Biochemical characterisation of chymotrypsin from the midgut gland of yellowleg shrimp, Penaeus californiensis.

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 2 h 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.

Purification and biochemical characterization of digestive lipase in whiteleg shrimp.

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 × 10(3) 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/ß-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.

Phenoloxidase activity of hemocyanin in whiteleg shrimp Penaeus vannamei: conversion, characterization of catalytic properties, and role in postmortem melanosis.

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.

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.

Penaeus vannamei isotrypsins: purification and characterization.

Three isotrypsins from digestive gland of Penaeus vannamei were purified and characterized by molecular, biochemical and kinetic parameters. Purified isotrypsins A, B, and C are glycoproteins with molecular masses between 30.2 and 32.9 kDa, and, therefore similar to other trypsins. The isoelectric points are anionic and different among the three isotrypsins: pH 3.5 for isotrypsin A, pH 3.0 for isotrypsin B, and pH 4.5 for isotrypsin C. Differences in the NH(2)-terminal amino acid sequences allowed us to define three different protein entities that match isotrypsins previously deduced by cDNA. Isoform C has higher physiological efficiency and specific activity, lower K(m), and requires higher concentrations of Ca(+2) to reach the same activity as the other two isotrypsins.

Characterization of proteases in the digestive system of spiny lobster (Panulirus interruptus).

Enzymes responsible for digestion of food protein were evaluated and characterized in red lobster (Panulirus interruptus). Several tissues, organs, and body fluids were analyzed. The same composition of proteases was found in gastric juice, midgut gland, and intestinal contents. Using specific substrates and inhibitors, we identified several isotrypsins and isochymotrypsins by gel electrophoresis. Protease activity was found at pH 3 and reduced by using pepstatin A. Operational variables of enzymes were characterized for management of future studies and potential biotechnologies. Types and activities of lobster digestive enzymes constitute background information to study the digestive abilities of the organism further, and will lead to understanding nutritional needs and feeding ecology, mainly because decapods display unique morphologic, metabolic, and behavioral changes during their life cycle. Also, such enzymes become alternative tools for use in biotechnologies.

Effects of dietary protein on the activity and mRNA level of trypsin in the midgut gland of the white shrimp Penaeus vannamei.

Protein food modulates the activity of proteases of the midgut gland of Penaeus vannamei. Shrimp fed with food containing 15, 30 and 50% protein exhibited differences in trypsin and chymotrypsin activity and trypsin mRNA levels. Shrimp fed with 30% protein showed higher trypsin and chymotrypsin activities than those fed 15 or 50% protein. An additional paralogue trypsin was observed with electrophoretic analysis in shrimp fed 30% protein. Shrimp fed 30% protein showed the highest trypsin to mRNA concentration, suggesting that trypsin genes expression is regulated transcriptionally.

Influence of molting and starvation on the synthesis of proteolytic enzymes in the midgut gland of the white shrimp Penaeus vannamei.

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.

Digestive proteinases of Brycon orbignyanus (Characidae, Teleostei): characteristics and effects of protein quality.

Juvenile piracanjuba, Brycon orbignyanus, in the wild consume protein from both plant and animal sources. Digestion of protein in piracanjuba begins in the stomach with pepsin, at low pH, and is followed by hydrolysis at alkaline pH in the lumen of the intestine. The digestive system in piracanjuba was evaluated to characterize the enzymes responsible for the digestion of feed protein and their composition. The gastric tissue synthesizes pepsin and the intestine tissues trypsin and chymotrypsin. Operational variables were evaluated and defined for future studies of the digestive system physiology. The enzymatic activity in the intestine and the relative concentration of enzymes were heavily influenced by the composition of the feed and the feeding regime, as detected by substrate-SDS-PAGE. Piracanjuba possess a mechanism of enzyme adaptation responding to food quality and regime, by varying the amount and composition of digestive proteases. This is a requisite study to determine the enzymes digesting protein in food and their characteristics and to gain some clues about the possible regulation mechanisms of enzyme synthesis in piracanjuba.