Ágústa Gudmundsdóttir

Atlantic Cod Trypsins: From Basic Research to Practical Applications

Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted group I trypsins, and the recombinant form of cod trypsin Y is the only biochemically characterized member of the novel group III trypsins. Trypsin Y is adapted to lower temperatures than all other presently known trypsins. This review describes the basic characteristics of and practical uses for trypsins of Atlantic cod, as well as those of other organisms. Overexpression of the recombinant forms of cod trypsins I and Y in microorganisms is explained as well as the advantages of using site-directed mutagenesis to increase their stability toward autolysis and thermal inactivation. Trypsins appear to play a key role in the nutrition and development of marine fish. We discuss the potential use of cod trypsins as biomarkers to evaluate the nutritional status of cod larvae and describe the industrial applications of cod trypsin I and other trypsins.

Isolation and characterization of two cDNAs from atlantic cod encoding two distinct psychrophilic elastases

The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50-64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.

Carnocin U149, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various Gram-positive bacteria including Listeria sp.

This paper describes a simple purification method for the purification of carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola UI49. The protocol was also applicable for the isolation of nisin Z, which is a biopreservative produced by Lactococcus lactis SIK-83. The protocol consists of only two purification steps, XAD chromatography and cation exchange chromatography. It is quick, easy, and can be used for large scale purification of these lantibiotics. The bactericidal activity of carnocin UI49 against carnobacteria, lactococci and Listeria was compared with that of nisin Z. The carnobacteria showed similar sensitivity towards carnocin UI49 and nisin. The nisin producing L. lactis strains were very sensitive towards carnocin UI49, while the non-producing L. lactis strains were more sensitive to nisin. The Listeria strains were weakly sensitive to carnocin UI49, lower concentrations of nisin were needed to inhibit growth.

Proteome analysis of abundant proteins in two age groups of early Atlantic cod (Gadus morhua) larvae.

The protein expression profiles of two different age groups of Atlantic cod larvae, at 6 days post hatch (dph) and 24 dph, were compared using 2-dimensional electrophoresis and MALDI-TOF mass spectrometry analysis. The life stage of the cod larvae under study covers the first feeding period, generally characterized by high mortality in cod hatcheries. Despite visible morphological and functional changes in larvae from 6 to 24 dph, the pattern of abundant proteins is largely conserved. Interestingly, more than half of the proteins identified in the present study corresponded to different isoforms of the same proteins. The keratins showed the most pronounced developmental stage specific expression pattern. Type II keratins were more dominant in younger larvae and type I keratins in the older larval group. Four isoforms of the fast skeletal muscle alpha-actin, 3 isoforms of beta-2 tubulin and 2 isoforms of alpha-actin were detected. The different isoforms may be either encoded by different genes or generated by post-translational modifications (PTMs) of the same gene product. The PTMs are largely overlooked by studies based on mRNA detection. Therefore, the proteome approach to understanding the cellular mechanisms underlying fish development is important.

Differential protein expression in early Atlantic cod larvae (Gadus morhua) in response to treatment with probiotic bacteria.

Proteome analysis was used to study the effects of probiotic bacteria treatment on the protein expression in early Atlantic cod (Gadus morhua) larvae. The main focus was on proteins connected to immune function, growth and development. The results demonstrated that none of the identified cod larvae proteins showing up-regulation after administration of probiotic bacteria are known to have a function in immunity. In contrast, several proteins exhibiting down-regulation after exposure to the probiotic bacteria may be related to stress and immune responses. This indicates that the probiotic treated larvae were experiencing a lower level of immune-stimulation than the control group. It is therefore tempting to postulate that the probiotic bacteria mixture used in the present study reduced the environmental stress by inhibiting the growth of pathogenic bacteria. Most of the identified up-regulated proteins in the probiotic group relative to the control may be linked to growth and development. The most pronounced up-regulation of proteins was found in several muscle α-actin isoforms indicating improved growth upon probiotic bacteria treatment.

Atlantic Cod Trypsin Y—Member of a Novel Trypsin Group

Abstract: A unique trypsinogen complementary DNA has been isolated from an Atlantic cod (Gadus morhua) cDNA library. Its predicted amino acid sequence contains 249 residues with a putative polypeptide of 227 residues. The distinctive features of this polypeptide, referred to as trypsin Y, are its low number of hydrogen-bond-forming residues, high content of Met and Pro residues, and lack of one conserved disulfide bond. Alignments show that cod trypsinogen Y has only approximately 45% identity to the two Atlantic cod trypsinogens I and X and most vertebrate trypsinogens. However, it has more than 70% identity to three other fish trypsinogens from two Pleuronectes and an Antarctic Notothenia species. These four trypsinogens share some unique characteristics and form a novel group, here referred to as group III.

Neonatal changes in fatty acid profile of phospholipids in rat heart muscle

The fatty acid composition of rat heart phospholipids was examined during the neonatal and postnatal period. The rats were killed on days 1, 7, 14 and 21 after birth and at the ages of 2 and 6 months. The fatty acyl chain composition of the two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) changed significantly during the first 2 months. In PC there was a marked and immediate increase in stearic acid, a significant but transient increase in arachidonic acid and late increase in linoleic acid content. In PE there was an immediate increase in stearic acid and docosahexaenoic acid, followed by a late increase in linoleic acid content. The observed alterations in fatty acid composition of heart muscle phospholipids resemble changes induced by repeated administration of norepinephrine and subsequent recovery. Neonatal stress and increased cardiac function play an important role in the modification of the fatty acid composition of rat heart muscle phospholipids during early development.

Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I - kinetic parameters, autolysis and thermal stability.

Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k(cat)/K(m)) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 degrees C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.

Cold-adapted and mesophilic brachyurins

Abstract Two different types of brachyurins, termed I and II, have been described in the literature. Within type I there are two subtypes, Ia and Ib. The prototype for the type I brachyurins is Fiddler crab collagenase I. Its coldadapted analogue from Antarctic krill, termed euphaulysin, shares many of its characteristics. Both enzymes are distinguished by their broad substrate specificity as well as the ability to cleave collagen. The precursor form of euphaulysin has been expressed in Pichia pastoris and processed to its fully active form using cod trypsin. A molecular model of euphaulysin, based on the known crystal structure of crab collagenase I, indicates that the core structure of these enzymes is almost identical. As a coldadapted enzyme, euphaulysin has a higher catalytic efficiency than crab collagenase I. It is also more sensitive to thermal inactivation and autolysis. Furthermore, euphaulysin has an increased length of several surface loops compared to crab collagenase I. Extended surface loops have been suggested to play a role in the cold activity of some bacterial enzymes. Sensitivity to autolysis is an important factor which contributes to the thermal instability of euphaulysin. Substitution of a highly exposed residue in the autolysis loop of euphaulysin resulted in an increased stability of the enzyme towards thermal inactivation without altering its catalytic efficiency.

Recombinant Trypsin Y from Atlantic Cod—Properties for Commercial Use

Abstract This paper describes the production of the novel recombinant trypsin Y polypeptide from Atlantic cod (Gadus morhua)in a Pichia pastorisexpression system and gives a characterization of the enzyme. It presents the first biochemical data supplementing the molecular description of the putative group III trypsins. Unexpectedly, the results demonstrate that the recombinant trypsin Y has a dual substrate specificity demonstrating both trypsin and chymotrypsin activities. It shows increasing proteolytic activity at temperatures between 2-21 °C, and it is completely inactivated at 30°C. These properties may be important for the commercial use of recombinant trypsin Y, as other cold-adapted proteolytic enzymes from the Aüantic cod have proven their usefulness in various industrial and medical applications.