• KSBB Journal
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• 제20권4호
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• pp.299-304
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• 2005

본 연구에서는 인공신경망 알고리즘을 이용하여 생물공정에서 수집된 형광스펙트럼 데이터를 분류, 분석하고 공정변수들을 예측하기 위한 공정의 모델링에 대해서 논의하였다. SOM에 의해 분류된 전파장 스펙트럼 데이터들은 발효공정의 변수와 형광데이터 사이에 비선형관계를 설명하기 위하여 사용되었다. BPNN알고리즘은 SOM에서 분류된 데이터들을 입력자료로 이용하여 공정에 대한 모델식을 세우고, 이를 이용하여 배출가스 내 $CO_2$ 농도 및 발효액 중 세포농도와 같은 공정변수들을 예측하는데 사용되었다. 또한 BPNN 모델은 강력하면서도 훈련데이터의 범위를 넘어서는 공정의 데이터들을 예측할 수 있기 때문에 폭넓은 활용가능성을 가지고 있다.

• KSBB Journal
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• 제16권1호
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• pp.1-10
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• 2001

Escherichia coli has been used as an expression work horse for foreign genes. This article summarized recent development in genetic engineering techniques for overproduction of medical proteins and industrial enzymes. Special emphasis was placed upon research activities concerning folding and refolding of inclusion bodies at genetic and fermentation levels. Plasmid and mRNA stabilization, development of strong inducible promoters, modification of translational elements and reduction of rpoteolytic degradation were carried out to elevate an expression level of a target protein. Optimization of culture conditions, improvement of denaturation and renaturation steps and coexpression of molecular chaperones or foldase were accomplished to produce active proteins in soluble form. Fusion protein systems with selective separation and surface display technology were also performed in an effort to make the E. coli expression system more effective and versatile.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1093-1100
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• 2015

Acetate and lactate in growth media are detrimental to the production of Thermus maltogenic amylase (ThMA), a heterologous protein, as well as to the growth of recombinant Escherichia coli. Only 50 mM of acetate or 10 mM of lactate reduced 90% of specific ThMA activity. In this study, mutant E. coli strains blocked in the ackA-pta or ackA-pta and ldh pathways were created, characterized, and assessed for their culture performace in 300 L-scale fermentation. The ackApta and ldh double-mutant strain formed significantly less lactate and acetate, and produced a concomitant increase in the excretion of pyruvate (17.8 mM) under anaerobic conditions. The ackA-pta mutant strain accumulated significant acetate but had an approximately 2-fold increase in the formation of lactate. The ackA-pta and ldh double-mutant strain had superior overall performance in large-scale culture under suboptimal conditions, giving 67% higher cell density and 66% higher ThMA activity compared with those of the control strain. The doublemutant strain also achieved a 179% improvement in volumetric ThMA production.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1136-1140
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• 2008

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'-phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.619-622
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• 2000

For the production of $B^{30}-homoserine$ human insulin precursor, four types of fusion peptides LacZ, MBP, GST, and His-tagged sequence were studied in this work. Recombinant E. coli JM 103 and E. coli JM 109 containing fusion peptides were cultivated at $37^{\circ}C$ for 1hr, and gene expression was occurred when 0.5mM of isopropyl-D-thiogalactoside(IPTG) was added to the culture broth, and followed by longer than 4hr fermentation respectively. DEAE-Sphacel and gel filtration chromatography, amylose and glutathione-Sepharose 4B affinity chromatography, and nickel-affinity chromatography system were employed as purification of $B^{30}-homoserine$ human insulin precursor. Recovery yields of His-tagged, LacZ, GST, and MBP fused $B^{30}-homoserine$ human insulin precursor resulted in 47%, 20%, 20%, and 18%, respectively.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권2호
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• Journal of Microbiology and Biotechnology
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• 제2권4호
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• pp.243-247
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• 1992

The thr operon of Escherichia coli TF427, an $\alpha$-amino-$\beta$-hydroxyvaleric acid (AHV)-resistant threonine overproducer, was cloned in a pBluescriptII $KS^＋$ plasmid by complementation of E. coli mutants. All clones contained a common 8.8 kb HindIII-generated DNA fragment and complemented the thrA, thrB, and thrC mutants by showing that these clones contained the whole thr operon. This thr operon was subcloned in the plasmid vectors pBR322, pUC18, and pECCG117, an E. coli/Corynebacterium glutamicum shuttle vector, to form recombinant plasmids pBTF11, pUTF25 and pGTF18, respectively. The subcloned thr operon was shown to be present in a 6.0 kb insert. A transformant of E. coli TF125 with pBTF11 showed an 8~11 fold higher aspartokinase I activity, and 15~20 fold higher L-threonine production than TF125, an AHV-sensitive methionine auxotroph. Also, it was found that the aspartokinase I activity of E. coli TF125 harboring pBTF11 was not inhibited by threonine and its synthesis was not repressed by threonine plus isoleucine.

• Journal of Microbiology
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• 제41권3호
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• pp.212-218
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• 2003

Citrus phytoene synthase (CitPsy) and ${\beta}$-carotene hydroxylase (CitChx), which are involved in caroteinoid biosynthesis, are distantly related to the corresponding bacterial enzymes from the point of view of amino acid sequence similarity. We investigated these enzyme activities using Pantoea ananatis carotenoid biosynthetic genes and Escherichia coli as a host cell. The genes were cloned into two vector systems controlled by the T7 promoter. SDS-polyacrylamide gel electrophoresis showed that CitPsy and CitChx proteins are normally expressed in E. coli in both soluble and insoluble forms. In vivo complementation using the Pantoea ananatis enzymes and HPLC analysis showed that ${\beta}$-carotene and zeaxanthin were produced in recombinant E. coli, which indicated that the citrus enzymes were functionally expressed in E. coli and assembled into a functional multi-enzyme complex with Pantoea ananatis enzymes. These observed activities well matched the results of other researchers on tomato phytoene synthase and Arabidopsis and pepper ${\beta}$-carotene hydroxylases. Thus, our results suggest that plant carotenoid biosynthetic enzymes can generally complement the bacterial enzymes and could be a means of carotenoid production by molecular breeding and fermentation in bacterial and plant systems.

• KSBB Journal
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• 제21권1호
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• pp.59-67
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• 2006

본 연구에서는 2차원 형광스펙트럼의 PCA 분석을 통하여 발효 공정을 모니터링하고 PCR과 PLS과 같은 다변량 분석기법을 이용하여 공정을 모델링하였다. 재조합 대장균 E. coli 와 효모 S.cerevisiae의 발효 공정 중에 얻어진 많은 양의 2차원 형광스펙트럼 자료는 우선 PCA를 통해 축소된다. 그리고 PCA에서 주성분점수와 적재 산점도는 발효 공정의 정성적 경향을 묘사하기 위해 사용되었다. 또한, PCR과 PLS는 2차원 형광스펙트럼의 분석을 위해 사용되었으며 PLS모델이 보정과 예측 능력에서 PCR모델보다 조금 더 우수한 성능을 나타냈다. 따라서 2차원 형광스펙트럼 자료를 이용하여 생물공정을 모델링 하고자 할 때는 PCR 방법보다는 PLS 방법을 사용하는 것이 유리할 것이다.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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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.