Characteristics of gelatin from lizardfish (Saurida micropectoralis) and threadfin bream (Nemipterus hexodon) skins as influenced by extraction conditions.

Gelatins from lizardfish and threadfin bream skins were extracted using distilled water at 45 and 60 °C and 4, 8 and 12 h. Gelatin recovered from both lizardfish and threadfin bream skins was in the range of 63.96-91.46%. As extraction temperature and duration increased, the turbidity of gelatin solution from both species increased. Gelatins isolated from either lizardfish or threadfin bream skins at 45 °C for 4 and 8 h showed the maximum bloom strength (245.03-320.85 g), which were also greater than commercial gelatin from bovine (208.55 g) (P < 0.05). The gelatin gels (6.67%, w/v) could set at 4 °C within 3 min and were able to set at room temperatures within 51.83 min. Gelatins extracted from both fish skins contained α1-, ß- and γ-chains as predominant protein components. The lightness of all gelatin gels faintly declined with an increase in extraction temperature and time. Among the various production conditions explored, lizardfish/threadfin bream skin gelatin developed at 45 °C and 8 h was found to be highly comparable to commercial bovine gelatin. Based on the results obtained, gelatin from both fish species could be used as a replacement for land animal counterparts and can be used in many different food and pharmaceutical products.

Bacillus thermoamylovorans-Related Strain Isolated from High Temperature Sites as Potential Producers of Medium-Chain-Length Polyhydroxyalkanoate (mcl-PHA).

Thermotolerant bacteria producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) were isolated from various thermal sites, including palm oil mill effluent, textile wastewater, and hot spring water, in Thailand. Fifteen strains were isolated at 45 °C using nutrient-rich (NR) medium. However, only six isolates produced mcl-PHA at 0.41 ± 0.01 g/L to 0.80 ± 0.01 g/L, representing a mcl-PHA content of 29.44% to 50.77% of the dry cell weight (DCW). The six strains of bacterial isolates could utilise a variety of substrates; all were identified as Bacillus thermoamylovorans. The highest mcl-PHA content (50.77% of the DCW) was accumulated by the B. thermoamylovorans strain PHA005 isolated from palm oil mill effluent. The mcl-PHA from strain PHA005 was composed of five different monomers, 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxytetradecanoate (3HTD), 3-hydroxyhexadecanoic acid (3HHD), and 3-hydroxyoctadecanoic (3HOD), with a monomer content of 24.12, 15.50, 13.00, 39.25, and 8.13 mol%, respectively. The optimum temperature for B. thermoamylovorans strain PHA005 growth is 45 °C, and it can survive at up to 60 °C. This is a first report of PHA synthesis by a thermotolerant B. thermoamylovorans. Moreover, the high content of 3HHD monomers (39.25 mol%) has never been reported in Bacillus.

A thermostable trypsin from freshwater fish Japanese dace (Tribolodon hakonensis): a comparison of the primary structures among fish trypsins.

Trypsin from Japanese dace (Tribolodon hakonensis) (JD-T) living in freshwater (2-18 °C) was purified. JD-T represented typical fish trypsin characteristics regarding the effects of protease inhibitor, calcium-ion, and pH. For the effect of temperature, JD-T quite resembled to the trypsins from tropical-zone marine fish and freshwater fish (the catfish cultured in Thailand), i.e., the optimum temperature was 60 °C, and it was stable below 60 °C at pH 8.0 for 15 min incubation. From the data, it seemed that the trypsin from freshwater fish is thermostable in spite of the fact that their habitat temperatures are low. So, we determined the primary structure of JD-T to discuss its thermostability-structure relationship. JD-T possessed basic structural features of fish trypsin such as the catalytic triad, the Asp189 residue for substrate specificity, 12 Cys residues forming six disulfide-bridges, and the calcium-ion-binding loop. On the other hand, the contents of charged amino acid residues in whole JD-T molecule (16.2%) and N-terminal region (13.8%) were similar to those of tropical-zone marine fish and other freshwater fish trypsins. Then, JD-T conserved the five amino acid residues (Glu70, Asn72, Val75, Glu77, and Glu80) coordinate with calcium-ion, and the proportion of negatively charged amino acids to charged amino acids in the calcium-ion-binding region of JD-T (75.0%) was equivalent to those of tropical-zone marine fish and freshwater fish trypsins. Therefore, it was suggested that the high thermostability of JD-T are stemmed from these structural specificities.

Pectin from plantain peels: Green recovery for transformation into reinforced packaging films.

Plantain peels as agro-waste are generated in the millions of tons per year with no profitable management strategies. On the other hand, the excessive use of plastic packaging threatens the environment and human health. This research aimed to address both problems via a green approach. High-quality pectin was recovered from plantain peels via an enzyme-assisted and ethanol-recycling process. The yield and galacturonic acid (GalA) content of the recovered low methoxy pectin was 12.43% and 25.0%, respectively, when cellulase was added at 50 U per 5 g peel powder, with a significantly higher recovery rate and purity than the pectin products extracted with no cellulase (P ≤ 0.05). The recovered pectin was further integrated and reinforced with beeswax solid-lipid nanoparticles (BSLNs) to fabricate films as a potential alternative packaging material to single-use plastics. The reinforced pectin films showed improved light barrier, water resistance, mechanical, conformational, and morphological properties. This study presents a sustainable strategy to transform plantain peels into pectin products and pectin-based packaging films with broad applications.

Partitioning and recovery of proteases from lizardfish (Saurida micropectoralis) stomach using combined phase partitioning systems and their storage stability.

Partitioning and recovery of proteases from stomach extract (SE) and acidified stomach extract (ASE) of lizardfish using a three-phase partitioning (TPP) system in combination with an aqueous two-phase system (ATPS) was optimized. The highest yield and purity were obtained in the interphase of the TPP system, which consisted of a SE or ASE to t-butanol ratio of 1.0 : 0.5 in the presence of 40% (w/w) (NH4)2SO4. Both TPP fractions were further subjected to ATPS. Phase compositions of ATPS including PEG molecular mass and concentrations as well as types and concentrations of salts influenced protein partitioning. The best ATPS conditions for protease partitioning into the top phase from TPP fractions of SE and ASE were 15% Na3C6H5O7-20% PEG1000 and 20% Na3C6H5O7-15% PEG1000, which increased the purity by 4-fold and 5-fold with the recovered activity of 82% and 77%, respectively. ATPS fractions of SE and ASE were subsequently mixed with several PEGs and salts for back extraction (BE). BE using 25% PEG8000-5% Na3C6H5O7 gave the highest PF and yield for both ATPS fractions. SDS-PAGE investigation revealed that the decrease in contaminating protein bands was observed after the combined partitioning systems. BE fractions of SE and ASE were quite stable at -20 and 0 °C up to 14 days. Therefore, the combination of TPP, ATPS and BE could be effectively applied to recover and purify proteases from the stomach of lizardfish.

Enzymological characteristics of pepsinogens and pepsins purified from lizardfish (Saurida micropectoralis) stomach.

One major pepsinogen, PG-I, and two minor pepsinogens, PG-II and PG-III were purified from lizardfish stomach by ammonium sulfate precipitation and two chromatographic columns. The three purified PGs migrated as single bands in native-PAGE gels with molecular weights (MW) ranging from 36 to 38 kDa. Each PG was converted to pepsin (P) at pH 2.0, and the MW were determined as 32 kDa (for P-I), 31 kDa (for P-II) and 30 kDa (for P-III). The optimum pH and temperature of pepsins were 2.0-3.5, and 40-50 °C. All 3 pepsins were strongly inhibited by pepstatin A. Divalent cations slightly stimulated the pepsin activities, but ATP had no effect on the pepsins. Purified pepsins were effective in the hydrolysis of various proteins. Km and kcat of the three pepsins for hemoglobin hydrolysis were 107.64-276.61 µM and 18.30-32.68 s-1, respectively. The new pepsins have potential for use in protein food procession and modification.

24kDa Trypsin: A predominant protease purified from the viscera of hybrid catfish (Clarias macrocephalus×Clarias gariepinus).

Trypsin was purified to homogeneity from the viscera of hybrid catfish (Clarias macrocephalus×Clarias gariepinus) through ammonium sulphate fractionation and a series of chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. It was purified to 47.6-fold with a yield of 12.7%. Based on native-PAGE, the purified trypsin showed a single band. The molecular weight of purified trypsin was estimated as 24kDa by size exclusion chromatography and SDS-PAGE. The optimum pH and temperature for N(α)-p-tosyl-l-arginine methyl ester hydrochloride (TAME) hydrolysis were 8.0 and 60°C, respectively. Trypsin was stable to heat treatment up to 50°C, and over a pH range of 6.0-11.0. Trypsin was stabilized by calcium ion. The trypsin activity was strongly inhibited by soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone and partially inhibited by ethylenediaminetetraacetic acid. Activity decreased continuously as NaCl concentration (0-30%) increased. Apparent Km value of trypsin was 0.3mM and Kcat value was 92.1S(-1) for TAME. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGYECQAHSQPPTVSLNA, which is highly homologous with trypsins from other species of fish.

Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization.

Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5'-4,4'-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 µM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.

Anionic trypsin from the spleen of albacore tuna (Thunnus alalunga): Purification, biochemical properties and its application for proteolytic degradation of fish muscle.

Anionic trypsin from albacore tuna spleen was purified by chromatographic separations on Q-Sepharose, Superdex 75 and Arginine Sepharose 4B. The trypsin migrated as single bands in both SDS-PAGE and native-PAGE. The molecular weight of purified trypsin was estimated to be 30 kDa using SDS-PAGE. The enzyme exhibited maximal activity at pH 9.0 and 55 °C for hydrolysis of Boc-Val-Pro-Arg-MCA. pH and temperature stabilities of the trypsin were well maintained in the pH range of 6-11 and over a temperature range from 20 up to 50 °C. The enzyme was effectively inhibited by soybean trypsin inhibitor, N­tosyl­l­phenyl­alanine chloromethyl ketone (TLCK) and Pefabloc SC. The N-terminal amino acid sequence of 20 residues of the purified enzyme was IVGGYECQAHSQPHQVSLNA, which is highly homologous to other fish trypsins. The kcat/Km of the enzyme for Boc-Val-Pro-Arg-MCA was 2.60 ±â€¯0.07 s-1 mM-1. Purified trypsin also hydrolysed fish muscle proteins, suggesting its effectiveness in degradation of food proteins.

Two trypsin isoforms from albacore tuna (Thunnus alalunga) liver: Purification and physicochemical and biochemical characterization.

Two trypsins (A and B) from the liver of albacore tuna (Thunnus alalunga) were purified to homogeneity using a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and Diethylaminoethyl-cellulose. Purity was increased to 80.35- and 101.23-fold with approximately 3.1 and 19.2% yield for trypsins A and B, respectively. The molecular weights of trypsins A and B were estimated to be 21 and 24kDa, respectively, by SDS-PAGE and size exclusion chromatography. Both trypsins showed only one band on native-PAGE. Trypsins A and B exhibited the maximal activity at 60°C and 55°C, respectively, and had the same optimal pH at 8.5 using Nα-p-Tosyl-l-arginine methyl ester hydrochloride (TAME) as a substrate. Stabilities of both trypsins were well maintained at a temperature up to 50°C and in the pH range of 7.0-11.0 and were highly dependent on the presence of calcium ion. The inhibition test demonstrated strong inhibition by soybean trypsin inhibitor and TLCK. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). The N-terminal amino acid sequence of 20 residues of the two trypsin isoforms had homology when compared to those of other fish trypsins.

29 kDa Trypsin from the pyloric ceca of Atlantic Bonito (Sarda sarda): recovery and characterization.

Trypsin from the pyloric ceca of Atlantic bonito (Sarda sarda) was purified and characterized with respect to its purity; molecular weight; sensitivity to temperature, pH, and inhibition; and N-terminal sequence. The purified trypsin had a molecular weight of 29 kDa as per sodium dodecyl sulfate polyacrylamide gel electrophoresis, and optimal activity was observed at pH 9 and 65 degrees C with BAPNA as a substrate. The enzyme was stable to heat treatment up to 50 degrees C and within the pH range of 7-12. It was stabilized by calcium ions, but its activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone, and phenyl methyl sulfonyl fluoride. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration (0-30%). The N-terminal 20 amino acid residues of Atlantic bonito trypsin were determined as IVGGYECQAHSQPWQPVLNS and were homologous with other trypsins.

Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis).

Two pepsins (A and B) were purified from the stomach of pectoral rattail (Coryphaenoides pectoralis) by acidification, ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography to obtain a single band on native-PAGE and SDS-PAGE. The purities of pepsin A and B were increased to 7.1- and 13.0-fold with approximately 5.7% and 2.2% yield, respectively. Pepsin A and B had the apparent molecular weights of 35 and 31 kDa, respectively, when analyzed using SDS-PAGE and Sephacryl S-200 gel filtration. Pepsin A and B showed maximal activity at pH 3.0 and 3.5, respectively, and had the same optimal temperature at 45 degrees C using hemoglobin as a substrate. Both pepsin A and B were stable in the pH range of 2.0-6.0 but were unstable at the temperatures greater than 40 degrees C. Activity of both pepsins was inhibited by pepstatin A and was activated by divalent cations, indicating pepsin characteristics. Activities of both pepsins continuously decreased as NaCl concentration increased (0-30%). The enzymes had high affinity and activity toward hemoglobin with Km and Kcat values of 98-152 microM and 32-50 S(-1), respectively. Purified pepsins generally showed the similar characteristics to other fish pepsins.

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 degrees 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 degrees 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 degrees 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.

Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol).

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.

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.

Interrelationship between myoglobin and lipid oxidations in oxeye scad (Selar boops) muscle during iced storage.

The interrelationship between myoglobin oxidation, lipid oxidation and discolouration in oxeye scad fish during iced storage were investigated. The myoglobin autoxidation rate increased with increasing storage time up to 12 days (p < 0.05) and remained constant thereafter (p > 0.05). Increase in metmyoglobin correlated well with a blue shift from 410 to 408 nm for myoglobin. The soret band of myoglobin decreased with a concomitant decrease in the redness index (p < 0.05). During storage, the extractable haem iron decreased (p < 0.05), while the non-haem iron increased (p < 0.05). Hydrogen peroxide and ferrylmyoglobin concentrations had increased at the end of storage (p < 0.05). The conjugated diene (CD) and peroxide value (PV) of oxeye scad lipids tended to stabilise during the initial phase of storage, increased in the differentiation phase and had declined at the end of storage. However, thiobarbituric acid reactive substances (TBARS) increased markedly (p < 0.05). Overall, lipid and myoglobin oxidations in oxeye scad occurred in a concurrent manner and each process appeared to enhance the other.

Characterisation of muscles from Frigate mackerel (Auxis thazard) and catfish (Clarias macrocephalus).

Frigate mackerel (Auxis thazard) and catfish (Clarias macrocephalus) can be used as alternative sources for surimi production. However, the functionality of surimi is species-dependent. This study aimed to characterise certain chemical and physical compositions of dark and ordinary muscles from these species. Catfish, particularly ordinary muscle, was composed of higher contents of lipid and carotenoid than Frigate mackerel muscle (p<0.05) but ordinary muscle from Frigate mackerel had the highest phospholipid content (p<0.05). Both dark and ordinary muscles of Frigate mackerel had greater contents of myofibrillar proteins than had catfish muscle (p<0.05). Myosin heavy chain and actin were predominant proteins found in both muscle types of both species. Dark muscle from Frigate mackerel had the highest sarcoplasmic protein content, especially extractable myoglobin (p<0.05). Muscles from Frigate mackerel had greater content of sodium chloride than had catfish (p<0.05). The highest contents of iron, copper and selenium were found in Frigate mackerel dark muscle (p<0.05). The pH of ordinary muscle from both species was higher than that of dark muscle (p<0.05). Frigate mackerel, especilly dark muscle, exhibited the most dark-red colour, as shown by the lowest L(*) and b(*) values with the highest a(*) value and redness index (a(*)/b(*)) (p<0.05).

Use of viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus) for the production of protein hydrolysate from toothed ponyfish (Gazza minuta) muscle.

Proteolytic activity of viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was studied. The optimal pH and temperature were 9.0 and 50°C, respectively, when toothed ponyfish (Gazza minuta) muscle was used as a substrate. When viscera extract from hybrid catfish was used for the production of protein hydrolysate from toothed ponyfish muscle, extract concentration, reaction time, and fish muscle/buffer ratio affected the hydrolysis and nitrogen recovery (NR) (p<0.05). Optimum conditions for toothed ponyfish muscle hydrolysis were 3.5% hybrid catfish viscera extract, 15 min reaction time and fish muscle/buffer ratio of 1:3 (w/v). High correlation between the degree of hydrolysis (DH) and NR (R(2)=0.974) was observed. Freeze-dried hydrolysate had a high protein content (89.02%, dry weight basis) and it was brownish yellow in colour (L(∗)=63.67, a(∗)=6.33, b(∗)=22.41). The protein hydrolysate contained a high amount of essential amino acids (48.22%) and had arginine and lysine as the dominant amino acids.

Mackerel trypsin purified from defatted viscera by supercritical carbon dioxide.

Viscera of mackerel (Scomber sp.) were defatted by supercritical carbon dioxide (SCO(2)) treatment. Trypsin (SC-T) was then extracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200 and Sephadex G-50. The purified SC-T was nearly homogeneous on SDS-PAGE, and its molecular weight was estimated as approximately 24,000 Da. N-terminal twenty amino acids sequence of SC-T was IVGGYECTAHSQPHQVSLNS. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of SC-T. The pH and temperature optimums of SC-T were at around pH 8.0 and 60°C, respectively, using N(α)-p-tosyl-L-arginine methyl ester as a substrate. The SC-T was unstable below pH 5.0 and above 40°C, and it was stabilized by calcium ion. These enzymatic characteristics of SC-T were the same as those of other fish trypsins, especially spotted mackerel (S. borealis) trypsin, purified from viscera defatted by acetone. Therefore, we concluded that the SCO(2) defatting process is useful as a substitute for organic solvent defatting process.

Simple preparation of pacific cod trypsin for enzymatic Peptide synthesis.

Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography on Benzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and their molecular masses were estimated 23,756.34 Da, 23,939.62 Da, and 24,114.81 Da by desorption/ionization time-of-flight mass spectroscopy (MALDI/TOF-MS) and their isoelectric points (pIs) were approximately 5.1, 6.0, and 6.2, respectively. The isolated Pacific cod trypsin showed high similarity to other frigid-zone fish trypsins. The kinetic behavior of tryptic hydrolysis toward N-p-tosyl-L-arginine methyl ester hydrochloride (TAME), N-benzoyl-L-arginine p-nitroanilide hydrochloride (BAPA), and p-amidinophenyl ester were also analyzed. In addition, the cod trypsin-catalyzed dipeptide synthesis was investigated using twelve series of "inverse subdtrates" that is p- and m-isomer of amidinophenyl, guanidinophenyl, (amidinomethyl)phenyl, (guanidinomethyl)phenyl, and four position isomers of guanidinonaphtyl esters derived from N-(tert-butoxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of the trypsin. All inverse substrates tested in this study undergo less enantioselective coupling reaction. The p-guanidinophenyl ester was most practical substrate in twelve series tested. The enzymatic hydrolysis of the resulting products was negligible.