A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

Purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

An aminopeptidase was purified from the skeletal muscle of common carp (Cyprinus carpio) to homogeneity, with 1160-fold purification and a yield of 4.3%. The purification procedure consisted of ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, Phenyl Sepharose and Macro-Prep High Q columns. The molecular mass of the enzyme was estimated to be 105 kDa and 100 kDa by SDS-PAGE under reducing conditions and gel filtration chromatography, respectively, suggesting it to be a monomer. The enzymatic activity was optimal at 35 °C and pH 7.0. The metal-chelating agents EDTA, EGTA and 1,10-phenanthroline specifically inhibited the enzyme activity while inhibitors of other proteinases did not show much effect, indicating that it was a metalloproteinase. Furthermore, bestatin, a specific aminopeptidase inhibitor strongly inhibited its activity. Divalent cations Mn²⁺, Mg²⁺ and Ba²⁺ slightly enhanced the enzymatic activity, while Co²⁺, Cu²⁺, Zn²⁺, Ca²⁺ and Fe²⁺ inhibited the activity to different extents. In addition, a sulfhydryl reagent was necessary to maintain its activity. Substrate specificity study revealed that the purified enzyme preferentially hydrolyzed Leu-MCA, followed by Arg-MCA, Ala-MCA and Tyr-MCA and it was thus regarded as a leucine aminopeptidase (LAP, EC 3.4.11.1). The apparent K_m and V_max values of the enzyme were 4.6 μM and 9.6 μmol min⁻¹ mg⁻¹, respectively for substrate Leu-MCA. This is the first report concerning purification and characterization of LAP from freshwater fish.     

Effects of metal ions on growth, β-oxidation system,and thioesterase activity of Lactococcus lactis

The effects of divalent metal ions (Ca²⁺, Mg²⁺, Fe²⁺, and Cu²⁺) on the growth, β-oxidation system, and thioesterase activity of Lactococcus lactis were investigated. Different metal ions significantly influenced the growth of L. lactis: Ca²⁺ and Fe²⁺ accelerated growth, whereas Cu²⁺ inhibited growth. Furthermore, Mg²⁺ inhibited growth of L. lactis at a low concentration but stimulated growth of L. lactis at a high concentration. The divalent metal ions had significant effects on activity of the 4 key enzymes of the β-oxidation system (acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and thiolase) and thioesterase of L. lactis. The activity of acyl-CoA dehydrogenases increased markedly in the presence of Ca²⁺ and Mg²⁺, whereas it decreased with 1 mmol/L Fe²⁺ or 12 mmol/L Mg²⁺. All the metal ions could induce activity of enoyl-CoA hydratase. In addition, 12 mmol/L Mg²⁺ significantly stimulated activity of L-3-hydroxyacyl-CoA dehydrogenase, and all metal ions could induce activity of thiolase, although thiolase activity decreased significantly when 0.05 mmol/L Cu²⁺ was added into M17 broth. Inhibition of thioesterase activity by all 4 metal ions could be reversed by 2 mmol/L Ca²⁺. These results help us understand the effect of metal ions on the β-oxidation system and thioesterase activity of Lactococcus lactis.     

Purification and characterization of a novel glucoamylase from Fusarium solani

Thermostable enzymes are currently being investigated to improve industrial processes of starch saccharification. A novel glucoamylase was purified to electrophoretic homogeneity from the culture supernatant of Fusarium solani on a fast protein liquid chromatographic system (FPLC). The recovery of glucoamylase after gel filtration on FPLC was 31.8% with 26.2-fold increase in specific activity. The enzyme had a molecular mass of 40 kDa by SDS-PAGE and 41 kDa by gel filtration. The glucoamylase exhibited optimum activity at pH 4.5. The K_cat and K_m were 441/min and 1.9 mg/ml, respectively, for soluble starch, specificity constant (K_cat/K_m) was 232. The enzyme was thermally stable at 50 °C and retained 79% activity after 60 min at this temperature. The half-life of the enzyme was 26 min at 60°C. The enzyme was slightly stimulated by Cu²⁺ and Mg²⁺ and strongly inhibited by Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe³⁺.     

Purification and characterization of chymotrypsins from the hepatopancreas of crucian carp (Carassius auratus)

Two chymotrypsins (chymotrypsin A and B) have been purified to homogeneity from the hepatopancreas of crucian carp (Carassius auratus) by ammonium sulphate fractionation and chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose and SP-Sepharose. The molecular masses of chymotrypsin A and B were approximately 28 and 27 kDa, respectively, by SDS–PAGE. Purified chymotrypsins also revealed single bands by native-PAGE. Optimum temperatures of chymotrypsin A and B were 40 and 50 °C, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Both enzymes were effectively inhibited by serine proteinase inhibitors and slightly activated by metal ions such as Ca²⁺ and Mg²⁺, while inactivated by Mn²⁺, Cd²⁺, Cu²⁺, Fe²⁺ to different degrees. Apparent K_ms of chymotrypsin A and B were 1.4 and 0.5 μM, and K_cats were 2.7 S⁻¹ and 3.4 S⁻¹, respectively. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A.     

Purification and characterisation of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06

Three exoinulinases (Exo-I, Exo-II, and Exo-III) and two endoinulinases (Endo-I and Endo-II) were purified from the culture broth of Aspergillus ficuum JNSP5-06 by ammonium sulphate precipitation, DEAE-cellulose column chromatography, Sepharose CL-6B column chromatography and preparative electrophoresis. The molecular weights of Exo-I, Exo-II, Exo-III, Endo-I, and Endo-II were determined to be 70 kDa, 40 kDa, 46 kDa, 34 kDa, and 31 kDa, respectively. Using inulin as the substrate, their K_m values were 43.1 mg/ml, 31.5 mg/ml, 25.3 mg/ml, 14.8 mg/ml, and 25.6 mg/ml, respectively. These five inulinases were stable below 50 °C with optimum activity at 45 °C, and were stable at a pH range of 4–8 with an optimum pH at 4.5 for exoinulinase and at 5.0 for endoinulinase. The inulinase activity was completely inhibited by Ag⁺ and strongly inhibited by Fe²⁺ and Al³⁺, whereas K⁺, Ca²⁺, Li²⁺, EDTA and urea had no significant influence on the inulinase activity.     

Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus

Transglutaminase (TGase, EC 2.3.2.13) from a Streptomyces hygroscopicus strain isolated from soil was purified from culture broth by ethanol precipitation, followed by successive chromatographies on CM-cellulose and Sephadex G-75 columns with a yield and purification-fold of 21.1% and 30%, respectively. The enzyme’s molecular weight was estimated as 38,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified microbial transglutaminase (MTG) exhibited optimum activity at 37–45 °C and in a range of pH 6.0–7.0 for hydroxamate formation from N-carboxybenzoyl-l-glutaminyl-glycine and hydroxylamine. The enzyme was not stable above 50 °C and was stable within a pH range of 5.0–8.0 at lower temperature. The MTG was not inhibited by Ca²⁺ and ethylenediaminetetraacetic acid, suggesting it was calcium-independent. Purified MTG was strongly inactivated by 5,5′-dithiobis (2-nitrobenzoic acid), Cu²⁺, Zn²⁺, Pb²⁺, and Hg²⁺, suggesting that this enzyme could possess a thiol group at the active site. The MTG stability was strongly affected by ethanol concentration. The enzyme activity was slightly elevated at a lower concentration of ethanol at 25 °C.     

Characterization of Ca-activated cell-bound proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation

Cell-bound proteinase from Virgibacillus sp. SK37 isolated from the first month of fish sauce fermentation was characterized. The enzyme showed the maximum activity at 65 °C, pH 7.0 and 9.5, using azocasein as a substrate. The enzyme required at least 10 mmol/l Ca²⁺ to effectively hydrolyze casein and the extent of casein degradation increased with Ca²⁺ concentration. Ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF) largely inhibited the activity, indicating a characteristic of Ca²⁺-activated serine proteinase. Among six synthetic substrates tested, the enzyme preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, indicating a subtilisin-like proteinase. Although activity towards actomyosin at 20 g/100 ml NaCl decreased to 63% compared to at 5 g/100 ml, the enzyme showed high stability at 25 g/100 ml NaCl, 30 °C. This was the first study to report biochemical characteristics of cell-bound proteinase from a moderately halophilic bacterium isolated from fish sauce.     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Antifungal and antibacterial activities of lectin from the seeds of Archidendron jiringa Nielsen

A Archidendron jiringa Nielsen lectin was purified by aqueous extraction, 90% ammonium sulphate precipitation and concanavalinA-Sepharose 4B affinity chromatography. Its specific activity was of 88.3 × 10² hemagglutination unit/mg protein for a yield of 51.6% total protein. The molecular weight is of 35.7 kDa. It has hemagglutinating activity against human blood group, rabbit, mouse, rat, guinea pig, geese and sheep erythrocytes. The hemagglutination activity of lectin was relatively insensitive to acidic pH above 2, had an optimal activity at pH 8, and stable below 45 °C for 30 min. The activity was stimulated by Ca²⁺, Mg²⁺ and Mn²⁺. The internal sequence indicated similarity with legume lectin family. Moreover, even at low concentrations antifungal activity was observed against Exserohilum turcicum, Fusarium oxysporum and Colletotrichum cassiicola. The minimal inhibitory concentrations were 0.227, 0.0567 and 0.0567 mg/ml for Bacillus subtilis, Staphylococcus aureus, and Candida albicans, respectively.     

Purification and some properties of α-amylase from post-harvest Pachyrhizus erosus L. tuber

α-Amylase, a starch splitting enzyme, was purified to homogeneity from post-harvest Pachyrhizus erosus L. tuber by successive chromatography on DEAE- and CM-cellulose columns. Purification achieved was 110 fold from the crude extract with a yield of 22.8%. SDS-PAGE showed a molecular weight of 40 kDa for the enzyme. The enzyme is of α-type as it lost total activity in the presence of EDTA, a chelating agent. It is a glycoprotein that contains 2.6% sugar as estimated by the phenol-sulfuric acid method. The enzyme displayed optimum activity at pH 7.3 and 37 °C with an apparent K_m value of 0.29% for starch as substrate. The enzyme was strongly inhibited by Cu²⁺, Fe²⁺ and Zn²⁺, moderately by Li²⁺, Hg⁺ and Cd²⁺ and slightly by Ag⁺, Mg²⁺ and K⁺. Calcium ion almost doubled the activity whereas Fe³⁺, Mn²⁺ and Na⁺ enhanced it appreciably.     

Helvellisin,a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa

A 33.5-kDa serine protease designated as helvellisin was isolated from dried fruiting bodies of the wild ascomycete mushroom Helvella lacunosa. It was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellulose, CM-Sepharose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was characterized by unique N-terminal amino acid sequence, thermostability and pH stability. The protease exhibited a pH optimum of 11.0 and a temperature optimum of 65 °C, with about 40% activity remaining at 87 °C and pH 5 and 13. Helvellisin demonstrated a protease activity of 14 600 U/mg toward casein. The K_m of the purified protease for casein was 3.81 mg/ml at pH 11.0 and 37 °C. The V_max was 5.35 × 10^− 2 mg ml^− 1 min^− 1. It was adversely affected by phenylmethylsulfonyl fluoride, suggesting that it is serine protease. The activity of the protease was enhanced by Mg²⁺, Fe²⁺ and Mn²⁺, but was curtailed by Cu²⁺, Hg²⁺ and Fe³⁺. It was devoid of antifungal and ribonuclease activities.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Purification and characterisation of polyphenol oxidase from red Swiss chard (Beta vulgaris subspecies cicla) leaves

We report purification and characterisation of a polyphenol oxidase from red Swiss chard (rcPPO). Our purification procedure resulted in a 39-fold enrichment in specific activity and 17% recovery of total enzyme activity. The purified rcPPO appeared as a monomeric protein of 41 kDa, with a specific conformation conserved in the Cu²⁺ combining region. It was optimally active at pH 7.5 and 45 °C. It had a diphenolase substrate preference towards l-DOPA, catechol and chlorogenic acid, but also exhibited weak monophenolase one toward 4-methoxyphenol and l-tyrosine. We also found that the enzyme was activated by K⁺, Na⁺, SDS and laurouyl sarcosine, but inhibited by divalent cations including Ca²⁺, Cu²⁺. Its activity was completely inhibited by ascorbic acid, cysteine, 1,4-dithiothreitol, β-mercaptoethanol, sodium diethyldithiocarbamate, sodium metabisulphite, sodium sulphite and thiourea. This first report on the purification and characterisation of red Swiss chard PPO provides a basis for understanding and use of this enzyme.     

Identification of a cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid (Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 °C and 40 °C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45 °C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca²⁺ and Mg²⁺ and inactivated by Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Al³⁺, Ba²⁺ and Co²⁺ to different degrees. Apparent K_m values of trypsin A and B were 2.18 μM and 1.88 μM, and K_cat values were 81.6 S⁻¹ and 111.3 S⁻¹ for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.     

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.