Latent phenoloxidase activity of hemocyanin (Hc) in whiteleg shrimp Penaeus vannamei was assayed to determine its potential involvement in postmortem melanosis. Conversion of pure 12-mer, but not ...6-mer, hemocyanin to phenoloxidase by endogenous (serine proteinases) and exogenous (SDS) effectors demonstrated the need of complex aggregation for displaying enzyme activity. Because Hc was converted to Hc-phenoloxidase (HcPO) by hemocytes extracts, the mechanism of conversion seems to be the same for polyphenoloxidases. HcPO has similar biochemical and kinetic properties as real polyphenoloxidases and uses mono- and diphenols as substrates. The kinetics of hydroxygenation of monophenols has a lag phase, typical for tyrosinases, contrary to oxidation of diphenols. Regardless of the structure of the substrate, melanin is finally formed. Because of the abundance, distribution, and resistance of Hc to freezing−thawing, involvement of Hc in black spot formation postmortem is suggested. This has important implications for commercialization of shrimp and related seafood.
Invertebrate trypsins: a review Muhlia-Almazán, Adriana; Sánchez-Paz, Arturo; García-Carreño, Fernando L
Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology,
08/2008, Letnik:
178, Številka:
6
Journal Article
Recenzirano
Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, ...in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.
•A chymotrypsin with collagenolytic activity from Penaeus californiensis is described.•The shrimp chymotrypsin is extensively compared to bovine chymotrypsin.•It is resistant to SDS and its mature ...protein consists of only one polypeptide chain.•Chymotrypsin is stable to temperature (50°C), sensible to low pH, possess acidic pI.
Chymotrypsin from shrimp, Penaeus californiensis, was compared to Bos taurus chymotrypsin, and its structure–function relationship was studied. Catalytic efficiency toward synthetic substrate is lower, but it has a broad specificity and higher activity toward protein substrates, including collagen. It is active at pH 4–10 and fully active up to 50°C for 2h and at least nine days at room temperature. The activation peptide is twice as long as bovine chymotrypsinogen, has less disulfide bridges, and is a single polypeptide. Only one activation step is necessary from chymotrypsinogen to the mature enzyme. Postmortem implications in muscle softening and melanisation, resistance to temperature and pH and efficiency with proteinaceous substrates make chymotrypsin useful as a biotechnological tool in food processing. This makes shrimp processing wastes useful as a material for production of fine reagents.
Chitin metabolism is of high relevance for shrimp growth because it has to be synthesized and cleaved in each molt. We studied chitin synthase and chitinase mRNAs from whiteleg shrimp Litopenaeus ...vannamei. For this, cDNA coding for cuticular chitin synthase (LvChS) and chitinase isoenzymes (LvChi1, LvChi2 and LvChi3) was amplified, sequenced and identified. In a qualitative analysis, LvChi1 and LvChi3 were detected only in the hepatopancreas and are probably involved in digestion of food chitin. LvChi2 transcript was found in pleopods, uropods, gills, eyestalk, and digestive tube; LvChi2 is likely to be involved in the hydrolysis of chitin from the exoskeleton and peritrophic membrane, but not in food chitin digestion. LvChS was found widely distributed in the organism, including the hepatopancreas. Thus, it seems to be involved in synthesis of chitin to build the exoskeleton and also the peritrophic membrane. Relative expression of LvChS and LvChi2 genes was evaluated by quantitative RT-PCR in the integument. These transcripts had a varying pattern of abundance during the molt cycle, based on the need of shrimp to synthesize or hydrolyze chitin from exoskeleton.
►In L. vannamei, ChS mRNA expresses in several organs. ►3 different Chi isoenzyme mRNAs have different putative roles. ►In the uropods, ChS mRNA is more abundant in postmolt stages. ►Chi2 mRNA is abundant in stages A and D0 with high variability among shrimps.
Penaeus vannamei lipase was purified from midgut gland of whiteleg shrimp. Pure lipase (E.C. 3.1.1.3) was obtained after Superdex 200 gel filtration and Resource Q anionic exchange. The pure lipase, ...which is a glycosylated molecule, is a monomer having a molecular mass of about 44.8 kDa, as determined by SDS-PAGE analysis. The lipase hydrolyses short and long-chain triacylglycerols and naphthol derivates at comparable rates. A specific activity of 1787 U mg⁻¹ and 475 U mg⁻¹ was measured with triolein and tributyrin as substrates, respectively, at pH 8.0 and 30°C in the absence of colipase. The lipase showed a K m, app of 3.22 mM and k cat, app/K m, app of 0.303 × 10³ mM⁻¹ s⁻¹ using triolein as substrate. Natural detergents, such as sodium deoxycholate, act as potent inhibitors of the lipase. This inhibition can be reversed by adding fresh oil emulsion. Result with tetrahydrolipstatin, an irreversible inhibitor, suggests that the lipase is a serine enzyme. Peptide sequences of the lipase were determined and compared with the full-length sequence of lipase which was obtained by the rapid amplification of cDNA ends method. The full cDNA of the pvl was 1,186 bp, with a deduced protein of 362 amino acids that includes a consensus sequence (GXSXG) of the lipase superfamily of α/β-hydrolase. The gene exhibits features of conserved catalytic residues and high homology with various mammalian and insect lipase genes. A potential lid sequence is suggested for pvl.
The aim of this work was to study mechanisms of homeostasis of the digestive system of the whiteleg shrimp Litopenaeus vannamei when fed anti-nutritional soybean trypsin inhibitor (SBTI). In a ...previous work, we tested if the shrimp could respond against SBTI in feed with the increase in the digestive proteolytic activity. This time we tested the time of response against the inhibitor and also how the two major digestive trypsin phenotypes change the proteolytic activity. In groups fed SBTI, the total proteolytic activity, trypsin and chymotrypsin decreased during food transit time (2–4h), but increased at 23h postprandial. Three metallo peptidases absent in the control group were identified in groups fed SBTI; individuals with trypsin phenotype CBA increased the activity of two metallo peptidases at 4h of SBTI ingestion, while, individuals with trypsin phenotype CB increased the activity of three metallo peptidases at 23h postprandial. The first evidence of feedback by the digestive system in the whiteleg shrimp fed SBTI was 1h postprandial, with a concomitant increased in metallo peptidases activity at 4h or 23h depending on the trypsin phenotype in each specimen.
Comparison of digestive proteinases in three penaeids Navarrete del Toro, María de los Angeles; García-Carreño, Fernando L.; Córdova-Murueta, Julio H.
Aquaculture,
07/2011, Letnik:
317, Številka:
1
Journal Article
Recenzirano
Recently, several groups of researchers reported that besides serine proteinases, other classes of proteinases may be involved in crustacean's food protein digestion, including cysteine and aspartic ...proteinases. In this paper, a comparative study of the class and type of digestive proteinases of whiteleg shrimp
Penaeus vannamei, blue shrimp
Penaeus stylirostris, and yellowleg shrimp
Penaeus californiensis is addressed, along with some operational characteristics. The substrate-SDS-PAGE zymogram of the three species showed varieties of different proteinases that were species-specific and proteinase composition that may be used for species identification or population studies. In the three species, trypsin and chymotrypsin were present as isoenzymes. Some active bands were active at acid pH and were partially inhibited by pepstatin A and based on bibliography information they are digestive enzymes. The involvement of these enzymes in food protein digestion is discussed and compared with digestive enzymes present in other decapod species. This and additional information expand the knowledge about enzyme food protein digestion in crustaceans, making clear that digestion process is more complex than previously alleged and if common patrons exist, there are some individualities. This information opens a line of research trying to fully understand the mechanism of protein digestion.
Lipase activity of the midgut gland during larval and postlarval stages of
Penaeus vannamei was assayed to determine its capability to digest lipids from feed and involvement in digesting lipids from ...reserves during fasting conditions. Lipase activity was detected at all larval stages, increasing from nauplii to protozoea. Lipase isoenzymes at larval stages were evaluated by SDS-PAGE using 4-methylumbelliferone butyrate as the substrate. Results showed that shrimp larvae possess a nearly complete set of lipases starting with the first larval stage. In addition, to understand the effects of fasting conditions as a stress factor on lipase activity, intermolt shrimp were fasted up to 5
days, a period corresponding to the normal time that shrimp starve during molting, in which they cannot eat. Digestive lipases were affected by fasting, increasing in activity after 24
h of treatment, suggesting that lipid is used as an energy reserve during fasting. Proteins with lipase activity were identified and characterized by zymograms; the presence of more than one lipase enzyme could be one way to hydrolyze triacylglycerides more efficiently as the first step of fat assimilation and to obtain energy from fatty acids under fasting conditions.
There is a growing interest in adding value to the jumbo squid
Dosidicus gigas fishery. This work describes two extraction procedures for processing muscle to obtain protein isolates with suitable ...functional properties. The effect on muscle protein solubility and protein recovery of combining freezing and grinding raw materials during storage was evaluated. Processes are based on extraction of protein at acid or alkaline pH and subsequent iso-electric precipitation. About 85% of the initial muscle protein was solubilized at pH 3 and 11. Regardless of the pH used for extraction, about 90% of the protein was obtained after precipitation at pH 5.5. The total yield from both procedures was 75%. Treatments during storage did not significantly affect solubility and yield of protein. Wastewater contained negligible amounts of protein and may be reused. Processing by acid and alkaline extraction are feasible alternatives for obtaining protein isolates either from fresh or frozen squid muscle, which is an important consideration when choosing the most appropriate and inexpensive method to scale up this technology.
We investigated the effect of starvation as a stimulant of the digestive system on digestive proteinase activities in the white shrimp Penaeus vannamei. The starved organisms were sampled ...periodically according to the molting stage and compared with a continuously fed group. Molting stage was included as an independent variable. Most analyzed variables, except for trypsin, were more affected by starvation than by molting, indicating that starvation is a stimulant that masks the effect of molting and showing that food or alimentary stress is more conspicuous than physiological ones. We found that starvation is a stimulant that surpasses the effect of molting, and because it affects the activity of digestive proteinases, studies of starving organisms in combination with tools of molecular biology, can be a helpful working model in the understanding of mechanisms of regulation of digestive enzyme activity. In the starved organisms, trypsin and chymotrypsin activities were similar, suggesting dependence of one to the other. Changes in proteolytic activities and the number of protein bands in electrophoresis showed evidence of synthesis regulation in the midgut gland of white shrimp.
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