The gene encoding the amylolytic enzyme Amo45, originating from a metagenomic project, was retrieved by a consensus primer-based approach for glycoside hydrolase (GH) family 57 enzymes. Family 57 contains mainly uncharacterized proteins similar to archaeal thermoactive amylopullulanases. For characterization of these family members soluble, active enzymes have to be produced in sufficient amounts. Heterologous expression of amo45 in E.coli resulted in low yields of protein, most of which was found in inclusion bodies. To improve protein production and to increase the amount of soluble protein, two different modifications of the gene were applied. The first was fusion to an N-terminal His-tag sequence which increased the yield of protein, but still resulted in high amounts of inclusion bodies. Co-expression with chaperones enhanced the amount of soluble protein 4-fold. An alternative modification was the attachment of a peptide consisting of the amino acid sequence of the mobile-loop of the co-chaperonin GroES of E.coli. This sequence improved the soluble protein production 5-fold compared to His6-Amo45 and additional expression of chaperones was unnecessary.
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In this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infecting Thermus strain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the species Thermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with an Mr or 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His30, Tyr58, His132, and Cys140) that form a Zn2+ binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, ie, T. scotoductus (100%), Thermus thermophilus (100%), and Thermus flavus (99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, ie, Escherichia coli (34%), Serratia marcescens (28%), Pseudomonas fluorescens (13%), and Salmonella enterica serovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception of Deinococcus radiodurans (25%) and Bacillus cereus (15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95 ° C. The optimum temperature range for the enzyme activity is 50 ° C to 78 ° C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl.
Nile tilapia (Oreochromis niloticus) farmed in recirculation aquaculture system (RAS) was filleted and packaged in 100% air and 50% CO2: 50% N2 modified atmosphere (MA) prior to storage at 1˚C and -1˚C for up to 27 days. Fillets were sampled regularly and analyzed for headspace gas composition, sensory and microbial changes. Shelf life varied with apparent relation to storage temperature, package atmosphere and microflora. Pseudomonads were reported as the main spoilage organisms in tilapia fillets during chilled storage conditions. Sensory analysis of cooked samples as well as microbial growth indicated fillets packaged in 100% air had a shelf life of 13-15 days during storage at 1˚C and 20 days at -1˚C. At the end of shelf life in 100% air packaged groups, TVC and pseudomonads counts reached log 7 colony-forming units g-1 in flesh. Whereas in 50% CO2: 50% N2 packaged fillets, the lag phase and generation time of bacteria was extended and recorded counts of <log 4 colony-forming units g-1 up to 27 days of storage at both 1˚C and -1˚C . However, 50% CO2: 50% N2 conditions restricted fillets shelf life to 23 days based on sensory changes mainly fillets color characteristics.
BACKGROUND
Although protein isolates have been proven as a potent raw material for protein hydrolyzate preparation, the fishy odor associated with lipid oxidation is still detected. The remaining hemoglobin (Hb) in protein isolates can effectively induce lipid oxidation, leading to the formation of fishy odor in the resulting hydrolyzate. The aim of this study was to elucidate the impact of Hb with different forms, oxyhaemoglobin (oxy-Hb) and methaemoglobin (met-Hb), on lipid oxidation and the development of fishy odor during hydrolysis of protein isolates.
RESULTS
During hydrolysis of protein isolate up to 120 min, non-haem iron content, peroxide value and thiobarbituric acid reactive substances slightly increased (P <0.05). When oxy-Hb or met-Hb was incorporated, the marked increases in all parameters were observed, especially within the first 60 min of hydrolysis. The higher increases were obtained with the latter, suggesting that met-Hb was more pro-oxidative than oxy-Hb. However, no differences in degree of hydrolysis of all samples were observed (P > 0.05). The marked increases in the b*, ΔE*, ΔC* values, fishy odor / flavor and volatile compounds were also found in the resulting hydrolyzate containing either oxy-Hb or met-Hb.
CONCLUSION
Hb, particularly met-Hb, induced lipid oxidation and the development of a fishy odor / flavor in fish protein hydrolysate. © 2013 Society of Chemical Industry
Chemical compositions and muddy compounds in dorsal and ventral muscles of Nile tilapia and broadhead catfish were comparatively studied. On a dry weight basis, Nile tilapia was rich in protein (93.1–93.8%), whilst broadhead catfish contained protein (55.2–59.5%) and lipid (36.6–42.4%) as the major constituents. Ventral portion had higher lipid or phospholipid contents with coincidentally higher geosmin and / or 2-methylisoborneol (2-MIB) contents. Geosmin was found in mince of Nile tilapia and broadhead catfish at levels of 1.5 and 3.2 μg / kg, respectively. Broadhead catfish mince had 2-MIB at level of 0.8 μg / kg, but no 2-MIB was detected in Nile tilapia counterpart. When pre-washing and alkaline solubilization were applied for preparing protein isolate (PI), lipid and phospholipid contents were lowered with concomitant decrease in geosmin and 2-MIB contents. Protein hydrolysate produced from PI had a lighter color and a lower amount of muddy compounds, compared with that prepared from mince. Therefore, PI from both Nile tilapia and broadhead catfish could serve as the promising proteinaceous material, yielding protein hydrolyzate with the negligible muddy odor and flavor.
To reduce the practice of discarding commercially fished organisms, several measures such as a discard ban and extra allowances on top of landings quotas (“catch quota”) have been proposed by the European Commission. However, for their development and successful implementation, an understanding of discard patterns on a European scale is needed. In this study, we present an inter-national synthesis of discard data collected on board commercial, towed-gear equipped vessels operating under six different national flags spanning from the Baltic to the Mediterranean Seas mainly between 2003 and 2008. We considered discarded species of commercial value such as Atlantic cod (Gadus morhua), haddock (Melanogrammus aeglefinus), European hake (Merluccius merluccius), and European plaice (Pleuronectes platessa). Comparisons of discard per unit effort rates expressed as numbers per hour of fishing revealed that in the Mediterranean Sea minimum size-regulated species such as hake are generally discarded in much lower numbers than elsewhere. For most species examined, variability in discard rates across regions was greater than across fisheries, suggesting that a region-by-region approach to discard reduction would be more relevant. The high uncertainty in discard rate estimates suggests that current sampling regimes should be either expanded or complemented by other data sources, if they are to be used for setting catch quotas.
Contaminants have been analyzed in cod (Gadus morhua) since 1990 as part of the national monitoring program for the environmental conditions in the sea around Iceland. The aim of this study was to determine the temporal trends of persistent organic pollutants (POPs: polychlorinated biphenyls (PCBs), p,p′-Dichlorodiphenyl dichloroethene (p,p′ -DDE), hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), chlordanes (CHLs) and toxaphenes (Tox)) and trace elements (As, Cd, Cu, Hg, Pb, Se and Zn) in cod over the last two decades at two different locations in the Arctic Ocean north of Iceland. The relationship between the contaminant concentrations and biological covariates was also determined. All of the POPs showed decreasing trends but the trace elements showed no clear signs of trend except arsenic which showed an increasing trend and zinc which showed a decreasing trend. The concentration of the POPs were lower or similar in the Icelandic cod compared to cod sampled in Norway, the Barents Sea and in the Baltic Sea, except for HCB which was higher in the Icelandic cod compared to the Norwegian cod. The concentration of the trace elements As, Cu, Hg and Zn were similar in the Icelandic cod compared to cod sampled in Norway and Greenland but the concentration of Cd was higher in the Icelandic cod. The inclusion of the biological covariates was found to be important for the statistical analysis. The POPs had a positive relationship with liver fat content but negative relationship with liver weight. The trace elements had a negative relationship with liver fat and liver weight except As which had positive relationship with liver weight. Only positive relationships were observed between the contaminant concentrations and length.
The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254 nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60 ° C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products.
The main objective of natural resource management is tocreate social and economic value while maintaining sustainability. In this paper, we introduce an enhanced method for simulating high-dimensionaltime series and apply it to Icelandic resource management data. Themethodology can be used in many contexts, but is particularly appropriate for simulating the many complex interactions involved in natural resource management. The simulations can be used to explore the sensitivity of re-source management policies to future changes using an a ffi nity parameter.A ffi nity, qualitatively similar to correlation, is an ordinal measure between –1and +1 that models one's belief how much the future might behave like , ordi ff erent from, the past. The main appeal of the method is its reliance ondata and relative independence from assumptions about that data. In the paper, we apply it on data on Icelandic cod with encouraging results.
Most of the Icelandic cod is caught in bottom trawlers or longliners. These two fishing methods are fundamentally different and have different economic, environmental, and even social effects. In this paper we present a hybrid-simulation framework to assess the impact of changing the ratio between cod quota allocated to vessels with longlines and vessels with bottom trawls. It makes use of conventional bioeconomic models and discrete event modeling and provides a framework for simulating life cycle assessment (LCA) for a cod fishery. The model consists of two submodels, a system dynamics model describing the biological aspect of the fishery and a discrete event model for fishing activities. The model was run multiple times for different quota allocation scenarios and results are presented where different scenarios are presented in the three dimensions of sustainability: environmental, social, and economic. The optimal allocation strategy depends on weighing the three different factors. The results were encouraging first-steps towards a useful modeling method but the study would benefit greatly from better data on fishing activities.