Purification and characterization of pancreatic elastase from Atlantic cod (Gadus morhua)

Gildberg, Asbjørn; Øverbø, Kersti

doi:10.1016/0305-0491(90)90122-a

Cited by 38 publications

(24 citation statements)

References 14 publications

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“…Common to the two fish trypsins is also a more hydrophilic surface and higher overall hydrophilicity, which are also observed for cold-adapted subtilisin (Davail et al, 1994), a-amylase (Feller et al, 1992), Antarctic fish trypsin (Genicot et al, 1996) and cod elastase (Gilberg et al, 1990). The effect of increased hydrophilicity is proposed to be improved solvent interactions and reduced compactness of the molecule (Davail et al, 1994).…”

Section: Comparison To Anionic Salmon and Bovine Trypsinmentioning

confidence: 83%

Structure of a Non-psychrophilic Trypsin from a Cold-Adapted Fish Species

1998

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The crystal structure of cationic trypsin (CST) from the Atlantic salmon (Salmo salar) has been refined at 1.70 ,A, resolution. The crystals are orthorhombic, belong to space group P212121, with lattice parameters a = 65.91, b = 83.11 and c = 154.79 ,~,, and comprise four molecules per asymmetric unit. The structure was solved by molecular replacement with AMoRe and refined with X-PLOR to an R value of 17.4% and Rfree of 21.5% for reflections IFI > 30"F between 8.0 and 1.7 A resolution. The four non-crystallographic symmetry (NCS) related molecules in the asymmetric unit display r.m.s, deviations in the range 0.31-0.74,~ for main-chain atoms, with the largest differences confined to two loops. One of these is the calcium-binding loop where the electrondensity indicates a calcium ion for only one of the four molecules. In order to find structural rationalizations for the observed difference in thermostability and catalytic efficiency of CST, anionic salmon trypsin (AST) and bovine trypsin (BT), the three structures have been extensively compared. The largest deviations for the superimposed structures occur in the surface loops and particularly in the so-called 'autolysis loop'. Both the salmon enzymes possess a high methionine content, lower overall hydrophobicity and enhanced surface hydrophilicity, compared with BT. These properties have so far been correlated to cold-adaptation features, while in this work it is shown that the non-psychrophilic cationic salmon trypsin shares these features with the psychrophilic anionic salmon trypsin.

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Section: Comparison To Anionic Salmon and Bovine Trypsinmentioning

confidence: 83%

Structure of a Non-psychrophilic Trypsin from a Cold-Adapted Fish Species

1998

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“…Kafienah et al (1998) isolated and purified human neutrophil elastase with the ability to cleave collagen type I which is resistant to attack by most proteolytic enzymes. Elastase has been isolated from marine and fresh water fish species (Cohen et al, 1981;Clark et al, 1985;Asgeirsson and Bjarnason, 1993;Gildberg and Øverbø, 1990;Raa and Walther, 1989). In some situations, collagen is considered to be a poor substrate for collagenase, so that the initiation of collagen breakdown is inhibited.…”

Section: Collagenase and Collagenolytic Enzymesmentioning

confidence: 99%

Extraction and Purification of Collagenase Enzymes: A Critical Review

Daboor¹,

Budge²,

Ghaly

et al. 2010

American Journal of Biochemistry and Biotechnology

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Problem statement: Enzymes have vital roles in several industrial processes (foods, cosmetics, nutraceuticals and pharmaceuticals) due to their highly selective nature and high activity at very low concentrations. Recent efforts to identify new sources of useful enzymes have been concentrated on the marine environment because of the potential to make use of processing wastes. About 35-50% of the mass of the fish caught is a waste that is disposed off at sea or in landfills. The extraction of enzymes from fish processing waste can reduce environment problems and improve the economics of the fish industry. Collagenases are a group of enzymes that can be extracted from fish waste. Approach: Comprehensive reviews of the literature on the extraction, purification, characterization and use of collagenases was carried out. Results: Collagenases have different molecular weights based on their types and sources. They have the ability to break down the peptide bonds in collagen at physiological pH. They are classified into two types: serine and metallocollagenase. Collagenolytic activities have been shown at a wide range of temperatures (20-40°C) and pH (6-8). Many activators can be used to achive collagenase activity including 4-Aminophenylmercuric Acetate (APMA), trypsin, potassium or sodium thiocyanate, iodoacetamide and potassium iodide. Dithiothreitol (DTT), mercaptoethanol, ethylendiaminetetracetic acid, ophenanthroline and cysteine inactivate the enzyme. Collagenases enzymes can be extracted with a variety of techniques using different buffering systems (tris-HCl, sodium bicarbonate, calcium chloride and cacodylate). All techniques involve the use of ammonium sulphate fractionation and centrifugation to precipitate the enzyme. Collagenases are normally purified using chromatographic techniques such as gel-filtration, ion-exchange and affinity column chromatography. Collagenase can be assayed with a number of methods, including: colorimetric absorbance, viscometry, radioactivity and fluorescence spectroscopy. Collagenases are partly responsible for toughness in red meats and are used as tenderizers in food industry, have application in the fur and hide tanning to ensure uniform dying of leather, used in medicine to treat burns and ulcers, eliminate scar tissues, transplantation of organs. Conclusion: Understanding of the nature of the enzymes and identifying the most suitable resources and the methods for their extraction and purification will have significant impact on the fish processing, food and medical industries.

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“…Trypsin activity was determined with the substrate alpha-N-benzoyl-dl-arginine-pnitroanilide (BAPNA) using a method modified by Gildberg and Øverbø [19]. BAPNA was used in the assay at a final concentration of 0.85 mM in 100 mM buffer Tris-HCl pH 8.20.…”

Section: Enzymes Quantificationmentioning

confidence: 99%