• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1026-1031
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• 2006

The optimization of the production of a fusion protein containing the antigenic domain 1 (AD-1) is of a great importance, considering its use in diagnostic tests. The fusion protein is produced by the fermentation of a recombinant strain of Escherichia coli containing the plasmid Mbg58, which expresses the AD-1 (aa 484-650) of human cytomegalovirus glycoprotein B as a fusion protein together with aa 1-375 of ${\beta}-galactosidase$. An important characteristic of promoters (lac and derivatives) used in recombinant protein production in E. coli is their inducibility. Induction by IPTG is widely used for basic research; however, its use in large-scale production is undesirable because of its high cost and toxicity. In this work, studies using different inducers and carbon sources for the production of a fusion protein containing the AD-l were performed. The results showed that lactose could be used as an inducer in the fermentation process for the production of this protein, and that expression levels could exceed those achieved with IPTG. The use of lactose for protein expression in E. coli should be extremely useful for the inexpensive, large-scale production of heterologous proteins in E. coli. Addition of sucrose to the fermentation medium improved the yield of recombinant protein, whereas addition of fructose or trehalose decreased the yield.

• Journal of Microbiology and Biotechnology
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• 제7권5호
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• pp.323-328
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• 1997

The recombinant Escherichia coli BL21(DE3)pLysE : pTCGT1 was grown to overproduce Bacillus macerans cyclodextrin glucanotransferase (CGTase) able to synthesize ${\alpha}$-cyclodextrin (CD) with a selectivity of 67%. A number of batch fermentations were performed to test the possibility of using lactose as an inducer of the E. coli T7 promoter system. A mixture of isopropyl ${\beta}$-D-thiogalactoside (IPTG) and lactose (1 : 1) gave a maximum CGTase activity of 2.4 U/ml, which was higher than the value obtained with induction by IPTG alone. Fed-batch fermentations involving a glucose-controlled growth period followed by a gene-expression phase with mixtures of IPTG and lactose were employed to achieve high cell density and thereby increase total CGTase activity. Optimized fed-batch fermentation using the modified inducer (IPTG : lactose=1 : 3) and 100 g/l yeast extract solution in the gene-expression phase resulted in a maximum CGTase activity of 62.9 U/ml and a final cell mass of 53.5 g/l, corresponding to a 31-fold increase in CGTase activity and a 29-fold increase in cell mass compared with the control batch fermentation.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1979년도 춘계학술대회
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• pp.113.2-114
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• 1979

$\beta-Galactosidase$ is an enzyme which catalizes hydrolysis of lactose, a natural substrate, to glucose and galctose and transferring some monosac-charide units to active acceptors as sugar or alcohol. The occurence of $\beta-Galactosidase$ is known in various microorganisms, animals and higher plants and has been studied by many investigatigators. Especially, a great deal of articles for the enzyme of E. coli have been presented in genetic control mechanism and induction-repression effects of proteins, On the other hand, in the dairly products industry, it is important to hydrolyes lactosd which is the principal sugar of milk and milk products. During the last few years, the interest in enzymatic hydrolysis of milk lactose has teen increased, because of the lactose intolerence in large groups of the population. Microbial $\beta-Galactosidases$ are considered potentially most suitable for processing milk to hydrolyse lactose and, in recent years, the immobilized enzyme from yeast has been examined. Howev, most of the microbial $\beta-Gal$ actosidase are intracellular enzymes, except a few fungal $\beta-Gala-$ ctosidases, and extracellular $\beta-Galactosidase$ which may be favorable to industrial applieation is not so well investigated. On this studies, a mold producing a potent extracellular $\beta-Galactosidase$ was isolated from soil and identified as an imperfect fungus, Beauveria bassians. In this strain, both extracellular and intracellular $\beta-Galactosidases$ were produced simultaneously and a great increase of the extracellular production was acheved by improving the cultural conditions. The extracellular enzyme was purified more than 1, 000 times by procedures including Phosphocellulose and Sephadex G-200 chromatographies. Several characteristics of the enzymewas clarified with this preparation. The enzyme has a main subunit of molecular weight of 80, 000 which makes an active aggregate. And at neutral pH range, it has optimum pH for activity and stability. The Km value was determined to be 0.45$\times$10$^{-3}$ M for $o-Nitrophenyl-\beta-Galactoside.$ In any event, it is interesting to sttudy the $\beta-Galactosidase$ of B. bassiana for the mechanism of secretion and conformational structure of enzyme.

• KSBB Journal
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• 제5권3호
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• pp.195-200
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• 1990

대장균의 lipoprotein promoter, lactose promotor 및 operator 와 lipoprotein의 signal sequence 를 가지는 vector에alpha-IFN 유전자가 cloning된 plasmid pIF-Ill-B를 여러종류의 대장균 숙주 세포에 형칠전환하여 alpha-IFN 의 생산성, 생장특성을 조사하였다. 또한 plasmid 자체와 cloning된 alpha-IFN 유전자의 발현유도가 세포생장에 미치는 영 향을 조사하였다.

• KSBB Journal
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• 제23권6호
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• pp.474-478
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• 2008

효율적인 UDP-glucose regeneration system을 구축하기 위해서 재순환 시스템에 관여하는 4가지 효소 (UDP-glucose pyrophosphorylase, UDP-Kinase gene, UDP-galactose 4-epimerase, and $\beta$-1, 4-galactasyltrasnsferase)들을 E. coli AD202에서 발현 시켜 Disaccharide 합성 정도를 보았다. Disaccharide는 0.5 mM IPTG 농도에서 가장 높은 농도를 나타내었다. 대조구와 비교한 결과 LacNAc 농도는 1.34 mM로 10배 정도 정가하였고, lactose 농도는 0.39 mM로 대조구보다 2.6배 증가하였다. 총 disaccharide 농도는 1.73 mM 이며, 대조구 보다 6.5배 높은 생산성을 보였다. 본 논문은 결과는 metabolic flux regeneration으로 disaccharides 합성을 증가시킬 수 있다는 것을 보여주었다.

• 한국균학회지
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• 제26권1호통권84호
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• pp.127-133
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• 1998

Trichoderma reesei QM 9414를 N-methyl-N'-nitro-N-nitrosoguanidine으로 처리하여 얻은 돌연변이주들을 여러 탄소원에서 배양했을 때 carboxymethylcellulose(CMC), fiter paper 등의 기질에 대한 활성측정결과 다음과 같은 결과를 얻었다. 모균주가 catabolite repression을 받는 반면에 돌연변이주들의 cellulase 활성은 glucose를 탄소원으로 사용한 배지에서 모균주에 비해 CMCase 8.4배, FPase가 $5.4{\sim}5.7$배 증가함으로써 glucose-derepression성질을 갖는 돌연변이주들을 얻었으며 glucose를 탄소원으로 사용하고 lactose로 효소의 생산을 유도시켜본 결과 모균주에 비해 효소의 생산에 안정성을 갖는 것으로 나타났으며 돌연변이주 1이 상대적으로 더 우수한 돌연변이주로 나타났다. 돌연변이주들에 의해 생산된 효소는 모균주와 마찬가지로 pH 4.8, $60^{\circ}C$에서 최적활성을 나타내었다.

• Journal of Microbiology
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• 제44권6호
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• pp.689-693
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• 2006

Pbh1, from the fission yeast Schizosaccharomyces pombe, is a baculoviral inhibitor of apoptosis (IAP) repeat (BIR) domain-containing protein. Its unique encoding gene was previously found to be regulated by nitric oxide and nitrogen starvation. In the current work, the Pbh1-lacZ fusion gene was used to elucidate the transcriptional regulation of the pbh1 gene under various carbon sources. When fermentable carbon sources, such as glucose (at a low concentration of 0.2 %), sucrose (2.0 %) and lactose (2.0 %), were the sole carbon source, the synthesis of $\beta$-galactosidase from the Pbh1-lacZ fusion gene was reasonably enhanced. However, the induction by these fermentable carbon sources was abolished in the Pap1-negative S. pombe cells, implying that this type of induction of the pbh1 gene is mediated by Pap1. Ethanol (2.0%), a nonfermentable carbon source, was also able to enhance the synthesis of $\beta$-galactosidase from the fusion gene in wild-type cells but not in Pap1-negative cells. The results indicate that the S. pombe pbh1 gene is up-regulated under metabolic oxidative stress in a Pap1-dependent manner.

• BMB Reports
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• 제38권5호
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• pp.609-618
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• 2005

$\gamma$-Glutamyl transpeptidase (GGT, EC 2.3.2.2.) catalyzes the transfer of the $\gamma$-glutamyl moiety from $\gamma$-glutamyl containing ompounds, notably glutathione (GSH), to acceptor amino acids and peptides. A second gene (GGTII) encoding GGT was previously isolated and characterized from the fission yeast Schizosaccharomyces pombe. In the present work, the GGTII-lacZ fusion gene was constructed and used to study the transcriptional regulation of the S. pombe GGTII gene. The synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene was significantly enhanced by NO-generating SNP and hydrogen peroxide in the wild type yeast cells. The GGTII mRNA level was increased in the wild-type S. pombe cells treated with SNP. However, the induction by SNP was abolished in the Pap1-negative S. pombe cells, implying that the induction by SNP of GGTII is mediated by Pap1. Fermentable carbon sources, such as glucose (at low concentrations), lactose and sucrose, as a sole carbon source, enhanced the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in wild type KP1 cells but not in Pap1-negative cells. Glycerol, a non-fermentable carbon source, was also able to induce the synthesis of $\beta$-galactosidase from the fusion gene, but other non-fermentable carbon sources such as acetate and ethanol were not. Transcriptional induction of the GGTII gene by fermentable carbon sources was also confirmed by increased GGTII mRNA levels in the yeast cells grown with them. Nitrogen starvation was also able to induce the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in a Pap1-dependent manner. On the basis of the results, it is concluded that the S. pombe GGTII gene is regulated by oxidative and metabolic stress.

• 한국미생물·생명공학회지
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• 제31권3호
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• pp.277-283
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• 2003

전통 발효식품인 된장으로부터 mannanas의 생산균으로 분리된 WL-7균주는 형태적 특성, 생화학적 성질 및 16S rRNA의 염기서열에 근거하여 Bacillus subtilis로 동정되었다. B. subtilis WL-7의 배양상등액으로 제조한 조효소액을 이용하여 mannanase 활성을 측정한 결과, 55$^{\circ}C$와 pH 6.0에서 최대활성을 보였으며 , 반응온도 $50∼60^{\circ}C$와 반응 pH 5.0∼7.5에서 최대활성의 90% 이상의 활성을 나타냈다. 분리균WL-7의 mannanase생산성을 높이기 위해 LB배지에 소당류와 고분자 탄수화물을 첨가하여 배양한 결과, lactose, 밀기울, avicel, $\alpha$-cellulose, locust bean gum(LBG), konjak 등에 의해 mannanase생산성이 상당량 증가되었다. 특히 0.5% LBG와 0.5% konjak이 동시에 첨가된 LB 배지에서 10시간 배양하였을 때 배양상등액의 효소 활성이 224 U/ml로 최대 효소생산성을 보였으며, 이는 LBC와 konjak을 첨가하지 않은 배지에서 보다 효소 생산성이 200배 이상이 증가하였다. LBG와 konjak을 첨가하였을 때 B. subtilis WL-7의최대성장도가 약간 증가한 것에 비해 mannanase생산성은 현저히 증가된 것으로 보아 배양액 중의 LBG와 konjak의 가수분해 산물이 mannanas리 생합성을 유도한 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• 제21권11호
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• pp.1174-1178
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• 2011

Exopolysaccharides (EPSs) are microbial polysaccharides that are released outside of the bacterial cell wall. There have been few studies on EPS-producing lactic acid bacteria that can enhance macrophage activity and the underlying signaling mechanism for cytokine expression. In the current study, EPS-overproducing Lactobacillus (L.) paracasei KB28 was isolated from kimchi and cultivated in conditioned media containing glucose, sucrose, and lactose. The whole bacterial cells were obtained with their EPS being attached, and the cytokine-inducing activities of these cells were investigated. Gas chromatography analysis showed the presence of glucose, galactose, mannose, xylose, arabinose, and rhamnose in EPS composition. EPS-producing L. paracasei KB28 induced the expression of tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-6, and IL-12 in mouse macrophages. This strain also caused the degradation of $I{\kappa}B{\alpha}$ and phosphorylation of the major MAPKs: Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2. The use of pharmacological inhibitors showed that different signaling pathways were involved in the induction of TNF-${\alpha}$, IL-6 and IL-12 by L. paracasei KB28. Our results provide information for a better understanding of the molecular mechanisms of the immunomodulatory effect of food-derived EPS-producing lactic acid bacteria.