Hepatopancreas Extracts Research Articles

Purification and characterization of leucyl aminopeptidase from the Todarodes pacificus hepatopancreas

Here, leucyl aminopeptidase (LAP) was purified from the hepatopancreas extract (HPE) of Todarodes pacificus, with 63.7-fold purification and a yield of 6.5%. Ammonium sulfate fractionation and sequential chromatographic steps, involving Sephacryl S-300, Toyopearl DEAE-650 M, DEAE-Sepharose CL-6B, and Sephadex G-50, were used for purification. The bitter-taste improvement of trypsin casein hydrolysate (TCH) by the purified LAP was also investigated. The LAP was characterized as a monomer; its molecular mass was 29.0 kDa under denatured conditions and 27.1 kDa under native conditions. With an optimum pH of 7.5 and optimum temperature of 45 °C, it remained stable up to ∼50 °C, within a pH range of 6–8. Its activity was inhibited by Cu2+ and Hg2+, bestatin, and the metal-chelating agents EDTA and 1,10-phenanthroline, and slightly enhanced by Fe2+, Zn2+, Mn2+, and Mg2+. Moreover, the purified enzyme was classified as a leucyl aminopeptidase, as it preferentially hydrolyzed Leu, followed by Met, Phe, Ala, and Lys p-nitroanilide. The apparent Km and Vmax values of the enzyme were 0.385 mM and 796.2 U/mg, respectively, for LeuPNA. Additionally, LAP-treated TCH showed lower bitterness than untreated TCH. The bitterness improvement effect of LAP could be attributed to the hydrolysis of hydrophobic amino acids at the N-terminus of the peptide; this confirms the substrate specificity of LAP. It has been confirmed that enzymes such as LAP that effectively reduce the bitter taste of bitter hydrolysates are distributed in the hepatopancreas of Todarodes pacificus, and this protein hydrolysate will be used as a bitter taste improver and food industry.

Proteases from Penaeus monodon Fabricius, 1798 (Decapoda, Penaeidae): partial characterization and localization

Abstract This study aimed to characterize and determine the distribution of proteases in various anatomical parts of Penaeus monodon using mainly electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels impregnated with casein (casein-SDS-PAGE) and specific protease inhibitors. Twelve caseinolytic bands designated A-L, with estimated molecular masses of 11.4-49.6 kDa were detected by SDS-PAGE, and were most abundant in the hepatopancreas. Among the 12 bands, E, J, K and L were predominant, A, G and H were identified as chymotrypsin-like proteases, D, E, I, J, K and L were identified as trypsin-like proteases, and B, C and F were identified as serine proteases. The proteases were most active at 50°C and pH 7.0. The proteolytic and trypsin-like activities of Penaeus monodon were most abundant in hepatopancreas extracts and least abundant in extracts from the haemolymph. Trypsin-like proteases in the hepatopancreas extract were found to activate prophenoloxidase (proPO) to form phenoloxidase (PO).

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B1 and fumonisin B1 in the hepatopancreas of coastal lagoon wild and farmed shrimp Litopenaeus vannamei.

This study aimed to establish the combined effect of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p < 0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB1 and FB1. The greatest inhibition occurred when AP was incubated with a mixture of AFB1 and FB1. The IC50 for AFB1 on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398μg/mL, respectively. The IC50 of FB1 was 0.87μg/mL for wild shrimp and 0.69μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB1 + FB1 suggesting a possible synergistic or potentiating inhibitory effect.

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family.

Expression of RXR, EcR, E75 and VtG mRNA levels in the hepatopancreas and ovary of the freshwater edible crab, Oziothelphusa senex senex (Fabricius, 1798) during different vitellogenic stages.

The objective of the present study was to investigate the expression profile of retinoid X receptor (RXR), ecdysone receptor (EcR) and ecdysone inducible gene (E75) in the hepatopancreas and ovary of Oziothelphusa senex senex during different vitellogenic stages. RXR, EcR and E75 complementary DNAs (cDNAs) were isolated from the ovaries, while vitellogenin (VtG) cDNA was isolated from the hepatopancreas of vitellogenic female crab. Deduced amino acid sequence of the messenger RNAs (mRNAs) of RXR, EcR and E75 showed more than 80% identity with their respective mRNAs of other brachyurans. VtG mRNA was not detected in the ovary throughout vitellogenic stages. RXR and EcR were significantly increased in the ovaries during vitellogenic stage I. The levels of EcR, E75 and VtG in the hepatopancreas elevated significantly during vitellogenic stages I and II, whereas the levels of RXR elevated only in vitellogenic stage I. During vitellogenic stage III, the levels of RXR, EcR and VtG in the hepatopancreas were significantly decreased. Immunoprecipitation analysis revealed the presence of VtG in the haemolymph, hepatopancreas and ovary extracts from the females but absent in haemolymph and hepatopancreas extract of males. It can be inferred that RXR, EcR and E75 are involved in the regulation of synthesis of VtG in hepatopancreas, whereas in ovary, it is hypothesized that they play an important role in the uptake of VtG from the haemolymph, probably by regulating the levels of vitellogenin receptor. These are the first data showing an association between the expression levels of RXR, EcR and E75 and vitellogenesis and provide an alternative molecular intervention mechanism to the traditional eyestalk ablation to induce vitellogenesis and ovarian maturation in crustaceans.

Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex

The objective of the present study was to explore the site of synthesis of vitellogenin (Vtg) in fresh water edible crab, Oziothelphusa senex senex. Vtg cDNA fragments were isolated from the hepatopancreas of female crabs using RT-PCR method, and the deduced amino acid sequence of O. senex senex showed more than 60% identity with other brachyuran Vtg sequences. RT-PCR analysis showed that Vtg mRNA can be detected only in hepatopancreas of female Oziothelphusa but not in other tissues including eyestalks, Y-organs, mandibular organs, thoracic ganglion, hypodermis and ovary. Antibodies were raised against vitellin purified from the ovary of O. senex senex. Immunoprecipitation analysis revealed the presence of Vtg in the hepatopancreas of vitellogenic stage I females and in the hemolymph, hepatopancreas and ovary extracts from vitellogenic stage II females but absent in hemolymph and hepatopancreas extract of males. These results suggest that Vtg is synthesized only in hepatopancreas but not in the ovaries of O. senex senex. In addition, Vtg synthesized in hepatopancreas is transported to ovary through hemolymph.

Fractionation and Enzymatic Characterization of Endoprotease and Exopeptidase from Crude Extracts of Cuttlefish Sepia officinalis Hepatopancreas

This study examines the optimal fractionation method and conditions for the isolation of endoprotease- and exopeptidase-active fractions from crude extracts of cuttlefish hepatopancreas (HP) using four fractionation methods: ammonium sulfate fractionation (ASF), polyethylene glycol fractionation (PGF), ion exchange chromatography (IEC), and gel filtration chromatography (GFC). Total endoprotease activity highest in the fraction II (concentrate of fractions 34-42; 842.60 U) of GFC, followed by fraction III (40-60% ammonium sulfate fraction; 670.25 U) of ASF, fraction I (concentrate of fractions 8-12; 436.89 U) of IEC, and fraction II (10-20% polyethylene glycol; 307.31 U) of PGF. Total exopeptidase activity of these fractions was highest in fraction II (2,704.70 U) of GFC, fraction III (2,110.50 U) of ASF, fraction III (1,605.60 U) of PGF, and fraction II (concentrate of fractions 38-44; 1,196.22 U) of IEC. These results showed that fraction II of GFC had the highest activity toward both exopeptidase and endoprotease, with exopeptidase activity being 3.21 times higher than of endoprotease. These results suggest cuttlefish HP could be used as a potential source for the extraction of exopeptidase, an enzyme capable of catalyzing the cleavage of N- and C-terminal amino acids in polypeptides, Like endoprotease, the most efficient method for separating exopeptide-active fractions was GFC.

Antioxidant enzyme activities in Pacific white shrimp ( Litopenaeus vannamei) in response to environmental hypoxia and reoxygenation

Hypoxia–reoxygenation represents a relevant physiological challenge for shrimp culture. The sudden input of oxygen (O 2) after environmental hypoxia increases reactive oxygen species (ROS) production in Pacific white shrimp ( Litopenaeus vannamei). It has been postulated that a relatively high activity of the main antioxidant enzymes allows several adapted invertebrate species to survive repetitive cycles of hypoxia–reoxygenation by counteracting ROS production and, thus, preventing oxidative stress. In this study, we evaluated the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in extracts of muscle and hepatopancreas from white shrimp exposed to short-term (24 h) hypoxic conditions (1 mg L − 1 of dissolved O 2), and subsequent reoxygenation cycles (1 to 5 h, in 1 h intervals). Results show differences in antioxidant enzyme activities among tissues, even under control conditions. SOD activity was higher in muscle than in hepatopancreas whereas CAT and GPx activities were higher in hepatopancreas. In muscle, SOD activity was higher during hypoxia and lower after 1 h of reoxygenation. In hepatopancreas, antioxidant enzyme activities were higher during hypoxia and lowest in the first hour of reoxygenation. Significant changes in SOD, CAT and GPx activities were also observed in both tissues after 1 h and 2 h of reoxygenation suggesting that hypoxia–reoxygenation affects the antioxidant enzymes. These changes in antioxidant enzyme activities in response to hypoxia–reoxygenation may be crucial to avoid oxidative damage and to preserve the quality of the aquaculture product.

Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated Octopus maya

Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9–10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction on enzyme activity of 70 and 20% was observed in HP and GJ extracts, respectively when protease inhibitor Pepstatin A was used. That result suggests that the main proteases in the HP were aspartic acid proteinases type (possibly Cathepsin D) and some of them were present in the GJ. Dissociating discontinuous polyacrylamide gel electrophoresis showed activity bands between 20 and 28, 30 and 34, 35 and 45, 60 and 70 kDa, and a last one between 75 and 100 kDa. We concluded that extracellular digestion of O. maya takes place in an acid environment, around pH 6. In contrast, intracellular digestion in the HP is developed at pHs between 3 and 4, where cathepsin D could be the most important enzyme for O. maya.

A proteomics based approach to assessing the toxicity of bisphenol A and diallyl phthalate to the abalone ( Haliotis diversicolor supertexta)

The contamination of marine ecosystems by endocrine disrupting compounds (EDCs) is of great concern. Protein expression profile maybe a good method to help us understand the molecular mechanisms of EDCs-toxicity to aquatic organisms. In this study, the abalone ( Haliotis diversicolor supertexta), was selected as the target organism. Toxicological effects of two reference endocrine disruptors: diallyl phthalate (DAP, 50 μg L −1) and bisphenol-A (BPA, 100 μg L −1) were investigated after a three months static-renewal exposure on abalones using proteomics to analyze their hepatopancreas tissues. Some enzyme activity parameters of hepatopancreas extracts were also performed, including Na +–K +-ATPase, Ca 2+–Mg 2+-ATPase, peroxidase (POD) and malondialdehyde (MDA) production. After analyzing the proteomics profile of hepatopancreas by 2D gel electrophoresis, we found that 24 spots significantly increased or decreased at protein expression level (⩾2-fold difference) in the 2D-maps from the treatment groups. Eighteen out of 24 protein spots were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI–TOF-MS). These proteins can be roughly categorized into diverse functional classes such as detoxification, oxidative stress, hormone regulating, cellular metabolism and innate immunity. In addition, the enzymatic results indicated that DAP/BPA exposure affected the oxidative stress status and the cellular homeostasis, which partly corroborated the proteomics’ results. Taken together, these data demonstrate that proteomics is a powerful tool to provide valuable insights into possible mechanisms of toxicity of EDCs contaminants in aquatic species. Additionally, the results highlight the potential of abalone as a valuable candidate for investigating EDCs impacts on marine ecosystems.

Duplex Real Time PCR for the detection of hepatitis A virus in shellfish using Feline Calicivirus as a process control

The consumption of bivalve shellfish is a common cause of foodborne outbreaks of viral origin. The evaluation of the sanitary quality of these products, however, is still based on bacterial indicators of fecal contamination (Reg. (EC) No. 2073/2005 and No.1441/2007) even if it is known that they are not reliable indicators of viral contamination. In this study a duplex Real Time PCR method for quantitative detection of hepatitis A (HAV) in shellfish was developed. Feline Calicivirus (FCV) was used as a control for assessing the effectiveness of the concentration and extraction process and the ability to eliminate PCR inhibitors present in the food matrix. The specific primers and probes for detection of HAV and FCV, chosen respectively from the 5′-UTR region and in the ORF1 region, were labeled with two different dyes and detected simultaneously. The method was applied on spiked and non-spiked shellfish from a local market. The amplification of HAV in the presence of FCV showed good linearity ( R 2 = 0.994) and the sensitivity limit of the reaction was at least 5 × 10 2 TCID 50 g −1 of an hepatopancreas extract.

Regulation of global protein translation and protein degradation in aerobic dormancy

We hypothesized that protein turnover would be substantially suppressed during estivation in the land snail, Otala lactea, as part of a wholesale move to conserve ATP in the hypometabolic state, and that decreased rates of protein synthesis and degradation would be mediated by altering the phosphorylation state of key proteins. Rates of protein translation, measured in vitro, decreased by approximately 80% in extracts of foot muscle and hepatopancreas after 2 days of estivation, and this reduction was associated with strong increases in the phosphorylation of ribosomal factors, eIF2 alpha and eEF2, as well as decreased phosphorylation of 4E-BP1. Reductions in levels of markers of ribosomal biogenesis and a tissue-specific reduction in the phosphorylation state of eIF4E and eIF4GI were also evident after 14 days of estivation. Activity of the 20S proteasome decreased by 60-80% after 2 days of estivation and this decrease was mediated by protein kinase G in vitro, whereas protein phosphatase 2A activated the proteasome. Levels of protein carbonyls did not change in snail tissues during estivation whereas the expression heat shock proteins increased, suggesting that protein resistance to damage is enhanced in estivation. In conclusion, protein synthesis and degradation rates were coordinately suppressed during estivation in O. lactea and this is associated with the phosphorylation of ribosomal initiation and elongation factors and the 20S proteasome.

The effect of proteinase inhibitors in food protein hydrolysis by digestive proteinases of white shrimp (Penaeus vannamei) larvae

AbstractThis study describes the digestive capacities of 10‐day‐old white shrimp postlarvae (PL10) and how some inhibitors of proteinases affect the digestion of protein in aquafeeds by using in vitro hydrolysis techniques. Biochemical data showed eight active proteinases in the PL10 hepatopancreas extract. Enzymes belong to the metallo‐ and serine‐proteinase classes. The effect of inhibitors present in protein ingredients and aquafeeds on PL10 proteinases showed that ovalbumin alone and in commercial microcapsules yielded a significant inhibition in proteolytic activity of PL10 hepatopancreas enzymes. The capacity of PL10 proteinases to hydrolyse the protein fraction within different sources and microcapsules was demonstrated by two in vitro approaches, the pH‐stat (degree of hydrolysis, DH) and electrophoresis (coefficient of protein degradation, CPD). It was shown that PL10 proteinases hydrolyse, in different extent protein sources and microcapsules. Casein, cuttlefish meal and feeds containing these ingredients are quickly hydrolysed. By contrast, ovalbumin and microcapsules containing ovalbumin are not hydrolysed. Copyright © 2006 Society of Chemical Industry

Alexandrium in the Black Sea — identity, ecology and PSP toxicity

Alexandrium cf. tamarense was recorded for the first time along the north-eastern coast of the Black Sea in July 2001. Since then, it has been observed annually between May and October. A maximum density of Alexandrium spp. of 9 000 cells l−1 was recorded during August 2004. Examination of the Black Sea Alexandrium spp. by scanning electron microscopy showed both A. tamarense and A. minutum to be present. Monitoring during 2000–2004 included the collection of mussel hepatopancreas samples and subsequent analysis for paralytic shellfish poisoning (PSP) toxins. A receptor-binding assay showed a maximum value of 14μg STXdiHCl equivalents 100g−1 tissue to correspond to ambient concentrations of A. tamarense of 300 cells l−1. Analysis of the PSP toxins by liquid chromatography with fluorescence detection showed the presence of GTX2, GTX3 and GTX5 in mussel hepatopancreas extracts. PSP in humans has not been reported in the Black Sea region of Russia. Nevertheless, the presence of Alexandrium spp and of PSP toxins in shellfish are compelling reasons for the introduction of phycotoxin control methods and monitoring in Russian waters.

Transglycosylation reactions performed by glycosyl hydrolases from the marine anaspidean mollusc Aplysia fasciata

The search for glycosyl hydrolytic activities from natural sources and the investigation of their transglycosylation potential can provide enzymes with new interesting catalytic activities for the synthesis of oligosaccharides. We focused our attention on the sea hare Aplysia fasciata Poiret, 1789, a common Mediterranean opistobranch mollusc, describing the results on the presence of different glycosyl hydrolases found in this organism. The activities of interest are present in the visceral mass and in the hepatopancreas extracts which were highly active with respect to those from other marine molluscs, namely an exo-α-d-glucosidase activity and a β-galactosidase, were investigated. The former is able to produce panose in high percentage by transglycosylation from maltose, or α-d-glucosides of α-methyl mannopyranoside and 2-deoxyglucose using p-nitrophenyl α-d-glucopyranoside as donor. The β-galactosidase from hepatopancreas produced in transgalactosylation reactions the β-Gal-(1-4)-GlcNAc as the most abundant product and Gal-Xyl disaccharides of biological importance using p-nitrophenyl β-d-galactopyranoside as donor and GlcNAc or benzyl α-xyloside as acceptors, respectively.

Catalase from the white shrimp Penaeus ( Litopenaeus) vannamei: molecular cloning and protein detection

Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide. It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited. A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus ( Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues. Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies. The nucleotide sequence is 1692 bp long, including a 72-bp 5′-UTR, a coding sequence of 1515 bp and a 104-bp 3′-UTR. The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354. The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (∼55 kDa) and the tetrameric protein (∼230 kDa) detected in hepatopancreas extracts under native conditions. Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle.

Transglycosylation reactions performed by glycosyl hydrolases from the marine anaspidean mollusc Aplysia fasciata

The search for glycosyl hydrolytic activities from natural sources and the investigation of their transglycosylation potential can provide enzymes with new interesting catalytic activities for the synthesis of oligosaccharides. We focused our attention on the sea hare Aplysia fasciata Poiret, 1789, a common Mediterranean opistobranch mollusc, describing the results on the presence of different glycosyl hydrolases found in this organism. The activities of interest are present in the visceral mass and in the hepatopancreas extracts which were highly active with respect to those from other marine molluscs, namely an exo-α- d-glucosidase activity and a β-galactosidase, were investigated. The former is able to produce panose in high percentage by transglycosylation from maltose, or α- d-glucosides of α-methyl mannopyranoside and 2-deoxyglucose using p-nitrophenyl α- d-glucopyranoside as donor. The β-galactosidase from hepatopancreas produced in transgalactosylation reactions the β-Gal-(1-4)-GlcNAc as the most abundant product and Gal-Xyl disaccharides of biological importance using p-nitrophenyl β- d-galactopyranoside as donor and GlcNAc or benzyl α-xyloside as acceptors, respectively.

Antioxidant activity in hepatopancreas of the shrimp (Pleoticus muelleri) by electron paramagnetic spin resonance spectrometry.

Free radical scavenging properties of hepatopancreas extracts of Pleoticus muelleri were evaluated by electron paramagnetic spin resonance spectrometry methods (EPR) against the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The present study was carried out to characterize different physiological stages of the shrimp under environmental and nutritional stress, evaluating the effect on growth, survival, and functional morphology of the hepatopancreas. Feeding trials were carried out on juveniles (1 g initial weight) held in aquaria. Each diet, with different concentrations of vitamins A and E, was tested in triplicate groups during 25 days. The control groups were fed with fresh squid mantle and with a vitamin-free diet. For all of the diets, the extracts exhibited strong DPPH radical scavenging activity, suggesting that the tissue is a powerful natural antioxidant. Individuals fed with different concentrations of vitamin E showed the strongest effect on the DPPH radicals, reducing the DPPH radicals to 50%, after an incubation period of 3 min. In contrast, the extracts of control animals, fed with squid mantle, had the weakest antioxidant activity (4%). These data indicated that the presence of vitamin E in the diet can provide immediate protection against free radicals.

Digestive enzymes present in crustacean feces as a tool for biochemical, physiological, and ecological studies

The effect of food composition on the digestive system of Penaeus vannamei shrimp was used to determine the suitability of feces for analysis of class, type, composition of digestive proteinases, and whether alterations in the digestive gland are mirrored in feces composition. Enzymes recovered from feces and the midgut gland of white shrimp P. vannamei were used for comparison purposes. Three groups of shrimp were assembled: two groups fed two different brands of commercial feeds (PI and SC) with different content of protein, and the last group fed 50% PI feed and 50% thawed giant squid. Composition of proteinases in the midgut gland and feces were identical, and trypsin and chymotrypsin paralogues were identified in both samples by substrate-electrophoresis. Total proteolytic, trypsin, and chymotrypsin enzyme activities were higher in both samples from organisms fed SC, than in the other two groups. In the hepatopancreas, trypsin activity was ∼30% higher in SC fed group. Final average weights of shrimp were close in three groups, but hepatopancreas weight was 20% higher in the SC group. The degree of protein hydrolysis (DH) in vitro for the SC and PI was evaluated by the pH-stat method, using enzymes from feces and hepatopancreas of each group. The DH of food was no different, but it was affected by enzyme source, hepatopancreas extract (HPE) or feces extract (FE). DH was always higher when FE was the enzyme source than when HPE was the source. The proposed methods for recovery of enzymes from shrimp feces can be applied to other crustaceans. Measurements were sufficiently sensitive to allow quantifying the effects of feed on digestion physiology and other ecological and physiological applications, without the necessity of killing specimens.

INFLUENCE OF HARVEST SEASON ON THE PROTEOLYTIC ACTIVITY OF HEPATOPANCREAS AND MANTLE TISSUES FROM JUMBO SQUID (DOSWICUS GIGAS)

Jumbo squid (Dosidicus gigas) size and protease activities were evaluated after harvest in April (A) and November (N). A were smaller (560±120 g vs 6040±1130 g) and hepatopancreas was a larger percent of body weight (8.3±2.5 vs 4.6±2.1). Mantle from A had lower water (73.9±l.l vs 79.1± 1.2) and higher protein (29.0±1.1 vs 23.1±0.8). Lipid and protein contents of N and A hepatopancreas tissues did not differ (P < 0.05). Azocaseinolytic, trypsin-like, chymotrypsin-like, aminopeptidase, and carboxypeptidase activities were detected in mantle (ME) and hepatopancreas extracts (HPE). HPE and ME from N had higher activity than A for all substrates (P < 0.05). With azocasein substrate, HPE activity from A had a pH optimum of 5 - 6 and a temperature optimum of 70C, whereas HPE from Nhad highest activity at pH 9 and 40–70C. ME from A had maximum proteolytic activity at pH 6 and 60C, whereas that from N had maximum activity at pH 10 and 40C. SDS-PAGE zymograms of HPE from A and N revealed different patterns of activity and 1 and 2 major protease zones, respectively.