Extraction of harp seal gastric proteases and their immobilization on chitin

doi:10.1016/0308-8146(94)P4183-G

Food Chemistry

Volume 52, Issue 1, 1995, Pages 71-76

https://doi.org/10.1016/0308-8146(94)P4183-G Get rights and content

Abstract

Gastric proteases from seal stomach were isolated following homogenization, extraction, centrifugation and ammonium sulfate precipitation/fractionation. The crude seal gastric proteases (SGP) were stable in acid conditions with optimum stability at pH 3.0, but were unstable in the alkaline range. The crude SGP possessed excellent temperature adaptability with relative activities of 70 and 90% at 5 and 25 °C, respectively. The activity of crude SGPs was retained up to about 40 min incubation at 70 °C. The isolated SGP were immobilized on glutaraldehyde-treated chitin. The immobilized SGP exhibited optimum performance at pH 2.0, were most stable at pH 4.0 and had a 90 h half-life in a column with continuous operation for haemoglobin hydrolysis at room temperature. The native SGP clotted milk rapidly at pH 5.8 to 6.6; however, immobilized SGP had a lower milk-clotting activity.

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The unique catalytic properties of enzymes have led to the production of useful medicinal intermediates, foods, and biofuels from sustainable sources. However, the instability of soluble/free enzymes under several challenging conditions (e.g., pH, proteolysis, temperature, ionic potential, chemical denaturants) restricts the use of enzyme-based biocatalysts. Encapsulation of enzymes on suitable carriers would mitigate the instability issues faced in robust biocatalysis. An “ideal” carrier material employed for protein immobilization should be nontoxic, scalable, biocompatible, and should not compromise the biological activity and structure of proteins/enzymes. Thus, biodegradable and renewable biomass-derived functional materials (BDFMs) are envisaged as promising carriers for enzymes. BDFMs have in-built chemical functionalities and desirable physicochemical properties that enable their use in enzyme catalysis at the industrial scale. Numerous BDFMs have been used as immobilization matrices to improve the biocatalytic activity and stability of various enzymes. These solid materials are renewable and environmentally friendly compared with synthetic polymers. This review highlights the advancements, challenges and prospects in the emerging field of BDFMs (cellulose, silk protein, chitin, chitosan, lignocellulose, and a combination of biopolymers such as chitin/lignin and chitosan/alginate) for immobilization of enzymes (e.g., α-chymotrypsin, cytochrome c, carbonic anhydrase, glucose oxidase, ribonuclease, cholesterol oxidase, alkaline phosphatase, β-glucosidase, lipase, horseradish peroxidase, catalase, tyrosinase, acetylcholinesterase, amylase, invertase, protease, laccase, β-galactosidase, and several others) for biocatalytic processes. This review also describes the relationship between the structural properties and functionality of several enzymes immobilized in BDFMs, and profiles the impact of pH, temperature, reusability, stability of storage, and the activity of these enzymes. Future perspectives in this promising field, as well as potential difficulties, are discussed. This review will help in refining biocatalysis technologies whereby biomass-derived, environmentally friendly materials are employed as enzyme supports.
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Chymosin, Pepsins and Other Aspartyl Proteinases: Structures, Functions, Catalytic Mechanism and Milk-Clotting Properties
2017, Cheese: Chemistry, Physics and Microbiology: Fourth Edition
This chapter comprehensively reviews the classification, structure, function, catalytic mechanism, and milk-clotting properties of chymosin, pepsin, and other aspartyl proteinases Hydrolysis of the principal bovine milk proteins (β-, α_s-, and κ-caseins) by calf chymosin is described in detail, as is the hydrolysis of caseins from the milk of other, nonbovine, species. Rennets, other than chymosin, are discussed and their hydrolysis of caseins is described. Interspecies milk coagulation is reviewed and includes detail on the chymosins of several nonbovine species, including, camel, pig, equine, New World monkies, rat, and seal. The milk-clotting ability of aspartyl proteinases from other sources, for example, plants, fish, and crustaceans is discussed.
Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant
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Miniature (20 g) Cheddar-type cheeses were manufactured using enzymes extracted from the crustacean Munida or chymosin as coagulant. Cheeses were ripened at 8 °C and samples were collected for analysis after 2, 6 and 12 weeks. Proteolysis was assessed by urea-polyacrylamide gel electrophoresis, which showed that cheeses manufactured with the Munida extracts had a higher extent of degradation of β-casein than cheeses made using chymosin as coagulant. Patterns of proteolysis were also obtained by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry. In general, the products of proteolysis were more complex in cheese made using the Munida extracts than in cheese made by chymosin as coagulant. Statistical analysis of results clearly discriminated the cheeses on the basis of coagulant used. Molecular mass of peptides found in cheese made using Munida extracts were similar to those of peptides commonly detected in cheeses made using chymosin as coagulant.
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Citation Excerpt :
Chymosin has certain properties which differentiates it from pepsin; these are relatively high ratio of milk clotting to proteolytic activity, a pH optimum of 2.2–3.5 for haemoglobin hydrolysis, inability to inactivate ribonuclease, low activity on N-acetyl-l-phenylalanyl-3,5-diiodo-1-tyrosine, instability in 6M urea and zymogen molecular weight of 33,800 Da (Haard & Simpson, 1984). Furthermore, Han and Shahidi (1995) reported that immobilized seal gastric protease had a somewhat lower milk clotting activity compared to the crude native seal gastric protease. Gastricsins are aspartyl proteases that possess similar enzymatic and chemical properties to pepsin (De-Vecchi and Coppes, 1996; Sanchez-Chiang & Ponce, 1981).
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.

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Food Chemistry

Abstract

References (26)

Application of a synthetic hexapeptide as a standard substrate for the determination of the activity of chymosin

Neth. Milk Dairy J.

Chicken pepsinogens

Methods Enzymol.

Gastric proteases—structure, function, evolution and mechanism of action

Assays Biochem.

Autolysis of fish tissue — general aspects

Aspartic proteinases in fishes and aquatic invertebrates

Comp. Bioche. Physiol.

Purification and characterization of pepsins from the Arctic fish capelin (Mallotus villous)

Comp. Biochem. Physiol.

Efficacy of chicken pepsin as a milk clotting enzyme

J. Food Protect

A review of proteolytic enzymes from marine organisms and their utilization in the food industry

J. Aquatic Food Prod. Technol.

Modification of Proteins with proteolytic enzymes from the marine environment

Recovery of digestive enzymes from Atlantic cod (Gadus morhua) and their utilization in food processing

Protein measurement with folin phenol reagent

J. Biol. Chem.

Optimization of the immobilization of milk-clotting proteases to granular bone

Food Biotechnol.

Enzyme technology in the meat and fish industries

Enzyme technology in the meat and fish industries

Cited by (21)

Biomass-derived functional materials as carriers for enzymes: towards sustainable and robust biocatalysts

Bioactives from seafood processing by-products

Chymosin, Pepsins and Other Aspartyl Proteinases: Structures, Functions, Catalytic Mechanism and Milk-Clotting Properties

Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant

Application of chitin- and chitosan-based materials for enzyme immobilizations: A review

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