Enzymatic methods have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. Aquatic environment contains the largest pool of diversified genetic material and, hence represents an enormous potential for different sources of enzymes. In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates has been achieved and some interesting and novel applications related to marine enzymes in food processing have emerged. This review summarizes information related to digestive and muscular enzymes in fish and aquatic invertebrates. In addition, important developments in food applications of marine enzymes are reported.

Enzymes from fish and aquatic invertebrates and their application in the food industry

Nutrition of Litopenaeus vannamei reared in tanks or in ponds

Ocean acidification, chemical changes to the carbonate system of seawater, is emerging as a key environmental challenge accompanying global warming and other human-induced perturbations. Considerable research seeks to define the scope and character of potential outcomes from this phenomenon, but a crucial impediment persists. Ecological theory, despite its power and utility, has been only peripherally applied to the problem. Here we sketch in broad strokes several areas where fundamental principles of ecology have the capacity to generate insight into ocean acidification's consequences. We focus on conceptual models that, when considered in the context of acidification, yield explicit predictions regarding a spectrum of population- and community-level effects, from narrowing of species ranges and shifts in patterns of demographic connectivity, to modified consumer-resource relationships, to ascendance of weedy taxa and loss of species diversity. Although our coverage represents only a small fraction of the breadth of possible insights achievable from the application of theory, our hope is that this initial foray will spur expanded efforts to blend experiments with theoretical approaches. The result promises to be a deeper and more nuanced understanding of ocean acidification'and the ecological changes it portends.

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Ocean Acidification Through the Lens of Ecological Theory

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.

Invertebrate trypsins: a review.

The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion, as found in the group III trypsinogens of cold-adapted fishes.

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Evolution of Trypsinogen Activation Peptides

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 NH2-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 Km, and requires higher concentrations of Ca+2 to reach the same activity as the other two isotrypsins.

Penaeus vannamei isotrypsins: purification and characterization

The purification and characterization of a chymotrypsin from the hepatopancreas of the white shrimp Penaeus vannamei is described. Only one chymotrypsin was detected in contrast to other shrimp that have two major forms. P. vannamei chymotrypsin has a molecular mass of 33.2 kDa and a pI of 3.1. The molecular mass is high relative to other penaeid chymotrypsins. The proteinase is acid labile and exhibits optimum activity at pH 8. The enzyme is thermostable both at 25 and 37C. It is a serine proteinase. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor blocked the activity of the enzyme, and it was not affected by chymotrypsin inhibitors such as tosyl-PheCH 2 Cl or benzyloxycarbonyl-Phe-CH 2 Cl. Protein profiles of the hepatopancreas from two populations varied and this is suspected to be caused by differences in the expression of chymotrypsin in the white shrimp.

Purification and characterization of chymotrypsin from Penaeus vannamei (Crustacea:Decapoda)

An immunological approach was used to elucidate whether trypsin is synthesized and stored as trypsinogen in the midgut gland of the shrimp Litopenaeus vannamei. Two peptides were constructed using sequences deduced from known shrimp genes: trypsinogen activation peptide and an internal sequence. These peptides were used as haptens to elicit antibodies in rabbits. Specific antibodies were used to detect trypsinogen by Western blot and in histological sections of the midgut gland. Trypsinogen was found by Western blot and was localized into the midgut gland B cells by using immunohistology. In fed shrimp, trypsinogen associated with food particles was found in the lumen of the midgut gland tubules as well. Our results show that regulation of shrimp trypsin activity is similar to that of frequent feeder species, in which trypsin is stored as a zymogen, waiting for secretion and activation.

Trypsin Synthesis and Storage as Zymogen in the Midgut Gland of the Shrimp Litopenaeus Vannamei

Juvenile white shrimp, Penaeus vannamei, were studied using tray feeders in relation to food ingestion and enzymatic activity, with data taken every two hours for five days. The feed ingestion was calculated by subtracting the uningested and the leached material from the food provided. Significant differences (P < 0.05) in the ingestion rate were found during the experiment. The greatest ingestion occurred between 2000 and 2200 hours. The largest feed ingestion coincided with the nocturnal activity of shrimp. Time-series analysis showed a cycle of increased ingestion before midnight and lower ingestion during daylight. Total protease, trypsin, and chymotrypsin activity were measured during the last 24 hours of the study and included both fed and starved organisms. The enzymatic pattern was similar for both groups. There was a decrease of the protease and trypsin activities before and after 1800 hours, just before the ingestion increased. The study of digestive enzymes provided evidence of a close...

Rate of Ingestion and Proteolytic Activity in Digestive System of Juvenile White Shrimp, Penaeus vannamei, During Continual Feeding

Trypsin was purified from crayfish, Pacifastacus leniusculus, hepatopancreas, and the gene that encoded this enzyme was cloned from a hepatopancreas cDNA library. Crayfish trypsin is synthesized as ...

Patricia Hernández-Cortés

Papers

Penaeus vannamei isotrypsins: purification and characterization

Purification and characterization of chymotrypsin from Penaeus vannamei (Crustacea:Decapoda)

Trypsin Synthesis and Storage as Zymogen in the Midgut Gland of the Shrimp Litopenaeus Vannamei

Rate of Ingestion and Proteolytic Activity in Digestive System of Juvenile White Shrimp, Penaeus vannamei, During Continual Feeding

Trypsin from Pacifastacus leniusculus hepatopancreas: purification and cDNA cloning of the synthesized zymogen.