• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.273-278
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• 2002

Two-step fed-batch fermentations were carried out to overproduce Bacillus licheniformis maltogenic amylase (BLMA) in recombinant Escherichia coli. The first step was to increase the cell mass by controlling the feeding of a glucose solution, while the second step was designed to improve the amylase expression efficiency by supplementing organic nitrogen sources. The linear gradient feeding method was successfully adopted to maintain the glucose concentration below 0.2 g/l during the fed-batch mode, as effectively minimizing acetic acid formation. When the dissolved oxygen (DO) level became limiting, an accumulation of acetic acid and drastic decrease in specific BLMA productivity were observed. Glucose and organic nitrogen sources consisting of yeast extract and casein hydrolysate were simultaneously supplied in the pH-stat mode to further increase the specific BLMA expression efficiency. An organic nitrogen source consisting of 200 g/1 yeast extract and 100 g/1 casein hydrolysate was found to be the best among the various combinations tested. The feeding of an organic nitrogen source in the second-step fed-batch period was highly beneficial in enhancing the BLMA production. The optimized two-step fed-batch culture resulted in 78 g/l maximum dry cell mass and 443 U/ml maximum BLMA activity, corresponding to 1.5-fold increase in the dry cell mass and 3.7-fold enhancement in BLMA production, compared with the simple fed-batch fermentation.

• Journal of Industrial Technology
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• v.39 no.1
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• pp.15-19
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• 2019

This work focused on characterization of the starch degradation activity from extremophile strain Deinococcus geothermalis. Glucoamylase gene from D. geothermalis was cloned and overexpressed by pET-21a vector using E. coli BL21 (DE3). In order to characterize starch degrading activity of recombinant glucoamylase, enzyme was purified using HisPur Ni-NTA column. The recombinant glucoamylase from D. geothermalis exhibited the optimum temperature as $45^{\circ}C$ for starch degradation activity. And highly acido-stable starch degrading activity was shown at pH 2. For further optimization of starch degrading activity with metal ion, various metal ions ($AgCl_2$, $HgCl_2$, $MnSO_4{\cdot}4H_2O$, $CoCl_2{\cdot}6H_2O$, $MgSO_4$, $ZnSO_4{\cdot}7H_2O$, $K_2SO_4$, $FeCl_2{\cdot}4H_2O$, NaCl, or $CuSO_4$) were added for enzyme reaction. As results, it was found that $FeCl_2{\cdot}4H_2O$ or $MnSO_4{\cdot}4H_2O$ addition resulted in 17% and 9% improved starch degrading activity, respectively. The recombinant glucoamylase from D. geothermalis might be used for simultaneous saccharification and fermentation (SSF) process at high acidic conditions.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.153-159
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• 2009

Recombinant Escherichia coli BLR(DE3) harboring the hemA gene from Rhodobacter capsulatus under the control of a constitutive promoter, which we constructed previously, was used for the extracellular production of 5-aminolevulinic acid (ALA). The effects of several factors on ALA production were investigated in flask culture. ALA production by the recombinant E. coli was more efficient at $30^{\circ}C$ than $37^{\circ}C$. The glycine concentration had an important effect on cell growth. Glycine and succinic acid concentration of 5-10 and 10-20 g/L, respectively, resulted in high ALA production. In addition, the partial replacement of succinic acid by sodium glutamate increased the ALA production. The ALA production was inhibited by the presence of glucose in the medium. Using the optimal conditions, an ALA concentration of 8.2 g/L was achieved in jar fermentation without an added inducer or ALA dehydratase inhibitor; this is the highest reported concentration.

• Journal of Microbiology and Biotechnology
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• v.3 no.1
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• pp.1-5
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• 1993

The gene for mannose enzyme II of phosphoenolpyruvate-dependent phosphotransferase system from Corynebacterium glutamicum KCTC 1445 was cloned into Escherichia coli ZSC113 using plasmid pBR 322. The recombinant plasmid, designated pCTS3, contained 2.2 kb DNA fragment, and the physical map of the cloned DNA fragment was determined. The E. coli ptsM ptsG mutant transformed with pCTS3 restored glucose and mannose fermentation ability, and grew well on these sugars as the sole carbon source in the minimal medium. The transform ant harboring pCTS3 showed a PTS-mediated repression of growth on maltose by mannose analogue, 2-deoxyglucose. The specificity of the response to 2DG therefore indicates that the cloned DNA fragment carries mannose enzyme II gene.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.157-160
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• 2003

ALA is an intermediate in the tetrapyrrole biosynthesis pathway and has extensive applications as a biodegradable herbicide and insecticide as well as medical applications including photodynamic therapy of cancers. For the development of mass production process of ALA it is necessary to on-line monitor some metabolites such as glycine, succinate, LA and ALA. In this study, medium compositions and fermentation conditions were investigated for enhancement of ALA production by recombinant E. coli. A 2-dimensional fluorescence sensor was employed to monitor the bioprocess of ALA production. The monitored data is analyzed using principal component analysis, a powerful tool for multivariate statistical analysis.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.514.3-515
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• 1986

λP$_{L}$ promoter와 Infiuenza virus의 NS1 Structural gene이 있는 pASl EH801 plasmid를 E. coli host N5' 과 AR120에 각각 transformation하여 온도와 nalidixic acid로 각각 induction 하여 보았다 N5151의 경우, O.D.600 1.2에서 42$^{\circ}C$로 induction하였을 때 maximum productivity를 보였으며 AR120의 경우는 O. D. 600 1.2, 37$^{\circ}C$, 40$\mu\textrm{g}$ nalidixic acid/$m\ell$ induction 하였을 때 maximum yield를 보여주었다. 이때 pH, DO, temperature, $O_2$%, $CO_2$%를 A/D converter통해 computer에 연결시켜 data acquision을 한 결과, 접종 후 ON-line induction이 가능함을 알 수 있었다.

• KSBB Journal
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• v.30 no.6
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• pp.345-349
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• 2015

Antifreeze proteins (AFP) inhibit growth and recrystallization of ice, and permit organisms to survive in cold environments. The AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris icebinding protein), was produced in E. coli using a cold shock induction system. The culture temperature was shifted from $37^{\circ}C$ to $15^{\circ}C$ and a 20 L culture scale was used. The final weights of dried cell and FfIBP were estimated to be 126 g and 8.4 g, respectively. The thermal hysteresis (TH) activity ($1.53^{\circ}C$) of the produced FfIBP was 3.6-fold higher than that of the LeIBP (Leucosporidium ice-binding protein) produced in Picha. The current study demonstrates that large-scale production of FfIBP was successful and the result could be extended to further application studies using recombinant AFPs.

• Journal of Life Science
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• v.19 no.7
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• pp.845-851
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• 2009

A vector harboring double cassettes; a heterologous gene expression cassette of pHCE-InaN-antigen and a ghost formation cassette of pAPR-cI-E lysis 37 SDM was constructed and introduced to E. coli DH5a. For the production of a bacterial ghost vaccine, bacterial ghosts from E. coli / Streptococcus iniae with four different types of antigens - enolase, GAPDH, sagA and piaA - were produced by the optimization of fermentation parameters such as a glucose concentration of 1 g/l, agitation of 300 rpm and aeration of 1 vvm. Efficiency of ghost bacteria formation was evaluated with cultures of OD$_{600}$=1.0, 2.0 and 3.0. The efficiency of the ghost bacteria formation was 99.54, 99.67, 99.99 and 99.99% with inductions at OD$_{600}$=3.0, 1.0, 2.0 and 1.0 for E. coli/S. iniae antigens enolase, piaA, GAPDH and sagA, respectively. Ghost bacteria as a vaccine was harvested by centrifugation. The antigen protein expressions were analyzed by SDS-PAGE and western blot analysis, and the molecular weights of the enolase, piaA, GAPDH and sagA were 78, 26, 67 and 26 kDa, respectively. The molecular weights of the expressed antigens were consistent with theoretical sizes obtained from the amino acid sequences.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.496-510
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• 2007

This paper describes the use of a discrete mathematical model to represent the basic mechanisms of regulation of the bacteria E. coli in batch fermentation. The specific phenomena studied were the changes in metabolism and genetic regulation when the bacteria use three different carbon substrates (glucose, glycerol, and acetate). The model correctly predicts the behavior of E. coli vis-a-vis substrate mixtures. In a mixture of glucose, glycerol, and acetate, it prefers glucose, then glycerol, and finally acetate. The model included 67 nodes; 28 were genes, 20 enzymes, and 19 regulators/biochemical compounds. The model represents both the genetic regulation and metabolic networks in an integrated form, which is how they function biologically. This is one of the first attempts to include both of these networks in one model. Previously, discrete mathematical models were used only to describe genetic regulation networks. The study of the network dynamics generated 8 $(2^3)$ fixed points, one for each nutrient configuration (substrate mixture) in the medium. The fixed points of the discrete model reflect the phenotypes described. Gene expression and the patterns of the metabolic fluxes generated are described accurately. The activation of the gene regulation network depends basically on the presence of glucose and glycerol. The model predicts the behavior when mixed carbon sources are utilized as well as when there is no carbon source present. Fictitious jokers (Joker1, Joker2, and Repressor SdhC) had to be created to control 12 genes whose regulation mechanism is unknown, since glycerol and glucose do not act directly on the genes. The approach presented in this paper is particularly useful to investigate potential unknown gene regulation mechanisms; such a novel approach can also be used to describe other gene regulation situations such as the comparison between non-recombinant and recombinant yeast strain, producing recombinant proteins, presently under investigation in our group.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1108-1113
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• 2015

Cadaverine (1,5-diaminopentane) is an important industrial chemical with a wide range of applications. Although there have been many efforts to produce cadaverine through fermentation, there are not many reports of the direct cadaverine production from lysine using biotransformation. Whole-cell reactions were examined using a recombinant Escherichia coli strain overexpressing the E. coli MG1655 cadA gene, and various parameters were investigated for the whole-cell bioconversion of lysine to cadaverine. A high concentration of lysine resulted in the synthesis of pyridoxal-5'-phosphate (PLP) and it was found to be a critical control factor for the biotransformation of lysine to cadaverine. When 0.025 mM PLP and 1.75 M lysine in 500 mM sodium acetate buffer (pH6) were used, consumption of 91% lysine and conversion of about 80% lysine to cadaverine were successfully achieved.