• Journal of Microbiology and Biotechnology
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• 제4권3호
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• pp.200-203
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• 1994

Spodoptera frugiperda IPLB-SF-21-AE cells were cultivated in a spin-filter bioreactor with continuous perfusion for the recombinant $\beta$-galactosidase production. At the perfusion rate of 0.06 $hr^{-1}$, the maximum cell density of insect cells in this bioreactor system reached 3.5$\times$$l0^6$ viable cells/ml using the Grace media containing 5% FBS and 0.3% Pluronic F-68. The recombinant $\beta$-galactosidase production of 8, 100 units per reactor volume was also achieved at this perfusion rate.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.434-442
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• 2008

We observed that an inclusion body (IB) of recombinant ${\beta}$-galactosidase that was produced by the araBAD promoter system in Escherichia coli (E. coil) showed enzyme activity. In order to improve its activity, the lowering of the transcription rate of the ${\beta}$-galactosidase structural gene was attempted through competition between an inducer (L-arabinose) and an inducer analog (D-fucose). In the deep-well microtiter plate culture and lab-scale fermentor culture, it was demonstrated that the addition of D-fucose caused an improvement in specific ${\beta}$-galactosidase production, although ${\beta}$-galactosidase was produced as an IB. In particular, the addition of D-fucose after induction led to an increase in the specific activity of ${\beta}$-galactosidase IB. Finally, we confirmed that the addition of D-fucose after induction caused changes in the structure of ${\beta}$-galactosidase IB, with higher enzyme activity. Based on these results, we expect that an improved enzyme IB will be used as a biocatalyst of the enzyme bioprocess, because an enzyme IB can be purified easily and has physical durability.

• 미생물학회지
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• 제30권6호
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• pp.539-545
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• 1992

Lactobacillus casei SM-M1 의 플라스미드로부터 phospho-$\beta$-galactosidase gene 을 갖는 DNA 를 E. coli 에 클로닝한 pPLac15(13kb) 의 재조합 플라스미드를 제조하였다.(15). pPLac15 DNA 를 분리하여 제한효소로 처리하여 제한효소 지도를 작성하였다. Phospho-$\beta$-galactosidase 유전자의 발현을 높이기 위하여 lac promoter 를 가진 pUC18 의 PstI 위치에 클닝하여 pPLac18 을 제조하였으며, 이것을 다시 EcoRI 으로 절단하여 pUC 18 에 클로닝하여 얻은 pPLac23 (7.6 kb) 를 얻었다. Phospho-$\beta$-galactosidase 효소활성은 pPLac23 의 형질전환주인 E. coli SW-23 에서는 pPLac15 를 가진 형질전환주인 E. coli SW-15 보다 약 1.8 배의 효소의 활성을 나타내었으며 pPLac18 을 가진 E. coli SW-18 보다는 약간 높은 활성을 나타내었다.

• Applied Biological Chemistry
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• 제38권6호
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• pp.485-489
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• 1995

T-flask와 air-lift 생물반응기를 이용하여 Autographa californica nuclear polyhedrosis virus (AcNPV)의 Tn5B1-4 세포에의 감염시 fatty acid 및 lipid, mannose, folic acid, $CaCl_2$등의 배지 첨가물이 재조합 ${\beta}-galactosidase\;({\beta}-gal)$ 생산에 미치는 영향을 조사하였다. Cholesterol, tocopherol, tricaprylin 또는 mannose를 첨가하거나 folic acid를 보강첨가 했을 때 AcNPV의 재조합 ${\beta}-gal$ 생산은 향상되었으나 $CaCl_2$의 보강 첨가는 ${\beta}-gal$ 생산을 높이는데 효과적이지 못했다. Air-lift 생물반응기에서 0.34 mM cholesterol, 2.2 mM mannose 및 0.045 M folic acid를 보강 첨가한 배지를 이용하였을 때 재조합 ${\beta}-gal$ 생산은 basal medium에 비해 약 2배 정도의 증진 효과가 있었다.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.227-231
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• 1991

A gene coding for a $\beta$-galactosidase of Bacillus subtilis HP-4 was cloned in E. coli JM109 by inserting HindIII digested fragment of B. subtilis HP-4 chromosomal DNA into the site of pBR322 and selecting recombinant transformant showing blue color on X-gal plate. The recombinant plasmid, named pBG109, was found to contain the 1.4 Kbp HindIII fragment originated from B. subtilis HP-4 chromosomal DNA by Southern hybridization. The cloned gene was stably maintained and expressed in E. coli JM109 and the pBG109 encoded $\beta$-galactosidase had the same enzymatic properties as those of $\beta$-galactosidase produced by B. subtilis HP-4.

• Asian-Australasian Journal of Animal Sciences
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• 제17권12호
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• pp.1751-1757
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• 2004

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble $\beta$-galactosidase, the gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase (KNOUC112 $\beta$-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 $\beta$-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The $\beta$-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion $\beta$ -galactosidase expressed via pThioHis C at 37$^{\circ}C$ was about 5 times higher than that of unfused $\beta$-galactosidase expressed via pET-5b at 37$^{\circ}C$. Inclusion body of $\beta$-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 $\beta$ -gal was expressed via pET-5b at 37$^{\circ}C$. Fusion $\beta$ -galactosidase expressed at 37$^{\circ}C$ via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25$^{\circ}C$ prevented the formation of inclusion body, optimally at 25$^{\circ}C$. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25$^{\circ}C$. The soluble production of Thermus thermophilus KNOUC112 $\beta$-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

• KSBB Journal
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• 제9권3호
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• pp.294-298
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• 1994

$CaCl_2$, glucose, frutose, glutamine, glutamate 그리고 Ii pids와 같은 배지 첨가물들이 회분식 그리고 연속식 2단계 생물반응기 시스템에셔 재조합 ${\beta}$-galactosidase (${\beta}$-gal) 생산을 증진시키는가를 조사하였다. Sf 21 세포에 AcNPV의 감염시 $CaCl_2$, frutose, glutamate, cholesterol 및 tocoph eral과 같은 배지 첨가물을 첨가하였을 때 ${\beta}$-gal 생산이 증진되었다. 30mM $CaCl_2$, 2.2mM frutose, 4.lmM glutamine, 그리고 O.34mM cholesterol이 보강된 감염 배 지를 이용한 재조합${\beta}$-gal 생산은 약 40% 의 증가를 보였다.

• 미생물학회지
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• 제27권3호
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• pp.188-193
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• 1989

Gene for lactose catabolism in Lactobacillus casei SW-M1 was encoded by a 60Kb metabolic plasmid. A derivative of only 10kb, pPlac 15 of recombinant plasmid, was constructed by introducing into pBR322 and was cloned into E. coli using restriction endonuclease Pst I. A 10kb insery DNA in plasmid pBR322 was identified as a gene encoded phospho-$\beta$-galactosidase by the determination of enzyme activity. Phospho-$\beta$-galactosidase was apparently expressed in E. coli. The enzyme activities of cell-free extract from transformant E. coli HB101 carrying pPLac 15 DNA were not different from that of L. casei as a donor strain on the basis of enzyme properites. However, specific activity of phospho-$\beta$-galactosidase in the cloned strain with Lac $Y^{-}$ phenotype of E. coli HB101 was lower than that in L. casei strain.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.274-280
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• 1991

Effects of IPTG induction on cell growth and production of ${\beta}$-galactosidase-preS2 fusion protein (${\beta}$gal-preS2) were studied in a defined medium using a recombinant Escherichia coli JM109/pCMHB30. IPTG was added (0.2 mM) to induce the cloned-gene expression in the early-, mid-, and late-log growth phases. The most serious decreases in growth rate and plasmid stability were observed for the induction in the early-log growth phase. The expression level of ${\beta}$gal-preS2 attained by the induction in the mid-log phase was about 0.51 mg fusion protein/mg total cellular protein, which was 2- and 5-fold improvement over the levels obtained with the inductions in the early- and late-log phases. Formation of acidic byproducts including acetate and pyruvate showed different profiles during the fermentation period for each cases of induction; pyruvate was the major byproduct for the induction in the early-log phase while acetate production became more significant for the cases of inductions in the mid- and late-log phases.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1392-1400
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• 2017

Galactooligosaccharides (GOSs) are known to be selectively utilized by Bifidobacterium, which can bring about healthy changes of the composition of intestinal microflora. In this study, ${\beta}-GOS$ were synthesized using bifidobacterial ${\beta}-galactosidase$ (G1) purified from recombinant E. coli with a high GOS yield and with high productivity and enhanced bifidogenic activity. The purified recombinant G1 showed maximum production of ${\beta}-GOSs$ at pH 8.5 and $45^{\circ}C$. A matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the major peaks of the produced ${\beta}-GOSs$ showed MW of 527 and 689, indicating the synthesis of ${\beta}-GOSs$ at degrees of polymerization (DP) of 3 and DP4, respectively. The trisaccharides were identified as ${\beta}-{\text\tiny{D}}$-galactopyranosyl-($1{\rightarrow}4$)-O-${\beta}-{\text\tiny{D}}$-galactopyranosyl-($1{\rightarrow}4$)-O-${\beta}-{\text\tiny{D}}$-glucopyranose, and the tetrasaccharides were identified as ${\beta}-{\text\tiny{D}}$-galactopyranosyl-($1{\rightarrow}4$)-O-${\beta}-{\text\tiny{D}}$-galactopyranosyl-($1{\rightarrow}4$)-O-${\beta}-{\text\tiny{D}}$-galactopyranosyl-($1{\rightarrow}4$)-O-${\beta}-{\text\tiny{D}}$-glucopyranose. The maximal production yield of GOSs was as high as 25.3% (w/v) using purified recombinant ${\beta}-galactosidase$ and 36% (w/v) of lactose as a substrate at pH 8.5 and $45^{\circ}C$. After 140 min of the reaction under this condition, 268.3 g/l of GOSs was obtained. With regard to the prebiotic effect, all of the tested Bifidobacterium except for B. breve grew well in BHI medium containing ${\beta}-GOS$ as a sole carbon source, whereas lactobacilli and Streptococcus thermophilus scarcely grew in the same medium. Only Bacteroides fragilis, Clostridium ramosum, and Enterobacter cloacae among the 17 pathogens tested grew in BHI medium containing ${\beta}-GOS$ as a sole carbon source; the remaining pathogens did not grow in the same medium. Consequently, the ${\beta}-GOS$ are expected to contribute to the beneficial change of intestinal microbial flora.