• Korean Journal of Food Science and Technology
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• v.31 no.2
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• pp.458-464
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• 1999

The value of lysozyme as a natural food preservative is continuously increased due to its unique antimicrobial activity. To determine the optimum separation concentration among the various hen egg white protein (HEWP) concentrations (0.25, 0.5, 1.0, w/v), protein concentrations, lysozyme concentrations, specific activities (SA), and purification factors of prefiltered solution (PFS) and PM30 permeate solution (PMS) were compared. The purity of lysozyme separated at each step was analyzed and confirmed by gel permeation chromatography and electrophoresis. The fouling deposits on membrane were observed by SEM. The non-enzymatic proteins were removed over 99% by ultrafiltration (UF). The increased feed concentration did not contribute to the increase of SA. SA of PMS was 18 to 31 times higher than that of PFS. The optimum feed concentration was decided as 0.25% based on SA and purification factor. The non-enzymatic region of gel chromatogram was proved to be ovalbumin. The thickness of deposit on the UF membrane was approximately $0.9{\mu}m$ and removed by cleaning with 0.1 N NaOH. Therefore, UF using PM30 membrane was very effective to separate the antimicrobial lysozyme from various HEWPs.

• Journal of fish pathology
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• v.18 no.1
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• pp.59-66
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• 2005

In the present paper, the immunostimulatory effects of Korean mistletoe (Viscum album Coloratum) on the non-specific immune responses of Japanese eel (Anguilla japonica) were examined. Eel were innoculated with mistletoe, Freund's complete adjuvant (FCA), or phosphate-buffered saline (PBS) as a control into their peritoneal cavities. The number of nitrobule tetrazolium (NBT)-positive cells in the head kidney of fish was significantly increased by the second day post-injection of mistletoe. ROI products were more enhanced in mistletoe-injected fish kidney leucocytes than in FCA-injected ones. The level of lysozyme activity detected in the serum of fish 2 days after injection with mistletoe was also significantly higher than that found in the serum of the control fish. The appropriate concentration of mistletoe to induce the highest level of serum lysozyme activity was revealed to 1000${\mu}g$/200 g of fish. In phagocytic activity assay, mistletoe-sensitized eel kidney phagocytes captured more zymosan than did the control fish. Korean mistletoe appeared to be a good activator of the non-specific immune responses of Japanese eel.

• Molecular & Cellular Toxicology
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• v.5 no.4
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• pp.273-282
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• 2009

Cadmium (Cd) and tributyltin (TBT) are common contaminants of marine and freshwater ecosystems, and can induce the formation of reactive oxygen species (ROS). These ROS can, in turn, cause oxidative stress. In the present study, we investigated time-related effects of Cd (0.05 and 0.1 ppm) and TBT (5 and 10 ppb) treatment on antioxidant enzyme activity, i.e., the activity of superoxide dismutase (SOD) and catalase (CAT) in the gills and digestive glands of the ark shell, Scapharca broughtonii. In addition, hydrogen peroxide ($H_2O_2$) concentrations, lysozyme activity, and glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels were measured in the hemolymph. We found that Cd and TBT treatment significantly increased antioxidant enzyme mRNA expression and activity in the digestive glands and gills in a time-dependent manner. In response to the Cd and TBT treatments, antioxidant enzymes mRNA expression and activity increased up to day 5 in the digestive glands and then decreased by day 7. In the gills, antioxidant enzymes mRNA expression and activity increased up to day 3 and then decreased by day 5. Likewise, $H_2O_2$ concentrations significantly increased up to day 5 and then decreased by day 7. Finally, lysozyme activity decreased during the experimental period, whereas GOT and GPT levels were significantly increased in a time-dependent manner. These results suggest that antioxidant enzymes play an important role in decreasing ROS levels and oxidative stress in ark shells exposed to Cd and TBT.

• BMB Reports
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• v.40 no.4
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• pp.539-546
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• 2007

The lysozymes encoded by bacteriophage T7 and K11 are both bifunctional enzymes sharing an extensive sequence homology (75%). The constructions of chimeric lysozymes were carried out by swapping the N-terminal and C-terminal domains between phage T7 and K11 lysozymes. This technique generated two chimeras, T7K11-lysozyme (N-terminal T7 domain and C-terminal K11 domain) and K11T7-lysozyme (N-terminal K11 domain and C-terminal T7 domain), which are both enzymatically active. The amidase activity of T7K11-lysozyme is comparable with the parental enzymes while K11T7-lysozyme exhibits an activity that is approximately 45% greater than the wild-type lysozymes. Moreover, these chimeric constructs have optimum pH of 7.2-7.4 similar to the parental lysozymes but exhibit greater thermal stabilities. On the other hand, the chimeras inhibit transcription comparable with the parental lysozymes depending on the source of their N-terminals. Taken together, our results indicated that domain swapping technique localizes the N-terminal region as the domain responsible for the transcription inhibition specificity of the wild type T7 and K11 lysozymes. Furthermore, we were able to develop a simple and rapid purification scheme in purifying both the wild-type and chimeric lysozymes.

• Journal of Life Science
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• v.27 no.10
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• pp.1145-1151
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• 2017

In this study, we report on a simple, efficient method for obtaining penicillin G amidase (PGA) from recombinant Escherichia coli using a formulation mixed with detergent and lysozyme. Research was conducted on the extraction efficiency of PGA from the periplasmic space in cells in terms of the type of detergent, detergent concentration, pH, reaction time, and temperature of permeabilization. The extraction yield of PGA in the formulated surfactant/lysozyme treatment was increased by approximately (55-65 U/ml) in comparison with that in the single surfactant treatment. The released PGA solution was concentrated and exchanged with buffer using an ultrafiltration (U/F) system. The yields of diatomite filtration, membrane filtration (M/F), and U/F were 69.7%, 93.8%, and 77.3%, respectively. A total of 212 KU of PGA was recovered. At the 25-L culture scale, the overall yield of extraction using the mixed surfactant/lysozyme method was 49.2%. The specific activity of extracted PGA was 11 U/mg in protein. The concentrated PGA solution was immobilized on microporous silica beads without further purification of PGA. The total immobilization yield of PGA on the resin was 48.7%, while the enzyme activity was 101 U/g. The immobilized PGA was successfully used to produce 6-APA from penicillin G. Our results indicated that a simple extraction method from periplasmic space in E. coli may be used for the commercial scale production of ${\beta}-lactam$ antibiotics using immobilized PGA.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.5
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• pp.726-731
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• 2003

The aim of this research was to identify candidate probiotic lactic bacteria among indigenous dadih lactic isolates. Dadih is an Indonesian traditional fermented milk of West Sumatra which is fermented naturally. Viability of the strain is critical in determining the capacity of lactic bacteria to induce immune stimulation as well as to colonize in the intestinal tract. Therefore, LAB are proposed to exert health promoting or probiotic effects in human, such as inhibition of pathogenic microflora, antimutagenic, and the reduction of cholesterol levels. This manuscript reports in vitro probiotic properties of indigenous dadih lactic bacteria, especially some important colonization factors in GI tract, such as lysozyme, acid and bile tolerance. Bile Salt Hydrolase (BSH) activity, spectrum of bacteriocin, and antimutagenic activity of bacterial cells were also assessed. Twenty dadih lactic isolates were screened further for their tolerance to low pH, at pH 2 and 3 as well as their bile tolerance. There were ten isolates classified as acid and bile acid tolerant, and further screened for lysozyme tolerance, BSH activity. The spectrum of bacteriocin activity of isolates was assayed using cell-free neutralized supernatants by agar spot test against variety of pathogens. Lc. lactis subsp. lactis IS-10285, IS-7386, IS-16183, IS-11857 and IS-29862, L. brevis IS-27560, IS-26958 and IS-23427, Leu.mesen.mesenteroides IS-27526, and L. casei IS-7257 each has good survival rate at low pH values and in the presence of lysozyme, and short lag time in the presence of 0.3 % oxgall. Lc. lactis subsp. lactis IS-11857 and IS-29862 each has high BHS activity, Lc. lactis subsp. lactis IS-10285 and IS-16183 each had a positive spectrum of bacteriocin activity against E. coli 3301 and Lysteria monocytogenes ATCC 19112, while L. brevis IS-26958 has high BHS activity as well as positive spectrum of bacteriocin against E. coli 3301, Lysteria monocytogenes ATCC 19112, and S. aureus IFO 3060. All of the ten dadih lactic strains performed in vitro acid and bile tolerance, indicating a possibility to reach the intestine alive, and display probiotic activities.

• Journal of fish pathology
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• v.21 no.3
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• pp.247-257
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• 2008

To understand the activity of non-specific defence factors in cultured Sebastes, the antibacterial effect of the serum, skin mucus and homogenate of various organs from cultured oblong rockfish (Sebastes oblongus) and rockfish(Sebastes schlegeli) against pathogenic bacteria, Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and Streptococcus sp. was compared with that of flounder(Paralichthys olivaceus) and seabass(Leteolabrax japonicus). And the activities of proteolytic enzyme, chitinolytic enzyme and haemolycin as non-specific defence factor were investigated on the oblong rockfish and rockfish. Samples from oblong rockfish showed the highest antibacterial activity by lysoplate assay on agar plate mixed with pathogens, followed in descending order by rockfish, seabass, and flounder. Turbidimetric assay was carried to evaluate the lysozyme activity of fish samples against lyophilized cells of Micrococcus lysodeiktikus. The serum, kidney, liver, stomach, intestine and eyeball of oblong rockfish and the mucus and gill of rockfish appeared to have the highest lysozyme activity among the fish strains investigated. All samples except skin mucus, liver, and eyeball of oblong rockfish and rockfish showed proteolytic enzyme activity. Chitinolytic enzyme activity was showed in random sampling and haemolytic activity was remarkable in oblong rockfish. Therefore, Sebastes strain was proved to have effective defense mechanisms based on the antibacterial activities, and lysozyme, proteolytic enzyme, chitinolytic enzyme, and haemolycin were considered to act as the non-specific defence factor of Sebastes.

• Fisheries and Aquatic Sciences
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• v.6 no.2
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• pp.74-80
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• 2003

Two cellular biomarker parameters of the farmed Pacific oyster Crassostrea gigas were studied in vivo and in vitro after exposure to concentrations of polychlorinated biphenyls in terms of neural red uptake (NRU) and lysozyme activity. The oysters exposed in vivo to the xenobiotic concentrations, 0, 30, 90, and 180 ng/g for 14 days, enhanced hemocyte NRU with occasional significant differences (P<0.05), depending on the chemical concentration and duration. An adverse tendency was manifest in the lysozyme activities both in the hemocyte and serum of the oyster treated with the chemical in a same manner, rendering these two cellular parameters as biomarker candidates against the chemical. The oysters exposed in vitro to the chemical concentrations, 0, 1, 5, 10, 100, 1,000, and 10,000 ng/g for 24 hrs at $10^{\circ}C$ showed a similar tendancy as those exposed in vivo to the chemical. Unlike in vivo response, however, the in vitro NRU was first influenced by very low concentration of the chemical. In in vitro results, marked but not significant increase of hemocyte NRU was noticed at the chemical concentration of 5 ng/g, where the value was almost as high as those exposed to higher chemical concentrations, up to 10,000 ng/g. An unusual result was observed in the in vitro lysozyme activity of hemocyte in which significant decrease was first noticed at the chemical concentration of 100 ng/g.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.1
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• pp.23-30
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• 2014

The present study investigated the effects of dietary Edwardsiella tarda (E. tarda) specific bacteriophage (phage) and Bacillus subtilis (B. subtilis) mixture on innate immune responses and antibacterial activity of Nile tilapia, Oreochromis niloticus. In a dietary experiment, tilapia were fed the control diet (C), a phage-only supplemented diet (P), a B. subtilis only supplemented diet (B), or a B. subtilis and phage mixed diet (B+P). A respiratory burst and significant increase in lysozyme activity (P<0.05) were noted in the B+P group, as compared to other groups after 4 days of feeding. The B group showed a significant (P<0.05) increase in respiratory burst and lysozyme activity versus the C and P groups, whereas no significant increases (P<0.05) were observed in the P and C groups. $ACH_{50}$ was significantly up-regulated in the B+P group versus other groups after 8 days of feeding (P<0.05). In vivo antibacterial activity was significantly enhanced in the B+P fed group, as compared to other groups (P<0.05) after 7 days of E. tarda challenge. A significant (P<0.05) increase in antibacterial activity was seen in the B group, as compared to C or P groups after 14 days of feeding. These results suggest that a B. subtilis and phage mixture could be utilized as an alternative to antibiotics in the control of fish diseases caused by E. tarda.

• Journal of fish pathology
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• v.22 no.3
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• pp.293-303
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• 2009

Effects of stress on the low salinity stress were examined in the pacific abalone Haliotis discus discus. Changes in survival rate, hemolymph count, antioxidant enzyme activities (catalase: CAT and superoxide dismutase: SOD), respiratory burst activity, phenoloxidase activity, lysozyme activity and expression of heat shock protein 70 (HSP70) mRNA were measured 0, 3, 6, 12, 24 or 48hours after low salinity treatment with 25, 30, 33 and 35 psu. Survival rates of pacific abalone were 100% at 33 and 35 psu, but 93 and 97% at 25 and 30 psu for 48 hours, respectively. Hemolymph counts decreased in the time elapsed-dependent way at all of the experimental groups. At low salinity, 25 and 30 psu, SOD and CAT activity increased compared to the experimental group of 33 psu. Moreover, respiratory burst activities of the pacific abalone seemed to have no effect on low salinity stress at any experimental group. However, phenoloxidase activity is an important component of the defence against pathogen that was decreased in a reduction of salinity dependent way. Lysozyme activity also immediately reduced at 25 psu experimental group for 48 h. The HSP70 mRNA was weakly expressed at 33 psu, but strongly detectable at 25 psu experimental group. The HSP 70 mRNA expression in gill increased in the time elapsed-dependent way at 25 psu experimental group and then recovered at 48 h. These results suggest that low salinity stress give rise to inhibitory action of immune system as a result of the decrease of phenoloxidase and lysozyme activity in the pacific abalone, especially.