• Clinical and Experimental Vaccine Research
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• v.11 no.1
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• pp.12-29
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• 2022

Purpose: In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli, the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods: The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results: The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion: This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.

• Korean Chemical Engineering Research
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• v.47 no.6
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• pp.768-774
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• 2009

To improve the productivity of 1,3-propanediol(1,3-PD) with K. pneumoniae DSM4799 using pure glycerol and crude glycerol derived from the biodiesel process, optimizing fermentation conditions was performed by changing environmental factors such as anaerobic/aerobic condition, temperature, glycerol concentration, and pH. When anaerobic conditions were maintained, there was an improved 1,3-PD production compared with that from aerobic/anaerobic 2-stage fermentation. From the results with temperature $26{\sim}37^{\circ}C$, the higher 1,3-PD production yield was observed at $30{\sim}33^{\circ}C$. For an initial glycerol concentration higher than 60 g/L, cell growth and 1,3-PD production were inhibited. When crude glycerol was used, the initial 1,3-PD production appeared to be inhibited. After 48 hr of incubation, however, 1,3-PD production with crude glycerol was even higher than that with pure glycerol, demonstrating the feasibility of 1,3-PD production using crude glycerol as a substrate. Fed-batch fermentation was applied for the high concentration of 1,3-PD without substrate inhibition. By regulating pH at 7 during the fed-batch with glycerol lower than 40 g/L, the yield of 1,3-PD was 25% higher than that without pH regulation(0.56 g/g vs. 0.45 g/g). In conclusion, based on our results, anaerobic conditions, temperature at $30^{\circ}C$, pure or crude glycerol lower than 40 g/L, and pH regulation at 7 were the optimized conditions for 1,3-PD production using K. pneumoniae DSM4799, making it more feasible to produce 1,3-PD at higher concentration and a lower price.

• Journal of Korean Society of Water and Wastewater
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• v.27 no.6
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• pp.689-701
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• 2013

Bacterial growth and corresponding consumption of carbon and phosphorus were examined in which tap water samples containing a very low concentration of free chlorine were supplemented with organic carbon and/or phosphorus. The experiments were performed in a fed-batch mode under a controlled temperature of $20^{\circ}C$. In the phosphorus alone-added water, there was no significant increase in bacterial numbers measured as heterotrophic plate count (HPC) in the bulk water. However, bacterial growth was stimulated by the addition of carbon (e.g., bulk HPC levels increased to $10^3CFU/mL$) and further stimulated by the combined addition of carbon and phosphorus (e.g., bulk HPC to $10^5CFU/mL$). The same effects were observed in biofilm HPC and biomass formed on polyethylene (PE) slide surfaces. In the water where organic carbon and phosphorus were added together, the highest biofilm HPC and biomass (measured as extracellular polymeric substance components) densities were observed which were $7.6{\times}10^5CFU/cm^2$ and $5.3{\mu}g/cm^2$, respectively. In addition to the bacterial growth, additions of organic carbon and/or phosphorus resulted in different bacterial carbon-to-phosphorus (C/P) consumption ratios. Compared to a typical bacterial C/P consumption ratio of 100:1, a higher C/P ratio (590:1) occurred in the carbon alone-added water, while a lower ratio (40:1) in phosphorus alone-added water. Comparative value (80:1) of C/P ratio was also observed in the water where organic carbon and phosphorus were added together. At the given experimental conditions, bacterial growth was deemed to be more sensitive to microbially available organic carbon than phosphorus. The effect of phosphorus addition, which resulted in a lower C/P consumption ratio, seemed to be tightly associated with the presence of microbially available organic carbon. These results suggested that the control of extrinsic carbon influx seemed to be more important to minimize bacterial regrowth in drinking water system, since even low content of phosphorus naturally occurring in drinking water was enough to allow a bacterial growth.

• KSBB Journal
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• v.21 no.3
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• pp.204-211
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• 2006

For the production of the recombinant human interferon-gamma(rhIFN-${\gamma}$) in Escherichia coli, human glucagon and ferritin heavy chain were used as fusion partners. Even though rhIFN-${\gamma}$ is expressed as an inclusion body form in E. coli because of strong hydrophobicity of itself, over 50% of fused rhIFN-${\gamma}$ was expressed as soluble form in E. coli $Origami^{TM}$(DE3) harboring pT7FH(HE)-IFN-${\gamma}$ which encodes ferritin heavy chain-fused rhIFN-${\gamma}$. In the case of using glucagon-ferritin heavy chain hybrid mutant as a fusion partner, 6X His-tag was additionally introduced to N-terminus of GFHM(HE)-IFN-${\gamma}$ for enhancing purification yields of rhIFN-${\gamma}$. Fusion protein HGFHM(HE)-IFN-${\gamma}$ with two 6X His-tag was more effectively bound to Ni-NTA agarose bead than GFHM(HE)-IFN-${\gamma}$ with a 6X His-tag. rhIFN-${\gamma}$ was completely purified from enterokinase-treated HGFHM(HE)-IFN-${\gamma}$ by Ni-NTA affinity column. For high-level production of rhIFN-${\gamma}$, glucose was used as the sole carbon source with simple exponential feeding rate($2.4{\sim}7.2g/h$) in fed-batch process. The effective lactose concentration for the expression of the rhIFN-${\gamma}$ was $10{\sim}20mM$. Under the fed-batch culture conditions, rhIFN-${\gamma}$ production yield reached 11 g DCW/L for 6 hours after lactose induction.

• Journal of Korean Society of Environmental Engineers
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• v.36 no.8
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• pp.561-569
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• 2014

This study was carried out to get the characteristics of anaerobic digestion for chemical/biological sludge produced from municipal sewage treatment plant for phosphorus. Anaerobic mesophilic batch tests showed that the ultimate biodegradability of waste activated sludge showed 31%, PACl sludge 24%, Alum sludge 26%, respectively. At the S/I 1.0, 75% of total biodegradable volatile solids (TBVS) of waste activated sludge was degraded with an initial rapid decay coefficient, k1 of $0.1129day^{-1}$ and 74% of TBVS of PACl sludge with k1 of $0.0998day^{-1}$, and 76% of TBVS of Alum sludge with k1 of $0.1091day^{-1}$ for 20 days. During the operation of SCFMRs, the 3 reactor (Control, PACl, Alum) pH maintained 6.7~7.0 and the reactor alkalinity maintained 1,800~ 2,200 mg/L as $CaCO_3$. The average biogas production rates of SCFMRs fed with PACl sludge and Alum sludge were 0.089 v/v-d and 0.091 v/v-d, respectively, which was 27~28% lower than that of the control (0.124 v/v-d) at an HRT (hydraulic retention times) of 20 days. And the methane content during the operation ranged 70~76% in 3 reactor. The average TVS removal efficiency of SCFMRs fed with PACl sludge and Alum sludge were 19.6% and 19.9%, respectively, at an HRT of 20 days, which showed 4% lower than that of the control (23.8%). The average BVS removal efficiency of SCFMRs fed with PACl sludge and Alum sludge were 25.8% and 26.9%, respectively, at an HRT of 20 days, which was 8~9% lower than that of the control (34.5%).

• Journal of the East Asian Society of Dietary Life
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• v.26 no.1
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• pp.88-98
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• 2016

The pharmacological effects of ginseng berry have been known to improve psychological function, immune activities, cardiovascular conditions, and certain cancers. It is also known that fermentation improves the bioavailability of human beneficial natural materials. Accordingly, we investigated the optimal fermentation conditions of ginseng berry extract with strain isolated from conventional foods. We also analyzed the fermentation product and its antioxidant activity. The bacterium isolated from fermented kimchi was identified as Lactobacillus sp. strain KYH. To optimize the process, fermentation was performed in a 5 L fermenter containing 3 L of ginseng berry extract at 200 rpm for 72 hr. Under optimized conditions, batch and fed-batch fermentations were performed. After fermentation, organic acids, amino acids, sugars, ginsenosides, and antioxidant activity were evaluated. The optimum fermentation conditions were determined as pH 7.0 and a temperature of $30^{\circ}C$, respectively. After fermentation, the amounts and compositions of organic acids, amino acids, sugars, ginsenosides, and antioxidant activity were altered. In comparing the distribution of ginsenosides with that before fermentation, the ginsenoside Re was a major product. However, amounts of ginsenosides Rb1, Rc, and Rd were reduced, whereas amounts of ginsenosides Rh1 and Rh2 increased. Total phenol content increased to 43.8%, whereas flavonoid content decreased to 19.8%. The DPPH radical scavenging activity and total antioxidant activity increased to 27.2 and 19.4%, respectively.

• KSBB Journal
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• v.22 no.1
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• pp.16-21
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• 2007

Although Energy demands of modern society increase rapidly, current energy would be exhausted shortly. Therefore development of bio-ethanol production process from cellulose containing materials was extremly demanded. Therefore development of highly functional cellulase is requisite for this purpose. In this study cellobio-hydrolase (CBH1) gene from Trichorderma reesei was used to increase cellulase activity by directed evolution and highly functional cellobio-hydrolase was obtained and characterized.

• Journal of Life Science
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• v.9 no.2
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• pp.153-159
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• 1999

To express constitutively the inulinase gene (INUl) of Kluyveromyces marxianus in Saccharomyces cerevisiae, three yeast promoters such as GAPDH, ADH1 and ENO1 were connected upstream of INUl. The resulting plasmids, pYIGP, pADHl-INU, and pENO-INU were introduced to S. cerevisiae SEY2102 host strain, respectively, and then each transformants were selected by staining of colonies on sucrose-agar plate. When the yeast transformants were cultivated on 2$\%$ dextrose media, the total expression levels of inulinase reached to 1.11 unit/mL, 0.88 unit/mL, and 0.69 unit/mL for respective GAPDH, ADH1, and ENO1 promoter systems. On 4% dextrose media, however, the inulinase activities were observed at 2.00 unit/mL for pYIGP, 0.71 unit/mL for pADH1-INU, and 1.40 unit/mL for pENO-INU. This result indicates that the constitutive expression of INUl was significantly affected by the initial concentration of dextrose and the promoter strength was in the order GAPDH, ENO1, and ADH1 promoter at high dextrose concentration. Taking into account the plasmid stability, however, it is suggested that the ENO1 promoter system is more suitable for the INU1 expression on high dextrose medium or in the fed-batch cultivation accumulating ethanol at high level.

• Korean Journal of Organic Agriculture
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• v.24 no.3
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• pp.453-463
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• 2016

This study was undertaken to investigate the effects of dicarboxylic acid supplementation, as replacement antibiotics, of on in vitro ruminal parameters and milk yield and milk composition in lactating cows. in vitro treatments were 1) Con (4 g of basal diet), 2) CM (4 g of basal diet + 0.05 ml of monensin), 3) CR (4 g of basal diet + 0.1 ml of dicarboxylic acid) and in vivo treatments were 1) Con (25 kg of basal diet/head/day), and 2) CR (25 kg of basal diet + 5 g of dicarboxylic acid/head/day), respectively. A total 10 lactating dairy cows ($649{\pm}19kg$ average body weight, $99{\pm}65$ average milking days) were divided in to two groups according to mean milk yield and number of days of postpartum. The cows fed a basal diet during adaptation (2 wk) and experimental diets during the treatment periods (4 wk). In the first in vitro experiment, there were no statistical differences between treatments in pH, gas production, and ammonia-N and lactic acid concentration during incubation. However, dry matter digestibility was significantly higher in CR treatment compared to control or CM treatment (P<0.05). Total VFA was tended to higher in CR treatment than those of control and CM treatment (P>0.05). In the second experiment, milk yield was significantly higher in treatment (40.39 kg) compared to control (35.19 kg), (P<0.05). Milk composition and MUN were not changed by dietary supplementing dicarboxylic acid. Therefore the present results reporting that supplementation of dicarboxylic acid might enhance the stabilization of ruminal fermentation and increase the milk yield of lactating cows.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.4
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• pp.479-485
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• 2017

Objective: The effects of particle size of processed barley grain, enzyme addition and microwave treatment on in vitro dry matter (DM) disappearance (DMD), gas production and fermentation pH were investigated for feedlot cattle. Methods: Rumen fluid from four fistulated feedlot cattle fed a diet of 860 dry-rolled barley grain, 90 maize silage and 50 supplement g/kg DM was used as inoculum in 3 batch culture in vitro studies. In Experiment 1, dry-rolled barley and barley ground through a 1-, 2-, or 4-mm screen were used to obtain four substrates differing in particle size. In Experiment 2, cellulase enzyme (ENZ) from Acremonium cellulolyticus Y-94 was added to dry-rolled and ground barley (2-mm) at 0, 0.1, 0.5, 1, and 2 mg/g, while Experiment 3 examined the interactions between microwaving (0, 30, and 60 s microwaving) and ENZ addition (0, 1, and 2 mg/g) using dry-rolled barley and 2-mm ground barley. Results: In Experiment 1, decreasing particle size increased DMD and gas production, and decreased fermentation pH (p<0.01). The DMD (g/kg DM) of the dry-rolled barley after 24 h incubation was considerably lower (p<0.05) than that of the ground barley (119.1 dry-rolled barley versus 284.8 for 4-mm, 341.7 for 2-mm; and 358.6 for 1-mm). In Experiment 2, addition of ENZ to dry-rolled barley increased DMD (p<0.01) and tended to increase (p = 0.09) gas production and decreased (p<0.01) fermentation pH, but these variables were not affected by ENZ addition to ground barley. In Experiment 3, there were no interactions between microwaving and ENZ addition after microwaving for any of the variables. Microwaving had minimal effects (except decreased fermentation pH), but consistent with Experiment 2, ENZ addition increased (p<0.01) DMD and gas production, and decreased (p<0.05) fermentation pH of dry-rolled barley, but not ground barley. Conclusion: We conclude that cellulase enzymes can be used to increase the rumen disappearance of barley grain when it is coarsely processed as in the case of dry-rolled barley. However, microwaving of barley grain offered no further improvements in ruminal fermentation of barley grain.