Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus 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.     

Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) Postlarvae

Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL₁₅ giant freshwater prawn ( Macrobrachium rosenbergii ). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0–73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.     

Myofibrillar proteolysis by myofibril-bound serine protease from white croaker <Emphasis Type="Italic">Argyrosomus argentatus</Emphasis>

ABSTRACT:   The modori phenomenon is defined as heat-induced myofibrillar degradation caused by endogenous serine protease(s) of fish muscle during Kamaboko fish meat gel production. This study was undertaken to analyze myofibrillar proteolysis of white croaker Argyrosomus argentatus muscle, which is an ingredient of high quality Kamaboko, by myofibril-bound serine protease (MBSP) under conditions corresponding to the modori phenomenon. White croaker MBSP was stable between pH 2–11 and below 65°C, and about 60% of its initial activity remained after incubation for 2 h under the conditions at 65°C and pH 7.5. About 60% of the enzyme activity was suppressed by 0.5 M NaCl. White croaker MBSP degraded various myofibrillar proteins between 40 and 70°C and pH 6.0–9.0, and preferentially degraded myosin heavy chain rather than other myofibrillar proteins. The enzyme degraded the myosin heavy chain most strongly at 55°C and pH 7.0, and a major part of the bands of myosin heavy chain and its degradation products disappeared for a period of 2 h. These degradation characteristics are very similar to those observed during the modori phenomenon, indicating that MBSP could be a modori-inducing protease involved in the modori phenomenon of white croaker Kamaboko production.     

Conditions for the induction of some selective enzymes from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and their hydrolysis ability against mackerel and gracilar (asparagus, <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>)

ABSTRACT:   This study was conducted to optimize the cultivation conditions for Bacillus subtilis to produce proteases, amylases and cellulase, and to further investigate the hydrolysis ability against mackerel and asparagus. The extracellular enzymes from B. subtilis after 2 and 4 days incubation in a modified medium, containing 1% skim milk, 1% soya meal, 0.25% starch, 0.25% K₂HPO₄, 0.5% NaCl and 0.05% MgCl₂ were collected for the hydrolysis of asparagus and minced mackerel, respectively. Except for the α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging ability of the hydrolyzed asparagus, the trolox equivalent antioxidation capacity and DPPH scavenging ability of both samples increased significantly ( P  < 0.05) after 1 h hydrolysis and further increased during elongated hydrolysis at 50°C. Sodium dodecylsulfate–polyacrylamide gel electrophoresis indicated severe degradation of muscle proteins during hydrolysis. Changes in reducing sugar, soluble proteins and peptides before/after hydrolysis suggested the extracellular enzymes from B. subtilis could effectively hydrolyze the mackerel or asparagus, and subsequently improve their antioxidation ability.     

Characterization of a Heat Stable Protease from Arrowtooth Flounder;

Sodium dodecyl sulfate gel electrophoresis revealed rapid proteolytic degradation of myosin heavy chain in heated arrowtooth flounder muscle. A proteolytic enzyme of approximately 32,000 molecular weight was extracted from the muscle and purified 125 fold. Activity of the semi-purified enzyme at 55°C was optimal against casein at pH 6.0-7.0. Incubation with chemical reagents indicated the involvement of sulfhydryl groups in enzyme activity.     

Partial characterization of the digestive enzymes of Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> under culture conditions

The digestive enzyme activities of Pacific bluefin tuna Thunnus orientalis were evaluated for specific activity and characterized for pH and temperature optima in crude extracts of stomach, caecal mass, and proximal, middle and distal intestine. A higher level of alkaline proteolytic activity was detected in the caecal mass than in the proximal intestine. Total alkaline proteases, trypsin, chymotrypsin and leucine aminopeptidase (LAP) were tested. The temperature and pH analyses showed that proteolytic activity as well as lipase were maximal in the alkaline range, with a maximum at pH 9.0 and at temperatures between 35 and 60°C, except for the pepsin, which showed maximum activity at the same temperatures but in the acid range (pH 3.0). The α-amylase activity showed a broader range in activity, both for pH and temperature, with higher activity over the alkaline pH values and higher temperature. The lipase activity seems to be nondependent on bile salts under our assay conditions, resulting in a significant activity reduction in the presence of bile salts. This knowledge will allow the development of a gastrointestinal model (everted intestine) where food or feed will be hydrolysed with the fish’s own enzymes, a project that is being undertaken in our laboratory as a contribution to the development of novel diets for tuna fish.     

TMAOase, trimethylamine-N-oxide demethylase, isa thermostable and active enzyme at 80°C

ABSTRACT:   Purified trimethylamine- N -oxide demethylase (TMAOase)from walleye pollack muscle is a thermostable protein that was notinactivated after heating at 80°C for 30 min.The heated enzyme was electrophoresed in the same manner as fornative enzyme. Circular dichroism (CD) spectra for purified enzymechanged reversibly in the temperature range of 10–80°C.As the enzyme was still active at 80°C, the CD spectralchange did not directly relate to enzyme activity. TMAOase activity inthe myofibrillar fraction decreased sharply above 30°C,but was extracted and recovered from the heated myofibrillar fraction,suggesting that the activity seemed to be interrupted and apparently inactivateddue to the thermal alteration of myofibrillar proteins or some unknownfactors. The complicated profile found in dimethylamine (DMA) formationfrom trimethylamine- N -oxide (TMAO) in walleye pollack muscleduring heating consisted of both enzymic and non-enzymic processes.Most DMA was produced enzymatically below 40°C and interruptedabove 40°C. Therefore, DMA and trimethylamine was formednon-enzymatically at high temperatures regardless of the presenceof native enzyme. A new, simple and easy purification method wasproposed based on the thermostable nature of the enzyme.     

Effect of plant protease inhibitors on digestive proteases in two fish species, Lutjanus argentiventris and L. novemfasciatus

This work provides a comparative study of the inhibitory effect of several plant protein sources on digestive proteases of two snappers: yellow snapper (Lutjanus argentiventris) and dog snapper (Lutjanus novemfasciatus). Seed extracts did not affect gastric proteases whereas they significantly inhibit intestinal proteases. Inhibition of alkaline proteases showed that pancreatic proteases of L. argentiventris were more sensitive to seed protease inhibitors than those of L. novemfasciatus. Legume seeds showed the highest inhibitory capacity on alkaline proteases causing inhibition higher than 50% in total proteolytic activity. Protease inhibition on digestive extracts was assessed using different relative concentration of seed extracts and represented by constructing dose response curves. In order to reduce the inhibitory effect, seed extracts were acid-treated before the inhibition assay. Results showed that acid treatment did not affect the inhibitory capacity of seeds on alkaline proteases in both species. However, when the action of gastric enzymes was simulated on seed extracts, the inhibitory capacity was reduced significantly, mainly in the case of L. novemfasciatus. The responses of fish enzymes to heat-treated seed extracts were also tested. Only higher temperatures were capable of reducing the inhibitory capacity of seed, with the specific response to the snapper species. The use of biochemical assays allows us to quantify the action of inhibitors on total proteolytic activity. In addition, zymograms obtained by substrate-SDS-PAGE provided qualitative information about the number and type of proteases affected by each inhibitor. Each seed extract produces a characteristic profile of inhibition on alkaline protease. The results obtained are important for future formulation of feeds for these snapper species.     

Functional and Biochemical Characterization of Proteins Remaining in Solution After Isoelectric Precipitation

ABSTRACT

The resulting supernatant from the isoelectric precipitation of “sawdust” from Cape hake portioning and blue whiting muscle was concentrated by ultrafiltration and the retentate spray-dried. Two main peaks corresponding to molecular weights of 15 kDa and 65 kDa were detected in the supernatant from both raw materials; in the retentate the main proteins had approximately 18 kDa and 73 kDa, and some proteins with 59 kDa were detected in the permeate. The protein content was 74.0% and 70.0% for hake by-products and blue whiting, respectively. Dried proteins had relatively low emulsifying and foaming capacity but high foaming stability and were whitish in color. Hydrophobicity and sulphydryl content were similar to those from other protein sources.     

Effect of water temperature and diet on growth and mortality of Neptunea arthritica juveniles

The growth and mortality of Neptunea arthritica juveniles hatched from different egg masses (controlled conditions and field) reared at 10 and 15 °C, and provided with sardine (D1) and polychaete (D2) as foods were evaluated. Results showed that diet type and water temperature significantly affect the growth and mortality. Interaction between these factors reflected robust relationship among them, but restricted exclusively to growth. Through the study period, juveniles treated at 10 °C showed maximum increments of 2.5 mm (shell length), 2.3 mm (shell width) and 0.25 g (body weight) from their initial size. While maximum values of those treated at 15 °C for the above-mentioned parameters were 7.6 mm, 5.5 mm and 1 g respectively. In both cases, these increments were recorded for individuals fed with sardine. Mortality was significantly affected by diets and water temperature, which significantly increased under D2 [10 °C (20–67%), 15 °C (70–87%)] or 15 °C [D1 (30–83%), D2 (70–87%)]. According to the results, juveniles of N. arthritica could be maintained under controlled conditions, which allows growth improvement. Mortality appears to be the limiting factor, but this could be improved by the implementation of efficient culture method, basically oriented to increase the water quality.     

Identification and characterization of proteases from skin mucus of tambacu,a Neotropical hybrid fish

Skin secretions of fishes constitute a rich source of proteins with a broad spectrum of antimicrobial properties. We report here the characterization of proteases from skin mucus of tambacu, an economically important Neotropical hybrid fish. The effects of pH on the proteolytic activities of the mucus acting on various substracts – hemoglobin, casein, bovine serum albumin (BSA) and ovalbumin (OVA) – were tested. Optimal pH values for protease activity on hemoglobin were 4.5 and 8.5, on casein, 8.5, on BSA, 5.0 and 7.5, and on ovalbumin, 4.5 and 6.5. The proteolytic activity was inhibited on all of these substrates in the presence of specific inhibitors: caseinolytic activity was inhibited by inhibitors of serine and metalloproteases; hemoglobinolytic activity was inhibited by serine, aspartic and metalloproteases inhibitors; albuminolytic activity on BSA was inhibited by serine and aspartic proteases inhibitors, and on ovalbumin, by cysteine and aspartic proteases inhibitors. Gelatin zymography revealed that the skin mucus of tambacu consisted primarily of serine and metalloproteases. Hemoglobin zymography showed one proteolytic band inhibited by EDTA, whereas casein zymography showed two proteases inhibited by serine proteases inhibitors. We were able to identify all classes of proteases in the mucus from the skin of tambacu. These, and these results suggest that the proteolytic activities of the skin mucus of fish may play an important role in the defense against microorganisms and ectoparasites.     

Egg and larval distributions of seven fish species in north-east Atlantic waters

The distribution of egg and larvae of mackerel, horse mackerel, sardine, hake, megrim, blue whiting and anchovy along the European Atlantic waters (south Portugal to Scotland) during 1998 is described. Time of the year, sea surface temperature and bottom depth are used to define the spawning habitat of the different species. Mackerel, horse mackerel, and sardine eggs and larvae presented the widest distribution, whereas megrim and anchovy showed a limited distribution, restricted to the Celtic Sea and the Bay of Biscay respectively. Correspondingly mackerel, horse mackerel and sardine showed the highest aggregation indices. Blue whiting larvae were found at the lowest temperatures, whereas anchovy eggs and larvae were found in the warmest waters. The analysis is a basis for evaluation of ongoing changes in the pelagic ecosystem of the north‐east Atlantic.     

Preliminary Characterization of European Sardine Transglutaminase

Abstract

The significant role of transglutaminase (TGase) in the setting of surimi led us to perform its preliminary characterization in sardine muscle. The time course of the reaction and the effect of enzyme level were studied. The temperature optimum was around 35°C and the thermal stability was studied in the 15-55°C range. Higher TGase activity was measured in sardine dark muscle than in the white muscle. A slow decrease of TGase activity during ice storage of sardine was recorded, but after eight days in ice it was about 50% of its initial activity. Cold storage of sardine for a short period did not affect TGase activity.     

In vitro assay of plant protease inhibitors from four different sources on digestive proteases of rohu, Labeo rohita (Hamilton), fingerlings

The in vitro inhibitory effect of protease inhibitors from four seed extracts (soybean, grasspea, black gram and horse gram) on digestive proteases of rohu was assessed by enzyme inhibition assay and substrate sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. High proteolytic activity was detected in the intestinal extract of rohu (Labeo rohita) fingerlings at two different pH ranges (8–8.5 and 10–11). That protein digestion occurs mainly in the alkaline condition in this fish without a stomach is evident from very high trypsin activity (0.95±0.04 benzoyl‐dl ‐arginine‐p‐nitroanilide U mg protein⁻¹) in the intestine. In case of grass pea seed, more than 50% inhibition of alkaline protease activity was recorded when the ratio of inhibitor to enzyme was 9.41 μg U⁻¹. More than 40% inhibition of protease activity was recorded in case of horse gram seed when the ratio of inhibitor to enzyme was 5.51 μg U⁻¹. Black gram at 11.0 μg U⁻¹ and soybean seed proteins at 62.75 μg U⁻¹ resulted in 50% and more than 30% inhibition of digestive protease activity in rohu fingerlings respectively. A plot of the inhibition values obtained by changing the relative concentrations of enzyme/inhibitor resulted in different dose–response curves for different protein sources. The use of substrate gel electrophoresis allowed the visualization of the aforementioned differences in inhibition. Each seed extract produced a characteristic profile of protease inhibition. It is concluded that protease inhibitors present in plant protein sources adversely affect the digestive proteases in fish and hence there is a need to eliminate/reduce the amount of such inhibitors through proper processing before incorporation into aquafeeds.     

Assessing the end-point temperature of heated fish and shellfish meats

ABSTRACT: Attempts have been made to assess the end-point temperature (EPT) of heated fish and shellfish meats by using the coagulation method together with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and enzyme activity determination. Unfrozen and frozen fish and shellfish meats were heat-treated at different selected temperatures with 0, 15 or 30 min holding times. Proteins were extracted with NaCl solution. The coagulation method was able to determine EPT of heated fish and shellfish meats between 60 and 67°C. SDS-PAGE patterns of the filtrates from heated meats were closely related to the results of the coagulation method and enzyme activity determination. Two proteins responsible for producing coagulum of fish meat extracts seem to be lactate dehydrogenase (LDH) and glyceraldehyde phosphate dehydrogenase (GAPDH). End-point temperatures determined by these methods were not significantly different between unfrozen and frozen samples. On the contrary, a highly thermostable protein with a molecular mass of 35 kDa was detected in heated shellfish meats up to 108°C. In scallop adductor muscle, this highly thermostable protein was found to be the tropomyosin subunit from its amino acid composition and their partial sequences. Tropomyosin could be used as an EPT indicator up to 108°C for heated shellfish meats.     

Digestive dynamics during chyme formation of Octopus maya (Mollusca,Cephalopoda)

In the present study, digestive dynamics, measured through the changes in the in situ pH along the digestive tract, was evaluated along with the glycogen concentration in the digestive gland (DG) and the proteolytic enzyme activity of the gastric juice (GJ) (both acid and alkaline proteases) to obtain useful information that will allow for the understanding of the DG's function and the role that it plays as an extracellular digestive enzyme source and for energy storage, providing new information on the digestive physiology of Octopus maya. The results showed that pH along the digestive tract changed according to the postprandial time following the food transit. At the beginning, the pH on crop (St) was 5, changing to 6 when the food arrived. Similar changes were observed on the caecum (Ce) and DG. Glycogen from DG is used as a source of energy during digestion and recovered 8 h after feeding. Maximum activities of digestive gland GJ enzymes were observed 6 h after feeding, indicating that chyme enzymes are still active when they arrive at the DG's lumen. The presence of food in the digestive tract modifies the pH, which, in combination with GJ, favours the activity of the released enzymes of O. maya.     

Qualitative and quantitative changes of F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATPase in Japanese flounder and red sea bream associated with rearing temperatures

ABSTRACT:   Fast skeletal muscles of Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major were examined for quantitative and qualitative changes of mitochondrial ATP synthase (F_oF₁-ATPase) in association with rearing temperatures. The specific activities of F_oF₁-ATPase from Japanese flounder reared at 10°C, 15°C and 25°C for 4 weeks were determined to be 81 ± 11, 74 ± 13 and 83 ± 11 nmol/min·mg mitochondrial protein, respectively. The corresponding activity from red sea bream reared at 8°C for 5 weeks was determined to be 65 ± 9 nmol/min·mg mitochondrial protein, which was higher than 33 ± 9 nmol/min·mg mitochondrial protein in fish reared at 23°C. The contents of α- and β-F₁-ATPase in total mitochondrial proteins were not significantly different between fish reared at different temperatures for the two fish species. However, the contents of β-F₁-ATPase in the total fast skeletal muscle extracts, prepared from Japanese flounder reared at 10°C, were 2.1- and 2.9-fold higher than those for fish reared at 15°C and 25°C, respectively. The corresponding content from red seabream reared at 8°C was 2.2-fold higher than that for fish reared at 23°C. Therefore, the changes in F_oF₁-ATPase depending on rearing temperatures were species-specific.     

Isolation and characterisation of two extracellular metalloproteases from Aeromonas salmonicida ssp. salmonicida

Two extracellular metalloproteases were purified from a culture filtrate derived from Aeromonas salmonicida ssp. salmonicida . One enzyme, leucine aminopeptidase (LAP), which had a molecular mass 37 kDa, hydrolysed aminoterminal l -leucine and l -phenylalanine. The activity was inhibited by 1,10-o-phenanthroline, but not by EDTA. The addition of excess Zn²⁺ to an o-phenanthroline-inhibited enzyme restored most of its activity. The peptidase was temperature stable, and had an optimum temperature and pH of 60 °C and 8, respectively. The other enzyme, metalloprotease 3 (MP3), which had a molecular mass 20 kDa, was an endoprotease, and hydrolysed azocoll and hide powder-azure, but not gelatine. The MP3 enzyme had an optimum temperature and pH of ≈40 °C and 7.5, respectively, and a cationic isoelectrical point.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

Comparative Study on Precipitation Techniques for Protease Isolation and Purification from Labeo rohita Viscera

Precipitation techniques play a vital role in the industrial extraction of enzymes. The present study aimed to extract the proteases from the viscera of Labeo rohita (commonly called Rohu) and to compare the precipitation techniques for the isolation and purification of the enzyme. The enzyme is usually discarded as tons of waste during processing. Hence, a trial has been carried out to isolate the protease enzymes from viscera of the freshwater fish Labeo rohita. The proteases were precipitated with ammonium sulfate, ethanol, and acetone. Acetone precipitation was found to be the best option for the recovery of enzymes (54%) from the viscera of Rohu, and two caseinolytic protease bands were shown in the zymogram. The precipitates with highest proteolytic activity were further subjected to dialysis, and their molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).