyst for detergents, foods, pharmaceuticals, and environmental applications.     

Anionic Trypsin from the Pyloric Ceca of Pacific Saury (Cololabis saira): Purification and Biochemical Characteristics

Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants K_m and K_cat were 0.19 mM and 210 s^?1, respectively, while the catalytic efficiency (K_cat/K_m) was 1105.26 s^?1 mM^?1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.     

Purification and characterization of trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides

Trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides, was purified by fractionation with ammonium sulfate, ionic exchange, and affinity chromatography. The protein was purified 161.85-fold with a yield of 4%. Purified trypsin had an apparent molecular weight of 24 kDa according to an SDS-PAGE analysis. Optimal profiles of temperature and pH of the enzyme were 50°C and 8–10, respectively, using Nα-benzoyl-l-arginine ethyl ester as the substrate. The results of thermal and pH stability assays showed that the enzyme was stable at temperatures of up to 50°C and in the pH range of 6–8. Trypsin activity decreased with an increasing NaCl concentration (0–0.6 M). The activity of purified trypsin was effectively inhibited by a soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone, and was slightly inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, 1-(l-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane, and pepstatin A. Protein identification of the purified protease showed that the sequences of two peptides, LGEHNI and NLDNDIML, were highly homologous to other fish trypsins. The measurement of trypsin activity in different tissues showed that the highest activity was detected in pyloric ceca, followed by anterior intestine, middle intestine, hind intestine and spleen, but very low activities were found in other tissues. An inverse relationship between the trypsin activity in four tissues of pyloric ceca, anterior intestine, middle intestine and hind intestine and fish body weight as a result of increased pepsin in stomach indicated grouper growth status was increased.     

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACT

The serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl₂ (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as K_m (0.104 mM), k_cat (44.25 s^?1), and catalytic efficiency (427.54 s^?1 mM^?1). Monovalent, bivalent, and trivalent ions (Li⁺, K⁺, Hg²⁺, Al³⁺, Mg^2+, Cd^2+, Co^2+, Zn²⁺, and Al³⁺) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-_L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.     

Influence of feeding habits of fish on the characteristics of their visceral alkaline proteases with special reference to detergent compatibility

Technical characteristics and detergent compatibility of visceral alkaline proteases of three freshwater fish, namely Labeo rohita, Pangasianodon hypophthalmus and Cyprinus carpio of different feeding habits, were studied. Crude enzyme extract was partially purified by (NH₄)₂SO₄ precipitation and dialysis. The molecular weight was in the range of 20–63 kDa. The enzyme purification folds post‐dialysis were found to be 1.55, 1.81 and 2.17 in case of Rohu, Pangas and Common carp respectively. The alkaline protease from Rohu, Pangas and Common carp exhibited maximum activity at pH 10.0, 9.0 and 11.0 respectively. The enzyme temperature optima observed were 60°C (Rohu and Pangas) and 70°C (Common carp). SBTI and EDTA inhibited more than 90% of the activity at conc. of 50 mM. Exposure of the proteases to non‐ionic surfactants like Tween 20–80 retained about 92%–100% and 76%–100% of their activity at conc. (v/v) of 1% and 5% respectively. Proteases were found less stable in the presence of SDS. There was moderate to lesser influence of H₂O₂ and sodium perborate on the proteolytic activity. The alkaline protease from omnivorous fish was found superior compared to the herbivore and carnivore in respect of pH and temperature optima and stability with detergents and oxidizing agents.     

Trypsin Enzyme from Viscera of Common Kilka (Clupeonella cultriventris caspia): Purification,Characterization, and Its Compatibility with Oxidants and Surfactants

The purification of trypsin from the common kilka (Clupeonella cultriventris caspia) viscera (pyloric caeca) resulted in a 28.3-fold increase and 12% yield by ammonium sulfate precipitation (30–60%), Sephadex G-75, and DEAE-cellulose chromatography. Trypsin showed a molecular weight of 23.2 kDa and appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, and zymography. The trypsin had optimal activity at pH 8.0 and 60°C for the hydrolysis of α-N-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) substrate. Trypsin was stable up to 50°C and at pH range of 7.0–10.0. Activity was significantly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) inhibitors (p < 0.05). The enzyme was relatively stable toward oxidizing agents, retaining 59.7 and 98.0% of its initial activity after 1 h incubation in the presence of 15% H₂O₂ and 1% sodium perborate, respectively. Trypsin was significantly activated by surfactants and Ca²⁺, Mg²⁺, and Mn²⁺ and inactivated by Fe²⁺, Zn²⁺, Cu²⁺, Al³⁺, Ba²⁺, and Co²⁺ (p < 0.05). Nevertheless, Na⁺ and K⁺ had no significant effect on trypsin activity (p > 0.05). The purified trypsin showed significantly higher catalytic efficiency (k_cat/K_m) than porcine pancreatic trypsin against BAPNA and N-α-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) substrates (p < 0.05).     

Partial characterization and activity distribution of proteases along the intestine of grass carp, Ctenopharyngodon idella (Val.)

A series of studies based on biochemical assays and electrophoretical observations has been conducted in order to investigate activity distributions and partially characterize various types of proteinases in the digestive tract of grass carp, Ctenopharyngodon idella (Val.). The casein digestion assays revealed that the presence of acidic proteinase had the highest activity at pH 2.5–3.0 and the alkaline proteinases at pH 10.0. The acidic proteinase activity distribution was found to decrease gradually from the oesophagus to the anus. Pepstatin A and EDTA inhibited the acidic proteinases activity. The SDS‐substrate‐PAGE showed that crude extraction of grass carp intestine contained an acidic proteinase active component with molecular mass of 28.5 ku. The substrate‐PAGE at neutral pH condition showed the presence of two acidic proteinase active components. The activity distribution of alkaline proteinase was found to slightly fluctuate along the intestine. And the whole intestine had very high activity. The inhibition assays and substrate specificity assays showed that trypsin was the main active component of the alkaline proteinases. The SDS‐substrate‐PAGE further showed that the crude extraction of grass carp intestine had four types of alkaline proteinase with molecular mass of 26.4, 30.8, 43.0 and 105.0 ku respectively. They were characterized to be trypsin (26.4, 30.8 and 43.0 ku) and un‐serine proteinase (105.0 ku) respectively. No chymotrypsin was detected.     

Digestive protease characterization, localization and adaptation in blacklip abalone (Haliotis rubra Leach)

Digestive protease in blacklip abalone (Haliotis rubra Leach) from whole gut extracts was found to have dual pH optima at pH 3 and 10 and an optimum temperature of 45 °C. Over the biologically relevant range of pH (5–8), protease activity dropped to a minimum at pH 5 (53% of the maximal rate at pH 3) rising gradually and continuously up to (and beyond) pH 8. Over the biologically relevant range of temperatures (9–24 °C), protease activity increased continuously with activity at 24 °C being 75% higher than activity at 9 °C. Protease digestion was relatively uniform in gut extracts from sections containing gut contents. Digestion thus appears to be significantly extracellular (in the lumen of the gut). Analysis of gut sections washed free of food matter suggests the anatomical origin of protease from activity of gut regions in the order: digestive gland » salivary gland ≈ stomach > crop > intestine > upper oesophagus. Whole gut protease levels did not alter in response to a high protein artificial diet.     

Some proteolytic properties of hatching liquid in the sea trout,Salmo trutta m. trutta L.

Proteolytic activity of sea trout hatching liquid was examined towards casein and azocazein as a function of pH and temperature. The optimum pH for caseinolytic and azocaseinolytic activities were 9.4, and 9.0, respectively. At alkaline pH the enzyme was activated by low concentrations of Zn²⁺ ions (10⁻⁵ M). Maximum proteolytic activity of the hatching liquid was observed at 25°C. Temperatures exceeding 30°C caused a rapid reduction in enzyme activity. Proteolytic activity observed at 10°C was approximately 50% of that observed at 25°C. In general, a pseudo-Arrhenius plot indicated a Q₁₀ of 1.6 between 6 and 25°C.     

Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein⁻¹ min⁻¹) followed by silver carp (1.084 ± 0.061 U mg  protein⁻¹ min⁻¹), and catla (0.193 ± 0.006 U mg  protein⁻¹ min⁻¹). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.     

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.