Specific Trypsin Inhibitor Research Articles

Proteases from the Harpacticoid Copepod Tisbe biminiensis: Comparative Study with Enzymes from Farmed Aquatic Animals

The harpacticoid copepod Tisbe biminiensis has been tested as live prey in marine crustacean and fish larviculture. The aim of the present study was to characterize the proteinases in the crude extract of T. biminiensis. Trypsin activity was assayed in the crude extract prepared by the homogenization of specimens reared under controlled laboratory conditions and fed on diatoms and commercial fish food. The physical-chemical and kinectics parameters were determined using BApNA as substrate. Optimal pH and temperature were 9.0 and 55°C, respectively. This enzyme was thermostable until 50°C. Using BApNA as substrate, the Km was 0.59 mM and the proteolytic activity was strongly inhibited by specific trypsin inhibitors. However, the presence of other proteases was observed using substrate-SDS-PAGE. Eight caseinolytic bands were observed in the zymogram, four of them were not inhibited by classical mammalian trypsin inhibitors. Trypsin from T. biminiensis showed similar properties to those described for species used in commercial aquaculture. These results demonstrate that T. biminiensis may be a source of proteases, including trypsin-like enzymes.

Digestive Proteolytic Activity in the Sunn Pest,Eurygaster integriceps

The Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae), is one of the most important pests of wheat and causes considerable damage to this valuable crop annually. Digestive proteinase activity of adult insects was investigated using general and specific substrates and inhibitors. Proteolytic activity was low when the common conventional substrates, azoalbumin, azocasein and hemoglobin were used to assay salivary glands and midguts. Using the fluorescent casein substrate (BODIPY FL casein), total proteolytic activity was measured at different pH. Maximum proteolytic activity was detected at pH 7 (100%) and 8(65%) which suggested the presence of serine proteinases in the salivary glands. There was no detectable proteolytic activity in midgut extracts. The inhibitors; PMSF (inhibitor of serine proteinases) and TPCK (a specific chymotrypsin inhibitor) showed greater than 50% inhibitory effect on total proteolytic activity, however, TLCK (specific trypsin inhibitor) and E-64(specific cysteine proteinase inhibitor) did not inhibit total proteolytic activity. Using fluorescent specific substrates for serine and cysteine proteinases (Z-Arg-AMC, Z-Arg-Arg-AMC, Z-Arg-Phe-AMC and Suc-Ala-Ala-Pro-Phe-AMZ) revealed the presence of tryptic and chymotryptic activity in the salivary gland extract. Zymogram analysis under non-reducing SDS-PAGE conditions and using the substrate APNE showed at least 8 tryptic and chymotryptic activity bands in salivary gland extracts. A single high molecular weight band with tryptic activity (165 kDa) was detected using the substrate BApNA in a zymogram analysis uisng native-PAGE. Kinetic studies showed a km value of 0.6 mM for this enzyme against the substrate BApNA .The inhibitor TLCK decreased activity of the trypsin-like enzyme up to 73% and almost completely eliminated the only band related to this proteinase in the zymogram. Soybean Kunitz type trypsin inhibitor showed no effect on proteolytic activity of the trypsin-like serine proteinase. In general, the results revealed the presence of chymotrypsin- and trypsin-like serine proteinases in the salivary gland of E. integriceps, and it seems that the major total proteolytic activity is due to chymotrypsin proteinases.

Purification and Characterization of Trypsin from the Intestine of Hybrid Tilapia (Oreochromis niloticus × O.aureus)

Trypsin from the intestine of hybrid tilapia (Oreochromis niloticus x O.aureus) was purified by the following techniques: acetone precipitation, ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and DEAE-sephacel ion exchange chromatography. The purified enzyme was determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight was estimated as 22,000 Da. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 9.0 and 60 degrees C, respectively. The enzyme was stable over a broad pH range from 7.0 to 12.0 at 30 degrees C, and the enzyme was inactive at temperatures above 50 degrees C. The behavior of the enzyme for the hydrolysis of casein followed Michaelis-Menten kinetics with Km of 0.46 mg/mL. The purified enzyme was inhibited by the general serine protease inhibitor phenyl methyl sulphonyl fluoride (PMSF) and also by the specific trypsin inhibitor N-p-tosyl-L-lysine chloromethyl ketone (TLCK) using Nalpha-CBZ-L-lysine p-nitrophenyl ester hydrochloride (CBZ-Lys.pNP) as a substrate. The protease was inhibited by the following ions in decreasing order: Zn2+>Fe3+>Cu2+>Al3+>Co2+=Pb2+>Cd2+>Mn2+. The ions Li+, Na+, K+, Mg2+, and Ba2+ had little effect on the enzyme, and Ca2+ can partially promote its activity at low concentration.

Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions

We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K(i) in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family.

Ovochymase in amphioxus Branchiostoma belcheri is an ovary-specific trypsin-like serine protease with an antibacterial activity

Ovochymases have been shown to be present in vertebrates; little information is available at present regarding ovochymase in invertebrates. Here we isolated a cDNA encoding an ovochymase homolog from amphioxus Branchiostoma belcheri, named BbOvc. The cDNA contained a 1248 bp open reading frame corresponding to a deduced protein of 415 amino acids with a predicted molecular mass of approximately 44.4 kDa. Phylogenetic analysis showed that BbOvc was located at the base of its vertebrate counterparts, suggesting that it represents the archetype of vertebrate ovochymases. BbOvc is found to display a tissue- and stage-specific expression pattern, with a predominant expression in the ovary of sexually matured females and in the early stage embryos (1–16-cell embryos). The recombinant ovochymase expressed in vitro shows a trypsin-like activity capable of hydrolysing the trypsin prototypic substrate N a -benzoyl- l-arginine ethyl ester (60 U BAEE/mg), which can be inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. It also exhibits an antibacterial activity capable of inhibiting the growth of bacteria like E. coli and V. parahaemolyticus. Taken together, these data indicate that BbOvc is a novel ovochymase with an antibacterial activity and offer first clues to its role as an immune-relevant molecule which may protect the early embryos from pathogenic attacks.

Digestive proteinases and peptidases in the hepatopancreas of the southern brown shrimp (Farfantepenaeus subtilis) in two sub-adult stages

The aim of this study was to examine proteinases and peptidases from the hepatopancreas of two sub-adult stages of Farfantepenaeus subtilis: SAS6 (5.93 ± 0.69 g wet weight) and SAS13 (13.26 ± 0.60 g wet weight). Trypsin and chymotrypsin activity was higher in the extract from the SAS6 individuals (P < 0.05). The highest activity among aminoacyl-β-naphthylamide substrates was found using alanine-, arginine-, leucine- and lysine-β-naphthylamide. There was a positive correlation between the recommended concentration of essential amino acids in penaeid shrimp feed and aminopeptidase activity in both sub-adult stages. Proteolytic activity of F. subtilis was strongly inhibited by specific trypsin inhibitors. The optimal temperature for trypsin, chymotrypsin and leucine aminopeptidase activity was between 45 and 55 °C. Six and seven bands were found in caseinolytic zymograms for SAS6 and SAS13 respectively. All bands were inhibited by phenylmethylsulfonyl fluoride in both sub-adult stages. The use of tosyl-lysine-chloromethyl-ketone and benzamidine caused strong inhibition of the proteolytic bands. Trypsin and chymotrypsin activity was the main difference observed between the protease pattern of SAS6 and SAS13F. subtilis.

Purification and characteristics of trypsins from cold-zone fish, Pacific cod (Gadus macrocephalus) and saffron cod (Eleginus gracilis)

Trypsins from the pyloric ceca of Pacific cod (Gadus macrocephalus) (GM-T) and saffron cod (Eleginus gracilis) (EG-T) were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparations were nearly homogeneous on SDS–PAGE and the molecular weights of both enzymes were estimated to be approximately 24 kDa by SDS–PAGE. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of GM-T and EG-T. The optimum pH and optimum temperature of both trypsins were around pH 8.0 and 50 °C, respectively, using Nα-p-tosyl-l-arginine methyl ester as substrate. The GM-T and EG-T were unstable above 30 °C and below pH 5.0, and they were stabilised by calcium ion. The N-terminal amino acid sequences of GM-T (IVGGYECTRHSQAHQVSLNS) and EG-T (IVGGYECPRHSQAHQVSLNS) were found. The percentage of hydrophobic amino acid in the N-terminal 20 amino acids sequences of these cold-zone fish trypsins was lower (28%) than those of temperate-zone fish trypsins (34%), tropical-zone fish trypsins (37%) and mammalian trypsins (34%). Whereas the content of charged amino acids in the GM-T and EG-T was relatively higher than those of trypsins from temperate-zone fish, tropical-zone fish and mammals. Moreover, the GM-T catalyzed synthesis of Nα-(tert-butoxycarbonyl)-l-alanyl-l-alanine-p-nitroanilide (Nα-Boc-l-Ala-l-Ala-pNA) has been studied by using Nα-(tert-butoxycarbonyl)-l-alanine-p-guanidinophenyl ester [Nα-Boc-l-Ala-OpGu (inverse substrate)] as acyl donor and l-alanine-p-nitroanilide (l-Ala-pNA) as acyl acceptor, respectively.

Digestive peptidases and proteinases in the midgut gland of the pink shrimpFarfantepenaeus paulensis(Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2–9.0) and 55 °C. CaCl2 did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P<0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.

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.

Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana

Cockroach allergens are associated with the development of asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a protease from Periplaneta americana and determine its allergenicity. A serine protease was isolated from P. americana extract using benzamidine sepharose column and characterized by immunobiochemical methods. Allergenicity of the protease was assessed by enzyme-linked immunosorbent assay, immunoblot, intradermal testing, histamine release and peripheral blood mononuclear cells (PBMCs) proliferation. Affinity purified protein of approximately 28 kDa (Per a 10) showed a single band of activity in gelatin zymogram and agarose plate assay. N-terminal sequence (IVGGRPAQI) revealed similarity with mite serine protease allergens and insect trypsins. It demonstrated proteolytic activity with azocollagen > gelatin > defatted-milk > casein including serine protease specific substrate, N-benzoyl-arginine-ethyl-ester-hydrochloride. It was inhibited by serine protease inhibitors, namely aprotinin > pefabloc > AEBSF > PMSF > benzamidine > antipain > leupeptin and trypsin-specific inhibitor (tosyl-lysyl-chloromethyl-ketone) suggesting it to be a trypsin-like serine protease. Per a 10 was recognized as a major allergen, showing IgE reactivity with >80% of cockroach sensitized patients by skin tests and immunoblot. It could induce significant histamine release (P < 0.05) in blood and secretion of interleukin-4 (IL-4) (P < 0.05) and IL-5 (P < 0.05) in culture supernatant of PBMCs from cockroach hypersensitive patients, suggesting a strong allergenic potency. A serine protease isolated from P. americana was demonstrated to be a major allergen (Per a 10). It has a potential for component-based diagnosis of allergy and will be useful in elucidating the mechanism of allergy.

Anionic Trypsin from North Pacific Krill (Euphausia pacifica): Purification and Characterization

An anionic trypsin (TRY-EP) was purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography. The purified enzyme was identified as a trypsin by LC-ESI-MS/MS analysis. The relative molecular mass of TRY-EP was 33 kDa, with isoelectric point of 4.5. The histidine, tryptophan, arginine, lysine, aspartic acid and glutamic acid residues were functional groups to TRY-EP. TRY-EP was activated by Ca2+ and Mg2+ and inhibited by some heavy metal ions (Zn2+, Cu2+, Pb2+ and Hg2+), organic solvents (ethanol, glycerin, DMSO and acetone) and specific trypsin inhibitors (benzamidine, CEOM, SBTI and TLCK). TRY-EP was active over a wide pH (6.0–11.0) and temperature (10–70°C) range, with optimum of pH 9.0 and 40–50°C. TRY-EP was stable between pH 6.0 and 11.0 and below 30°C. Compared with some trypsins from the Temperate and Tropical Zone organisms, TRY-EP and other trypsins from the Frigid Zone organisms have higher affinity to substrate and 2–42-fold physiological efficiency.

Pancreatic secretory trypsin inhibitor acts as an effective inhibitor of cysteine proteinases gingipains from Porphyromonas gingivalis

Porphyromonas gingivalis has been implicated as the major pathogen of periodontitis in adults. This organism produces an array of virulence factors, of which cysteine proteinases, referred to as gingipains K and R, are believed to play a crucial role in pathogenicity. The aim of this study was to investigate the susceptibility of gingipains K and R to inhibition by a pancreatic secretory trypsin inhibitor. Enzyme activities were measured spectrophotometrically using chromogenic turnover substrates. To estimate the value of the association constant (Ka), constant amounts of enzyme were reacted with increasing amounts of inhibitor to reach equilibrium. The Ka was calculated by fitting the experimental data to the given equation. In this study it was shown that gingipains are susceptible to pancreatic Kazal-type trypsin inhibitors (pancreatic secretory trypsin inhibitors). Bovine pancreatic secretory trypsin inhibitor, having an Arg residue at the P1 position of the reactive site, specifically inhibited the activity of the Arg-specific cysteine proteinase gingipain R, whereas porcine inhibitor, possessing a Lys residue at the P1 position, exhibited activity only against the Lys-specific cysteine proteinase gingipain K. The Ka values for the inhibitor-proteinase interaction were 1.6 x 10(6) m(-1) and 2.0 x 10(4) m(-1) for gingipain R and gingipain K, respectively. This finding is the first demonstration of the inhibitory potency of the Kazal-type specific trypsin inhibitors against cysteine proteinases. These discoveries open new possibilities for the use of naturally occurring inhibitors, displaying activity across enzyme families, as a model in designing new molecules of therapeutic significance.

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). CaCl 2 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.

Induction of IL‐6 release from human T cells by PAR‐1 and PAR‐2 agonists

Proteinase-activated receptors (PAR) have been recognized as playing an important role in inflammation and immune response. However, little is known of the expression and function of PAR on human T cells. In this study, the expression of PAR on highly purified human T cells was determined and the secretion of IL-6 from cultured T cells in response to serine proteinases and agonist peptides of PAR was examined. The results showed that T cells express PAR-1, PAR-2 and PAR-3 proteins and genes. Thrombin, trypsin and tryptase, but not elastase, were able to stimulate concentration-dependent secretion of IL-6 from T cells following a 16 h incubation period. The specific inhibitors of thrombin, trypsin and tryptase inhibited the actions of these proteinases on T cells, indicating that the enzymatic activity is essential for their actions. Agonist peptides of PAR SFLLR-NH2, TFLLRN-NH2 and SLIGKV-NH2, but not TFRGAP-NH2, GYPGQV-NH2 and AYPGKF-NH2, are also capable of inducing IL-6 release from T cells. In conclusion, induction of IL-6 secretion from T cells by thrombin, trypsin and tryptase is probably through the activation of PAR, suggesting that serine proteinases are involved in the regulation of immune response of the body.

Prevalence of the N34S Mutation in Patients with Acute Pancreatitis

Purpose: Serine Protease Inhibitor Kazal type I (SPINK-1) is a specific trypsin inhibitor expressed in the pancreas. It has been hypothesized that SPINK-1 acts as the first-line of defense against premature trypsin activation within the pancreas. Mutations in the SPINK-1 gene are associated with chronic pancreatitis however their role in acute pancreatitis remains unclear. This study aims at studying the prevalence of the N34S mutation in a cohort of patients with acute pancreatitis. Methods: Blood was collected from 102 patients with acute pancreatitis and 239 controls. We tested for the presence of the N34S mutation using validated genotyping methods. Results: The N34S mutation was detected in 5 patients (3 heterozygotes and 2 homozygotes) all of which had recurrent acute pancreatitis. 7 controls (all heterozygotes) tested positive. The calculated Odds Ratio was 2.391 with a 95% confidence interval of 0.828 – 6.906. Conclusions: The SPINK-1 N34S mutation does not seem to play an important role in the pathogenesis of acute pancreatitis. This suggests that the previously proposed mechanism of SPINK-1 associated protection needs to be reconsidered.

Variation in SPINK-1 N34S Effect Size Is Population-Dependant

Purpose: SPINK-1 is a specific trypsin inhibitor expressed in the pancreas and currently thought of as the first-line of defense against premature pancreatic trypsin activation. Mutations in the SPINK-1 gene are associated with pancreatitis but their reported effect varies widely among studies. Our aim is to systematically review the effect size of the SPINK1 N34S high-risk haplotype in patients with acute pancreatitis (AP) and chronic pancreatitis (CP) in different populations using meta-analysis. Methods: Case-control studies published by October 2005 were identified by Medline search. The significance of the association of the N34S mutation with pancreatitis was evaluated for each study separately [OR (95% CI)]. Separate meta-analyses were carried for the different disease subtypes. Results: The N34S mutation was associated with CP in general (OR: 10.50, 95% CI: 6.92–15.92). Patients with ACP had a weaker association (OR: 5.13, 95% CI: 3.26–8.08). The strongest association was with TCP (OR: 31.15, 95% CI 13.21–73.47). The association with AP was weak (OR:2.98, 95% CI: 1.71–5.22). There was no evidence of publication bias. Conclusions: SPINK-1 N34S has only a small effect in AP and ACP, but is strongly associated with young-onset chronic pancreatitis. The marked differences between the effect size in the disease subsets suggest that the previously proposed mechanism of SPINK-1 associated protection must be reconsidered.Table

Purification and characterization of hatching enzyme from flounder Paralichthys olivaceus

Using chorion of Paralichthys as a specific substrate, hatching enzyme (HE) from Paralichthys olivaceus (PHE) was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular size of PHE is about 34.8 kDa in SDS-PAGE. The PHE had obvious choriolytic activity, which was optimal at pH 7.0 and temperature of 35 degrees C, respectively. The Km value of the PHE for casein was 4.28 mg ml(-). The PHE was very sensitive to trypsin-specific inhibitors, especially serine protease-specific inhibitors, such as LBTI, SBTI, bestatin and p-APMSF, leupeptin, ovomucoid, PMSF, pepstatin A and TLCK, indicates that it is a trypsin-type serine protease. The PHE was also extremely sensitive to Cu(2+) and Ca(2+), combined with the results that it was inhibited by EDTA in a dose-dependent manner, indicates this PHE is also a kind of metalloprotease.

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon ( Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon ( Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl- dl-arginine- p-nitroanilide ( dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.

Digestive enzyme patterns and evaluation of protease classes in Catla catla (Family: Cyprinidae) during early developmental stages

Digestive enzymes of Catla catla were studied during ontogenic development. Specific amylase activity was 0.12 ± 0.01 mg maltose mg protein − 1 h − 1 in fish 4 days after hatching (DAH) and reached a maximum on (0.41 ± 0.12 mg maltose mg protein − 1 h − 1 ) 34 DAH. Total protease activity was minimum (123.2 ± 16.5 mU mg protein − 1 min − 1 ) on day-8 and reached its highest level (2713 ± 147.2 mU mg protein − 1 min − 1 ) on day-32. Trypsin activity showed constant increasing trend from day-16 onwards and was maximum on day-34 (118.1 ± 7.09 mU mg protein − 1 min − 1 ). Highest chymotrypsin activity was found on day-32 (1789.0 ± 111.7 mU mg protein − 1 min − 1 ). Lipase activity was detected in 4 DAH catla. Lipase activity increased steadily from day-22 onwards. SDS-PAGE of crude enzyme extracts showed that high molecular mass bands (41.8–127.8 kDa) appeared during the early stages followed by low molecular mass bands (17.8–37.2 kDa). The number of protease activity bands in substrate SDS-PAGE increased with age of fish. During ontogenesis of carp, soybean trypsin inhibitor (SBTI), PMSF and TLCK inhibited 75.5 ± 1.19% to 92.8 ± 0.85%, 53.3 ± 9.47% to 90.5 ± 2.6% and 39.8 ± 3.8% to 84.7 ± 1.54% of total protease activity, respectively. There was only 2.58 ± 0.66% to 10.21 ± 0.09% inhibition of protease activity with EDTA. SBTI and PMSF inhibited 8 and 4 activity bands, respectively. TLCK, a specific trypsin inhibitor, inhibited four trypsin-like enzymes in carp during ontogenesis.

Study of digestive proteinases and proteinase inhibitors of Daphnia carinata

Quantification of proteases activities and their class structure have been studied in a cladoceran, Daphnia carinata. Protease activity ranged from 0.28 to 0.55 Unit mg −1 protein min −1 with an average value of 0.42±0.06 Unit mg −1 protein min −1. Chymotrypsin activity was more than twofold higher (0.49±0.09 Unit mg −1 protein min −1) than the trypsin activity (0.21±0.02 Unit mg −1 protein min −1). Protease activity and reduction of activity in bands of samples treated with specific inhibitors were documented in photometric assay and substrate SDS–PAGE. Proteinase activity against azocasein was inhibited (91.4±1.5%) with SBTI. PMSF reduced the enzyme activity by 53.1±6.5%, and the azocasein hydrolysis was reduced up to 64.6±3.8% by the specific inhibitor of trypsin, TLCK. In the present investigation, the molecular weight of various activity bands ranged from 16.3 to 51.1 kDa. The molecular weights of several protein bands are similar to protease activity zones. The knowledge of digestive enzyme profiles of fish food organisms generated in the present study may assist in the formulation of age-specific feed.