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The catalytic properties of chitin deacetylase from Mucor rouxii were studied with the aim of using the results for control of the properties of chitosan prepared by enzymatic deacetylation. A crude deacetylase extract from the mycelium of Mucor rouxii exhibited maximal activity at pH 5.8 with both water-soluble and acid-soluble chitosan. The extracellular enzyme of the culture medium exhibited maximal activity at pH 4.8. The deacetylase in the crude extract was stable over the pH range of 4.1-8.9 at 25C, retaining 85-100% of maximal activity at 40C. The extract was most active towards acid-soluble chitoson at 50C and pH 5.8. Chitin deacetylase was stable to 40C when incubated without substrate, with 85% of the activity retained at 50C. Ca 2+ , Mn 2+ , and Zn 2+ had no effect on activity, while EDTA, Fe 2+ , and Fe 3+ caused partial inhibition in extracts incubated without substrate for 1 h at 25C and pH 5.8. The deacetylase in the mycelial extract and in the culture medium was slightly activated by Co 2+ . The differences in temperature optima and thermal stability of deacetylase and hydrolytic enzymes can be used for controlling the degree of hydrolysis of chitosan, while the difference in pH stability has been used for prepurification, resulting in 3-fold increase in specific activity of deacetylase.

Muscle proteases active at neutral and alkaline pH's include calcium-activated neutral proteases, heat-activated thiol proteases, serine proteases, and metallo-proteases. They participate to different extents in postmortem degradation offish muscle myofibrillar and scaffold proteins. Their activity in fish muscle is varied and depends on location in the muscle, the life cycle of the fish, the presence of endogenous activators and inhibitors, pH, and temperature. Recent studies indicate that neutral and alkaline proteases have more impact on the postmortem deterioration in quality of fish muscles than the cathepsins active at acid pH. Significant quality losses are caused by enzymatic collagen degradation in raw tissues and by heat-activated enzymes in fish gels.

The liver extract of squid Illex argentinus has a proteolytic activity at pH 2–8, with optimum at pH 2.6–3.1. At pH 6 the enzymes retain about 30 and 55% of the maximum proteolytic activity towards hemoglobin and casein, respectively. In squid mantle meat treated with the extract 24 h at 4C and 20C, the sarcoplasmic and myofibrillar proteins were hydrolyzed to low molecular fractions, as was shown by SDS-PAGE. Soaking isolated collagen fibers in the liver extract resulted after 24 h in almost complete solubilization of the fibers in buffered SDS/urea solutions. The solubilized product contained only low molecular weight components. A significant hydrolysis of the proteins in the squid mantle, treated with the liver extract at 20 or 4C, was accompanied by a decrease in toughness of the cooked product by about 65 and 40%, respectively, as compared to the toughness of untreated cooked samples. Cooking of the mantle directly in the liver extract had practically no tenderizing effect.

Many nitrogenous compounds are involved in postmortem reactions in the muscles of animals, primarily as enzymes catalyzing the catabolysis of different tissue components. Thus, they contribute to changes in color, flavor, and rheological properties of the products. Furthermore, proteins and NPN participate also as substrates in reactions leading to the formation of products affecting the pH in the fish tissues. On the other hand, the rate and extent of changes in proteins are influenced by the products of degradation of saccharides and organic phosphates, as well as by the concentration of free ions, whereas these depend on the antemortem condition of the fish and on the temperature in the tissues after catch. A positive relationship of the enthalpy of activation of fish myofibrillar ATPase and the habitat temperature was demonstrated, from large negative values for cold-adapted species to high positive values for tropical species (Johnstone and Goldspink, 1975).

Frozen fish stored several months at about −20°C may, after cooking, become tough, chewy, rubbery, stringy, or fibrous. This is accompanied by a loss in functional characteristics of the muscle proteins, mainly solubility, water retention, gelling ability, and lipid emulsifying properties. Freezing and thawing may result in lysis of mitochondria and lysosomes and a change in distribution of enzymes (Karvinen et al., 1982). A gradual decline in the activities of various muscle enzymes has also been observed during storage at freezing temperatures. The loss of ATPase activity, both in meat homogenates and in protein solutions, may reach 50–80% (Buttkus, 1967). The total solubility of proteins in neutral 5% NaC1 solution may decrease to about 30%, whereby the main loss regards the contractile proteins, mainly myosin heavy chain, M-proteins, tropomyosin, and troponins I and C in descending order (Owusu-Ansah and Hultin, 1992). It was shown by Jarenback and Liljemark (1975b), that the myosin microfibrils in fresh cod muscle could be almost totally extracted, whereas myosin in frozen muscle after prolonged storage was resistant to extraction. Significant changes in the sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS—PAGE) and HPLC profiles of cod sarcoplasmic proteins due to frozen storage have been shown recently by LeBlanc and LeBlanc (1992b).

Storage of frozen fish brings about a decrease of extractability of myofibrillar proteins. There is also deterioration of the texture and functional properties of the flesh. In model systems, aggregation of myosin, actin, tropomyosin, and whole myofibrils have been described. These changes are caused by concurrent action of partial dehydration due to the freezing out of water, exposure of the proteins to inorganic salts which are concentrated in the remaining nonfrozen fluid, interactions with free fatty acids liberated from phospholipids and with lipid oxidation products, and cross-linking by formaldehyde produced in some species of fish as a result of enzymic decomposition of trimethylamine oxide. The extent of protein alterations increases with time and temperature of storage as well as with advanced disintegration of the tissues and intermixing of their components. The role played by the individual factors and the significance of different types of bonds, i.e., hydrophobic adherences, ionic bonds, and covalent cross-links in particular cases are not yet fully disclosed. Retardation of the deteriorative changes of proteins in frozen fish is possible by avoiding high storage temperatures and oxidation of lipids, removing hematin compounds and other constituents promoting cross-linking reactions, and by adding cryoprotectors like sugars, several organic acids, amino acids, or peptides.

Krill, a small shrimp-like crustacean, is actually being exploited commercially in the Antarctic waters. In order to evaluate the offals generated during the processing of krill to food and feed products, as commercial sources of chitin, three kinds of shell waste were analysed. Depending upon the method of krill processing applied the shell waste contained 49–60% of proteinaceous material. The content of crude chitin in the dried deproteinised shells ranged from 34 to 49%. About 60% of the salt mixture impregnating the shells was phosphates and carbonates of calcium and magnesium.

The suitability of krill chitin, prepared by using different concentrations of KOH and HCI for deproteinization and demineralization, respectively, was investigated. The activity of enzymes immobilized on such supports depends on the degree of deproteinization of chitin, availability of amino groups, content of minerals, mesh size, structure of the surface, and conformation of the chitin molecules. It was found that invertase and amyloglucosidase achieved high activity after immobilization on chitin obtained at not too rigorous conditions of deproteinization. However, the activity of immobilized α-amylase and diastase increased significantly with the increase in concentration of KOH used for deproteinization. High content of minerals and proteins in chitin preparation causes a loss of immobilized enzyme activity.

Changes in the thiol groups were determined in the mantle meat of the squid (Illex argentinus) after heating at 98°C for 45 min. The contents of -SH groups in the raw mince was about 90 μmoles/g protein and after heating decreased by about 30%. The total contents of -SH groups after reduction of disulfide bridges by NaBH4 was 120 μmoles/g protein in the raw sample and did not change significantly in the cooked meat. After 45 min of cooking the mince contained about 18 times more dimethylamine and only twice as much free formaldehyde as the raw samples. The correlation coefficients between free -SH groups contents and the solubility, degree of thermal coagulation of proteins as well as the water holding capacity of the meat were +0.936, -0.947, and +0.880, respectively.

By simple adsorption or covalent binding, enzymes were immobilized on chitin isolated from the shells of edible shellfish. It is reported that the best results were obtained by immoblizing diastase on krill chitin by adsorption at pH 6.7 and an ionic strength of 0.05.