• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2141-2150
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• 2017

This study was conducted to develop a screening method using Colilert-18 and a device for the detection of E. coli from agri-food production environments and fresh vegetables. The specificity and sensitivity of Colilert-18 by temperature ($37^{\circ}C$ and $44^{\circ}C$) were evaluated with 38 E. coli and 78 non-E. coli strains. The false-positive rate was 3.8% (3/78) and 0% (0/78) at $37^{\circ}C$ and $44^{\circ}C$, respectively. The detection limit of E. coli at $37^{\circ}C$ at <1.0 log CFU/250 ml was lower than that at $44^{\circ}C$. The efficiency of the developed device, which comprised an incubator equipped with a UV lamp to detect E. coli in the field, was evaluated by measuring the temperature and UV lamp brightness. The difference between the set temperature and actual temperature of the developed device was about $1.0^{\circ}C$. When applying the developed method and device to various samples, including utensils, gloves, irrigation water, seeds, and vegetables, there were no differences in detection rates of E. coli compared with the Korean Food Code method. For sanitary disposal of culture samples after experiments, the sterilization effect of sodium dichloroisocyanurate (NaDCC) tablets was assessed for use as a substitute for an autoclave. The addition of one tablet of NaDCC per 50 ml was sufficient to kill E. coli cultured in Colilert-18. These results show that the developed protocol and device can efficiently detect E. coli from agri-food production environments and vegetables.

• KSBB Journal
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• v.10 no.1
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• pp.38-45
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• 1995

The expression of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) and trpC promoters in A. oryzae were compared using E. coli lacZ gents fusions. The specific activities of the expressed E. coli $\beta$-galactosidase in A. oryzae transformants containing the A. nidulans gpdA promoter were around 2,000 units per ug of protein. The specific activities of transformants containing the A. nidulans trpC promoter were very low, ranging from 10.5 to 52.3 units per ug of protein. These results showed that the expression of the A. nidulans gpdA promoter in A. oryzae was approximately 70 times greater than the A. nidulans trpC promoter. In western blot analysis, immunoreactive bands of a imlilar molecular weight as the E. coli $\beta$-galactosidase were observed in A. oryzae carrying the gpdA-lacZ fusion and to a lesser intensity in those carrying the tvpC-lacZ fusion. Southern analysis showed that the higher expression of the gpdA-lacZ fusion as compared to the trpC-lacZ fusion was not due a greater number of integrated plasmids.

• Journal of Life Science
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• v.14 no.1
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• pp.121-125
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• 2004

The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at $25^{\circ}C$ with groEL/ES expression improved the activity of CGTase by two fold, compared to $37^{\circ}C$ cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at$25^{\circ}C$, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at $37^{\circ}C$ without chaperone. The amount of soluble CGTase from $25^{\circ}C$ culture with chaperone was 3.5-fold higher than that of $37^{\circ}C$ culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.28-33
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• 1994

In normal cells tau protein is associated with axonal microtubules, whereas in Alxheimer's disease it is immobilized in the somatodendritic compartment of certain nerve cells as a major component of the paired helical filament. As a part of the study to analyze the nature of the paired helical filament (PHF) deposits and some related factors in brain, we have cloned and expressed a human tau gene cDNA in Escherichia coli to obtain the recombinant human tau protein in abundance.

• Food Science of Animal Resources
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• v.32 no.1
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• pp.62-67
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• 2012

This study calculated kinetic parameters of Escherichia coli O157:H7 and developed a probabilistic model to estimate growth probabilities of E. coli O157:H7 on polyethylene cutting boards as a function of temperature and time. The surfaces of polyethylene coupons ($3{\times}5$ cm) were inoculated with E. coli O157:H7 NCCP11142 at 4 Log $CFU/cm^2$. The coupons were stored at 13 to $35^{\circ}C$ for 12 h, and cell counts of E. coli O157:H7 were enumerated on McConkey II with sorbitol agar every 2 h. Kinetic parameters (maximum specific growth rate, Log $CFU/cm^2/h$; lag phase duration, h; lower asymptote, Log $CFU/cm^2$; upper asymptote, Log $CFU/cm^2$) were calculated with the modified Gompertz model. Of 56 combinations (temperature${\times}$time), the combinations that had ${\geq}$0.5 Log $CFU/cm^2$ of bacterial growth were designated with the value of 1, and the combinations that had increases of <0.5 Log $CFU/cm^2$ were given the value 0. These growth response data were fitted to the logistic regression to develop the model predicting probabilities of E. coli O157:H7 growth. Specific growth rate and growth data showed that E. coli O157:H7 cells were grown at $28-35^{\circ}C$, but there were no obvious growth of the pathogen below $25^{\circ}C$. Moreover, the developed probabilistic model showed acceptable performance to calculate growth probability of E. coli O157:H7. Therefore, the results should be useful in determining upper limits of working temperature and time, inhibiting E. coli O157:H7 growth on polyethylene cutting board.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.1045-1048
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• 2007

Two genes, dps encoding decaprenyl diphosphate synthase and dxs encoding 1-deoxy-D-xylulose 5-phosphate synthase, were isolated from Rhizobium radiobacter ATCC 4718. DNA sequencing analysis of the dps and dxs genes revealed an open reading frame of 1,077 bp and 1,920 bp, respectively. The heterologous expression in Escherichia coli BL21(DE3) was carried out in order to identify their functions. Recombinant E. coli BL21(DE3) harboring the dps gene produced $CoQ_{10}$ as well as $CoQ_8$ and $CoQ_9$, whereas E. coli harboring only the dxs gene produced more $CoQ_8$ compared with the wild-type E. coli. Additionally, the coexpression of dps and dxs genes in E. coli was carried out. The recombinant E. coli harboring only the dps gene produced $0.21{\pm}0.04\;mg/l$ of $CoQ_{10}$, whereas the coexpressed E. coli with dps and dxs genes produced $0.37{\pm}0.07\;mg/l$ of $CoQ_{10}$. HPLC analysis also showed that the $CoQ_{10}$ fraction (100% of the total CoQs distribution) was increased from $15.86{\pm}0.66%$ (only dps) to $29.78{\pm}1.80%$ (dps and dxs).

• Journal of Periodontal and Implant Science
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• v.38 no.1
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• pp.41-50
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• 2008

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

• Food Engineering Progress
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• v.14 no.3
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• pp.202-207
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• 2010

Low-pressure plasmas (LPPs) were generated with different gases such as air, oxygen and nitrogen, and their inactivation effects against Escherichia coli were compared in order to evaluate the potential as a non-thermal microbial disinfection technology. Homogeneous plasmas were generated under low pressure below 1 Torr at gas flow rate of 350 mL/min regardless the types of gases. Temperature increases by LPPs were not detrimental showing less than ${10^{\circ}C}$ and ${25^{\circ}C}$ increases after 5 and 10 min treatments, respectively. The smallest temperature increase was observed with air LPP, and followed by oxygen and nitrogen LPPs. More than 5 log reduction in E. coli was achieved by 5 min LPP treatment but the destruction effect was retarded afterward. The LPP inactivation was represented by a iphasic first order reaction kinetics. The highest inactivation rate constant was achieved in air LPP and followed by oxygen and nitrogen LPPs. The small D-values of the LPP also supported its potentialities as a non-thermal food surface disinfection technology in addition to the substantial microbial reduction of more than 5 logs.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1835-1841
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• 2015

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

• Microbiology and Biotechnology Letters
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• v.15 no.6
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• pp.436-440
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• 1987

Pullulanase gene (pul) of Klebsiella pneumoniae NFB-320 which was cloned previously in Escherichia coli with plasmid pBR322. The gene was analyzed with various restriction enzymes. The cloned gene was contained within n 10 kb BamHI DNA fragment. We constructed the restriction map of the hybrid plasmid pYKL451. The optimum temperatures for pullulanases produced in E. coli (pYKL451) and K. pneumoniae NFB-320 were almost the same, 50-55 $^{\circ}C$. The optimum pHs for the reaction of the enzymes produced by E. coli (pYKL451) and K. pneumoniae NFB-320 was 6.0. Both enzyme preparations were stable under the range of pH 5.0 to 10.0 when those were kept at 40 $^{\circ}C$ for 90 min and were stable until 40 $^{\circ}C$ when allowed to stand for 1hr at various temperatures.