Squid Hepatopancreas Research Articles

Utilisation of collagenolytic enzymes from sierra fish (Scomberomorus sierra) and jumbo squid (Dosidicus gigas) viscera to generate bioactive collagen hydrolysates from jumbo squid muscle.

Crude extracts of collagenases from jumbo squid (Dosidicus gigas) hepatopancreas and sierra fish (Scomberomorus sierra) viscera were used to hydrolyse squid muscle collagen into peptides with inhibitory capacity over angiotensin I-converting enzyme (ACE) and ABTS free radicals [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)], as a measure of their antihypertensive potential and antioxidant activity, respectively. Proteins from 20 to 200kDa were found in both enzyme extracts; however, in comparison to the jumbo squid extract (JSE), the extraction yield and specific activity of the enzymatic sierra fish extract (SFE) were ≈ 40% greater, suggesting the presence of enzymes with different collagenolytic activity. Moreover, the utilised collagen was obtained with a yield of 0.98 ± 0.09g/100g muscle from jumbo squid arms, which after an incubation with JSE and SFE generated peptides with different biological activity. However, the collagen hydrolysates from the enzymatic SFE contained a higher proportion of low-molecular-weight peptides than that obtained from JSE (15.2 and 7.9% of < 3kDa peptides, respectively). Finally, the antioxidant potential and ACE-inhibitory activity were increased after hydrolysis, being the SFE the one that showed a greater increase of both biological activities (82.28% of ACE inhibition and 64% of ABTS inhibition).

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

Chymotrypsin isolation from jumbo squid (Dosidicus gigas) hepatopancreas: Partial characterization and effect on muscle collagen.

Chymotrypsin was purified from jumbo squid hepatopancreas (HP) with 2.4-fold and yield 1.9%, and characterized with a molecular weight of 31 kDa, as estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin effect over collagen extracted from the mantle, fins and arms of the jumbo squid was evaluated. The enzyme exhibited the maximum activity at pH 7 and 65°C using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as a substrate and it was identified using the specific inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and phenyl methyl sulfonyl fluoride (PMSF), showing residual activities of 6% and 0%, respectively. Furthermore, high activity was observed in the pH range of 4.0 to 8.0. Purified enzyme showed a moderate in vitro activity using muscle collagen as a substrate. Although further research is needed, the results suggest that the enzyme has a potential application where acidic or slightly alkaline conditions are needed.

Comparison of S, Se, and 210Po accumulation patterns in common squid Todarodes pacificus from the Yellow Sea and East/Japan Sea

Activities of 210Po and concentrations of the chalcogen elements S and Se, were measured in muscle, stomach, gill, and hepatopancreas tissues of two groups of the common squid, Todarodes pacificus, from common spawning grounds but different feeding grounds: The Yellow Sea and the East/Japan Sea. All elements displayed lowest concentrations in the muscle tissues, but while S was highest in stomach and gills tissues, Se and 210Po were highest in hepatopancreas tissues, probably due to compartmentalization based on their toxicity. Whole body burden calculations based on organ weight contributions confirmed that the majority of the squids total S is contributed by the muscle while the majority of 210Po is contributed by the hepatopancreas. Overall, squids from the East/Japan Sea displayed significantly higher 210Po activities than those from the Yellow Sea, in line with previous reports on higher activities of dissolved 210Po in the water column of the former compared to the latter, which likely affects bioaccumulation by the foodweb. Tissue-based correlation patterns of the three chalcogens with each other and with the toxic heavy metal Cd suggest S- and Se-containing detoxifying mechanisms for 210Po and Cd in stomach and gills, but not in the hepatopancreas, which supports previous hypotheses that both 210Po and Cd could be bound to S- and Se-depleted compounds in the squid hepatopancreas.

Endoprotease and Exopeptidase Activities in the Hepatopancreas of the Cuttlefish Sepia officinalis, the Squid Todarodes pacificus, and the Octopus Octopus vulgaris Cuvier

This study examined and compared the exopeptidase and endoprotease activities of the hepatopancreas (HP) of cuttlefish, squid, and octopus species. The protein concentration in crude extract (CE) from octopus HP was 3,940 mg/100 g, lower than those in CEs from squid HP (4,157 mg/100 g) and cuttlefish HP (5,940 mg/100 g). With azocasein of pH 6 as a substrate, the total activity in HP CE of octopus was 31,000 U, lower than the values for cuttlefish (57,000 U) and squid (69,000 U). Regardless of sample type, the total activities of the CEs with azocasein as the substrate were higher at pH 6 (31,000-69,000 U) than at pH 9 (19,000-34,000 U). With L-leucine-p-nitroanilide (LeuPNA) of pH 6 as the substrate, the total activity of the HP CE from octopus was 138,000 U, higher than values from both cuttlefish HP (72,000 U) and squid HP (63,000 U). Regardless of sample type, the total activities of the CEs with LeuPNA as the substrate were higher at pH 6 (63,000-138,000 U) than at pH 9 (41,000-122,000 U). With LeuPNA as the substrate, the total activities of the CEs from octopus HP and cuttlefish HP were higher at pH 6 than at pH 9. However, there was no difference in total activity between pH 6 and 9 for squid HP CE with LeuPNA as the substrate. These results suggest that the octopus HP is superior to the cuttlefish HP and squid HP as a potential resource for extracting exopeptidases. Exopeptidases from octopus HP have potential industrial applications and their use might aid in reducing pollution related to the octopus industry.

Identification of a Novel Selenium-containing Compound, Selenoneine, as the Predominant Chemical Form of Organic Selenium in the Blood of Bluefin Tuna

A novel selenium-containing compound having a selenium atom in the imidazole ring, 2-selenyl-N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine, 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate, was identified from the blood and other tissues of the bluefin tuna, Thunnus orientalis. The selenium-containing compound was purified from the tuna blood in several chromatographic steps. High resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the exact mass of the [M+H](+) ion of the compound was 533.0562 and the molecular formula was C(18)H(29)N(6)O(4)Se(2). Its gross structure was assigned as the oxidized dimeric form of an ergothioneine selenium analog in which the sulfur of ergothioneine is replaced by selenium. Therefore, we named this novel selenium-containing compound "selenoneine." By speciation analysis of organic selenium compounds using liquid chromatography inductively coupled plasma mass spectrometry, selenoneine was found widely distributed in various tissues of the tuna, with the highest concentration in blood; mackerel blood contained similar levels. Selenoneine was measurable at 2-4 orders of magnitude lower concentration in a limited set of tissues from squid, tilapia, pig, and chicken. Quantitatively, selenoneine is the predominant form of organic selenium in tuna tissues.

Three types of proteinases in Japanese common squid Todarodes pacificus hepatopancreas as studied by using carp myofibrils as substrate

Three types of proteinases, namely cysteine, metallo, and serine proteinases, were found in squid hepatopancreas by studying the inhibition spectra using carp myofibril as substrate. The cysteine, metallo, and serine types showed the highest activities at 50, 35, and 40°C, respectively. The optimal pHs were 5, 7, and 9 for the cysteine, metallo, and serine types, respectively. When assayed at 20°C and pH 7.5, the metallo type showed the highest activity. The metallo type was characterized by a high selectivity in the digestion of myosin. Among the three enzymes, the cysteine type was found to be the most stable against thermal and acid treatments. Heat-treated myofibrils were more susceptible to the cysteine and serine types, but less susceptible to the metallo type. Acid treatment of myofibrils also enhanced the digestibility by the cysteine type. The results indicated that the cysteine type seemed to be the most suitable enzyme to produce peptides from denatured myofibrils by their random digestion.

Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: purification, characterisation, and casein hydrolysis

SummaryAn aminopeptidase (AP) was partially purified from jumbo squid (Dosidicus gigas) hepatopancreas with 154.24‐fold and yield of 6.15%. The purification procedure consisted of ammonium sulphate fractionation and DEAE‐Sephacel chromatography. The enzyme was approximately 48–53 kDa as estimated by SDS‐PAGE. With l‐leu‐p‐NA, it had optimum activity at pH 8.0 and 30 °C. The Km and Vmax/Km values of the enzymes for l‐leu‐p‐NA were 0.326 mm and 2787 at 37 °C, respectively. Activation energy (Ea) of the enzyme was 53.50 kJ M−1.The AP showed activity against seven synthetic substrates: l‐proline&gt;l‐methionine&gt;Ac. l‐γ‐glutamic&gt;l‐glycine&gt;l‐leucine&gt;l‐alanine&gt;l‐lysine‐p‐NA. The enzyme was strongly inhibited by Bestatin, partially inhibited by a metal‐chelating agent and by PCMB, a cystein protease inhibitor. Zn2+ and (or) Ca2+ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%.

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.

CYSTEINE PROTEINASE ACTIVITY IN JUMBO SQUID (DOSIDICUS GIGAS) HEPATOPANCREAS EXTRACTS

Jumbo squid specimens were captured, dissected, and the hepatopancreas was freeze dried for the extraction of proteolytic enzymes. An autolysis experiment conducted at 25C showed two peaks with maximum proteolysis at pH 3 and 5. The proteinase activity of the extract was measured using azocasein, BANA, Z-PAAFC and Z-AAAFC substrates. Activity of the extract with azocasein (pH 5) had a maximum at 55C and increased threefold with the inclusion of cysteine or DTT. The proteinase activity remained at least at 60% of the original after 45 h at 4C in the pH range of 3–8. Activity was inhibited 70–85% when extracts were treated with cysteine proteinase specific inhibitors. The proteinases extracted from jumbo squid hepatopancreas are mainly of the cysteine type and have significant activity towards a cathepsin L specific substrate. The understanding of proteinases from this tissue could have implications for quality control of jumbo squid food products.

Production of Water Soluble Antioxidative Plastein from Squid Hepatopancreas

To utilize a typical squid processing by-product, hepatopancreas was enzymatically hydrolyzed, then subjected to a plastein reaction. The substrate concentration that gave the highest plastein yield was 30% by using Alkalase. The optimum pH for the plastein synthesis with Alkalase ranged from 6 to 9. The dominant molecular size of the formed plastein was about 2000. Alkalase mediated plastein reaction seemed beneficial for enriching aspartic acid and glutamic acid, and eliminating hydrophobic amino acids such as phenylalanine, tyrosine, leucine, and isoleucine. Despite the heat pretreatment, hydrolysate powder and plastein retained their antioxidative affect, but a discouraging drawback of the hydrolysate was an unacceptable stimulating taste. For this reason, plastein was considered to be a much better choice to use as an antioxidant because it was tasteless. Plastein from squid hepatopancreas may be a useful antioxidant because it is stable against heat, dissolves easily in water and suppresses the proxidative effect of metals on lipid oxidation.

Accelerated Cheddar Cheese Ripening with an Aminopeptidase Fraction from Squid Hepatopancreas

ABSTRACT: An aminopeptidase (AP) fraction from squid (Illex illecebrosus) hepatopancreas was added to Cheddar cheese at 2 levels, and its influence on ripening indices was determined for up to 3‐mo storage at 11 °C. Two commercial enzymes (Neutrase ® and Flavourzyme ®) were similarly tested. Cheese with the higher level of squid AP contained more soluble N, amino acids, and Cheddar flavor after 1 mo, but it developed defects in texture and bitterness as ripening progressed. Cheese with less squid AP did not differ from the control with respect to all ripening indices over 3‐mo storage. Ripening Cheddar contains cysteine protease inhibitor(s) that inhibit low levels of squid AP but not Neutrase ® and Flavourzyme ®.

スルメイカ肝すい臓ならびに遡上シロサケ筋肉のプロテアーゼ加水分解によるアンジオテンシンＩ変換酵素阻害ペプチドへの変換

スルメイカ肝すい臓ならびに遡上シロサケ筋肉のプロテアーゼ加水分解によるアンジオテンシンＩ変換酵素阻害ペプチドへの変換

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus).

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.

Isolation and characteristics of trypsin inhibitor from the hepatopancreas of a squid ( Todarodes pacificus)

Trypsin inhibitor was purified from the hepatopancreas of squid ( Todarodes pacificus). The final inhibitor preparation was nearly homogeneous by SDS-PAGE with an estimated molecular weight of approximately 6300. The squid trypsin inhibitor was acid- and heat-stable, and active against trypsins from the pyloric ceca of starfish ( Asterias amurensis) and saury ( Cololabis saira) and porcine pancreatic trypsin. Amino acid composition of the squid trypsin inhibitor was compared with other invertebrate trypsin inhibitors. The squid trypsin inhibitor inhibited the autolysis of walleye pollock ( Theragra chalcogramma) myofibrillar proteins.

PURIFICATION AND CHARACTERIZATION OF AMINOPEPTIDASE FRACTIONS FROM SQUID (ILLEX ILLECEBROSUS) HEPATOPANCREAS

Atlantic short finned squid hepatopancreas (HP) aminopeptidases (APs) were partially separated from endoproteinases to determine their usefulness in accelerating Cheddar cheese ripening. Squid HP endoproteinases were predominantly cysteine proteases. The main peptidases identified in squid HP were APs. Several of the APs identified were metalloproteases and were activated by Ca 2+ , Mn 2+ , Zn 2+ or Mg 2+ salts. Squid HP homogenate was held at pH 7 at 0C for 20 h, incubated with 5 mM ZnSO 4 , fractionated by ammonium sulfate (20-80%) and dialyzed against 25 mM ZnSO 4 . The procedure resulted in a 3-186 fold increase in the ratio of exopeptidase to endoproteinase activity with different AP substrates. Recovery of specific APs ranged between 14-47%. The partially purified squid HP peptidases had a higher ratio ofexopeptidase to endoproteinase activity than two commercial products with AP activity, Flavozyme and Neutrase.

Lipase from neon flying squid hepatopancreas: purification and properties

Lipase from hepatopancreas of the neon flying squid ( Ommastrephes bartramii) has been partially purified. The molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33 000. Optimum pH was around 7.0 and the enzyme was relatively stable between pH 6.0 and 9.0 for 6 h at 25 °C. Optimum temperature of the enzyme reaction was around 25 °C. The enzyme was tolerably stable up to 37 °C. Several triglyceride (TG) molecular species were used as substrates for investigating the positional and fatty acid specificities of the enzyme. With respect to the hydrolysate of TG, the enzyme examined was not an sn-1,3 or an sn-2 positionally specific lipase. The enzyme appeared to have specific activity on monounsaturated and saturated chain TGs, most likely oleic and/ or palmitic acid. In this study, colipase was also separated from the enzyme.

Comparison of organophosphorous acid anhydrolases from different species using monoclonal antibodies

1. Monoclonal antibodies were raised against squid hepatopancreas organophosphorous acid (OPA) anhydrolase (EC 3.1.8.2) and were used to study structural similarities with OPA anhydrolases isolated from different sources. 2. Common epitopes were identified in OPA anhydrolases with diverse origins, and with different substrate specificities. 3. Epitopes unique to the squid hepatopancreas OPA anhydrolase were identified; optic ganglion and hepatopancreas contain different enzymes which can be distinguished by their epitopes.

Polyclonal antibody generation in rabbit by administration of an Organophosphorus Acid Anhydrolase (OPAA) from squid

When a nerve gas hydrolyzing enzyme [organophosphorus acid anhydrolase (OPAA), formerly DFPase] purified from squid hepatopancreas was injected into rabbits, the resulting sera (RAS) inhibited OPAA purified from either squid hepatopancreas or squid optic ganglia. The inhibition was non-competitive, with 50% inhibition at a 1:1,000 serum dilution, and with the limit of inhibition (in effect, a “titer”) at approximately 1:10,000. This RAS did not inhibit the distinctly different OPAAs from a mammalian and two bacterial sources. The hepatopancreas-generated RAS also reacted positively to the appropriate enzyme-linked immunosorbent assay (ELISA) at a titer of 1:100,000. In marked contrast, when OPAA purified from squid optic ganglion was injected into rabbits, the resulting sera did not inhibit squid OPAA, and did not give a positive ELISA. Control sera taken from the same rabbits prior to any injection (RS) did not inhibit the OPAAs. These results show another major difference between squid type OPAAs and the OPAAs from other sources, sometimes termed “Mazur type” OPAAs.

The Encapsulation of Squid Diisopropylphosphorofluoridate-Hydrolyzing Enzyme within Mouse Erythrocytes

The Encapsulation of Squid Diisopropylphosphorofluoridate-hydrolyzing Enzyme within Mouse Erythrocytes. McGuinn, W. D., Cannon, E. P., Chui, C. T., Pei, L., Petrikovics, I., and Way, J. L. (1993). Fundam. Appl. Toxicol. 21, 38-43.This study describes the entrapment of squid-type diisopropylphosphorofluoridate-hydrolyzing enzyme (DFPase) within mouse red blood cells. These erythrocytes thereby gain the ability to rapidly hydrolyze alkylphosphate cholinesterase (ChE) inhibitors such as diisopropyl fluorophosphate (DFP). DFPase rapidly hydrolyzes DFP to diisopropyl phosphate. Resealed erythrocytes provide a stable carrier system that can preserve the activity of encapsulated enzymes against otherwise rapid in vivo degradation; thus, ChE inhibitors can be degraded to relatively nontoxic metabolites by these erythrocyte carriers. Squid DFPase was purified from the hepatopancreas of Atlantic squid and DFPase activity was determined by measuring changes in fluoride ion concentration using a fluoride ion selective electrode. Mouse erythrocytes in suspension with excess squid DFPase were dialyzed against hypotonic buffer to allow the encapsulation of the enzyme to occur. Cells were then resealed by returning the suspension to isosmotic with saline. Rate of DFP hydrolysis observed with these cells was much greater that the rate of nonenzymatic hydrolysis and was directly proportional to the amount of the erythrocyte suspension added to the assay solution. The rate of hydrolysis was first order in substrate. Erythrocyte controls showed no endogenous DFPase activity. These results suggest that enzyme entrapment may be developed as a method to prevent and antagonize organophosphate poisoning.