• Journal of Microbiology and Biotechnology
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• 제3권3호
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• pp.168-173
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• 1993

The expression kinetics of human lipocortin I (LCI), a potential anti-inflammatory agent, was studied in the shake-flask and fermenter cultures of Saccharomyces cerevisiae carrying a galactose-inducible expression system. The cell growth, expression level of LCI, and the plasmid stability were investigted under various galactose induction conditions. The expression of LCI was repressed by the presence of a very small amount of dextrose in the culture medium, but it was induced by galactose after dextrose became completely depleted. The optimal ratio of dextrose to galactose for lipocortin I production was found to be 1.0 (10 g/l dextrose and 10 g/l galactose). With optimal D/G ratio of 1.0 and the addition of galactose prior to dextrose depletion, LCI of about 100~130 mg/l was produced. LCI at a concentration of 174 mg/l was porduced in the fed-batch culture, which was nearly a twice as much of that produced in the batch culture. The plasmid stability was very high in all culture cases, and thus was considered to be not an important parameter in the expression of LCI.

• 한국미생물·생명공학회지
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• 제32권2호
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• pp.135-141
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• 2004

인간 과립구 성장인자(hG-CSF)는 골수에서 생산되는 단백질로 호중구의 분화 및 생성을 촉진시키는 역할을 한다. 현재 재조합 hG-CSF는 암화학요법에 의한 호중구감소증, 골수이식시 호중구 감소증, 재생불량성 빈혈에 수반되는 호중구 감소증 등으로 적응증이 확대되고 있다. 본 연구에서는 OmpA signal sequence를 삽입하여 인간 과립구 성장인자(hG-CSF)가 분비발현되도록 고안된 T7 promoter 에 의하여 발현되는 pYRCl 발현백터를 제조하였다. E. coli BL2l (pYRCl) 발현시 $37^{\circ}C$에서 배양하는 경우 많은 양의 봉입체(aggregates)를 형성한다. 이에 비하여 $10\mu$M ucose를 포함하는 변형된 MBL배지에서 10 g/$\ell$IPTG를 유도물질로 7시간동안 $25^{\circ}C$에서 배양하였을 때 전체 periplasm단백질의 15%가 soluble rhG-CSF이었다. 또한, 유가식 배양방법을 사용하여 E. coli BL2l(pYRCl)에서 soluble rhG-CSF의 생산조건을 조사하였다. 유가식 배양에서 rhG-CSF의 발현량이 비증식속도를 $0.43 h^{-1}$ 에서 0.14 $h^{-1}$ 으로, 유도 배양시간을 최적화함으로써 rhG-CSF의 발현량이 4.4mg/$\ell$에서 24mg/$\ell$ 로 증가하였다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2005년도 2005 Annual Meeting & International Symposium
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• pp.230-232
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• 2005

The formation of insoluble aggregation of the recombinant kringle fragment of human apolipoprotein(a), rhLK8, in endoplasmic reticulum was identified as the rate-limiting step in the rhLK8 secretion in Saccharomyces cerevisiae. To analyze the protein secretion pathway, some of yeast genes closely related to protein secretion was rationally selected and their oligomer DNA were arrayed on the chip. The expression profiling of these genes during the induction of rhLK8 in fermentor fed-batch cultures revealed that several foldases including pdi1 gene were up-regulated in the early induction phase, whereas protein transport-related genes were up-regulated in the late induction phase. The coexpression of pdi1 gene increased rhLK8-folding capacity. Hence, the secretion efficiency of rhLK8 in the strain overexpressing pdi1 gene increased by 2-fold comparing in its parental strain. The oligomer DNA chip arrayed with minimum number of the genes selected in this study could be generally applicable to the monitoring system for the heterologous protein secretion and expression in Saccharomyces cerevisiae. With the optimization of fed-batch culture conditions and the alteration of genetic background of host, we obtained extracellular rhLK8 at higher yields than with Pichia pastoris systems, which was a 25-fold increased secretion level of rhLK8 compared to the secretion level at the initiation of this study.

• 한국미생물·생명공학회지
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• 제26권1호
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• pp.61-67
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• 1998

재조합 인간상피세포 성장인자(rhEGF)가 E. coli BL21(pYHB101) 균주를 사용하여 발현되었다. 10g/L glucose를 첨가한 변형된 MBL 배지를 사용하여 10 $\mu\textrm{m}$ IPTG/lactose로 2시간 동안 유도배양한 후 27$^{\circ}C$에서 48시간 동안 배양하였을 때 44.5 mg/L의 rhEGF가 발현되었다. 상기의 결과는 E. coli BL2l(pYHB101)를 사용하여 rhEGF를 발현시 lactose를 IPTG와 동일한 유도 물질로 사용 가능하다는 것을 시사하는 것이다. 회분식 배양에서 glucose를 10 g/L 첨가한 변형된 MBL 배지에 유도물질로 10 $\mu\textrm{m}$ lactose를 사용하였으며 28시간 동안 배양하였을 때 최대 45 mg/L의 rhEGF가 발현되었다. 유가식 배양에서 정지기에 0.5%(w/v) lactose와 0.25%(w/v) yeast extract를 첨가하였을 때 160mg/L의 rhEGF가 발현되었으며 94.3%가 분비되었다. 이에 비하여 유도기에 lactose를 첨가한 경우는 120 mg/L의 rhEGF가 발현되었으며 cytoplasm으로 발현된 불용성 봉입체의 비율은 20.9%에 달하였다. 이것은 lactose의 첨가시기가 E. coli BL2l(pYHB101)로부터 soluble rhEGF의 생성에 중요하다는 것을 확인한 결과이다.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1473-1481
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• 2018

A cellulase hyperproducing mutant strain, JNDY-13, was obtained using the ARTP mutation system and with Trichoderma reesei RUT-C30 as the parent strain. Whole-genome sequencing of JNDY-13 confirmed that 105 of the 653 SNPs were point mutations, 336 mutations were deletions and 165 were insertions. Moreover, 99 mutations were insertions and duplications. Among all the mutations, the one that occurred in the galactokinase gene might be related to the production of cellulases in T. reesei JNDY-13. Moreover, the up-regulation of cellulase and hemicellulase genes in JNDY-13 might contribute to higher cellulases production. Under optimal conditions, the highest cellulase activity by batch fermentation reached 4.35 U/ml, and the highest activity of fed-batch fermentation achieved was 5.40 U/ml.

• 생명과학회지
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• 제14권2호
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• pp.235-238
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• 2004

The possibility for developing multistage culture system to cultivate larvae by computer simulation with basic experiments was investigated. This culture system was composed of 3 stages. At the 1st stage, Chlorella sp. were cultivated and at 2nd stage Chlorella sp. were supplied to rotifer (Brachionus plicatilis), and rotifer were supplied to larvae at the 3rd stage. In this study, Chlorella sp. were cultivated by batch culture to search for the possibility of continuous feeding to rotifer at 2 nd stage. The maximum specific growth rate ($\mu$$_{max}$) of Chlorella sp. at the logarithmic phase was 0.56 [1/day]. Rotifer was cultivated by fed-batch culture at the feeding rate of Chlorella sp., $10^3$, $10^4$, $10^{5}$ and $10^{6}$ [cells/rotiferㆍh] to search for the relation between the feeding rate of Chlorella sp. and the growth rate of rotifer. As the results, the minimum feeding rate of Chlorella sp. was 2.8 ${\times}$ $10^4$ [cells/rotiferㆍday] in the multistage culture system, then the change of rotifer concentration at 2nd stage was simulated by computer. The required amount of rotifer for the growth of larvae was also increased as the growth of larvae. On the 9th day of the culture, the rotifer uptake rate of larvae was 250 [cells/rotiferㆍday]. Based on these basic experiments and results, It was suggested that the possibility of multistage culture system to cultivate larvae with continuous feeding of Chlorella sp. and rotifer.r.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.99-102
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• 2001

본 연구에서는 히루딘을 생산할 수 있는 재조합 S. cerevisiae 에서 ‘히루딘 유전자의 copy number 와 히루딘 발현양과의 관계를 규명하였으며 , ${\delta}$ 서열을 이중으로 사용한 히루딘 발현벡터를 제조하여 히루딘 유전자의 효모염색체로의 도입효율을 증가시켰다. 숙주세포인 효모의 GALl 유전자를 파쇄하여 균체에 의한 갈락토스 소모를 방지하여 보다 경제적으로 히루딘을 생산할 수 있는 시스템을 개발하였으며, 재조합 H. polymorpha을 이용한 발효공정에서 히루딘 생산을 위한 최적의 메탄올 농도를 결정하였다.

• 공업화학
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• 제7권4호
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• pp.743-749
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• 1996

본 연구에서는 고밀집 섬유 다발체 내에 수용상과 유기상을 동시에 주입시켜 나타나는 모세관 현상을 이용하여 최소 공간에서 최대한의 물질 전달 면적을 갖게 한 정적 액-액 접촉 장치가 개발되었다. 이 장치의 특성과 안정성을 평가하기 위해 TBP와 우라닐 이온을 함유한 질산계의 연속 추출 실험이 수행되었으며, 추출 성능은 동일 상비 조건에서 수행된 회분식 성능과 동일하였다. 초기 수용액에 대한 추출 수율을 증가 시키기 위해서는 일정한 수용액 유속에서 유기상의 유속 증가가 필요하였고, 같은 상비 조건의 회분식의 추출수율을 갖기 위해서는 수용액이 장치 내에서 일정한 체류 시간을 유지해야함을 알 수 있었고 본 추출계에서는 약 1.9분이 필요함을 알 수 있었다. 본 연구에서 개발된 고 밀집 섬유체 접촉기는 안정성과 재현성이 뛰어 났으며, 일반 물질 분리용 뿐만 아니라, 액-액계면 넓고 안정되어 있어 용매 추출 속도론 해석에도 사용될 수 있음을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1898-1903
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• 2007

We attempted to modulate the overall protein expression rate through the addition of a repressor against the araBAD promoter system of Escherichia coli, in which glucose was used as a repressor. Therefore, 0.5% L-arabinose was initially contained as an inducer in culture medium, and either 2% glucose or 2% glycerol was used as a carbon source, and it was found that the expression of recombinant interferon-${\alpha}$ could be observed at the beginning of the batch culture when glycerol was used as a carbon source. However, when glucose was used, the initiation of recombinant interferon-${\alpha}$ expression was delayed compared with that when glycerol was used. Furthermore, when the addition of 0.5% glucose was carried out once or twice after 0.5% L-arabinose induction during DO-stat fed-batch culture, the distributions of soluble and insoluble recombinant interferon-${\alpha}$ were modulated. When glucose was not added after the induction of L-arabinose, all of the expressed recombinant interferon-${\alpha}$ formed an inclusion body during the later half of culturing. However, when glucose was added after induction, the expressed recombinant interferon-${\alpha}$ did not all form an inclusion body, and about half of the total recombinant interferon-${\alpha}$ was expressed in a soluble form. It was deduced that the addition of glucose after the induction of L-arabinose might lower the cAMP level, and thus, CAP (catabolite activator protein) might not be activated. The transcription rate of recombinant interferon-${\alpha}$ in the araBAD promoter system might be delayed by the partial repression. This inhibition of the transcription rate probably resulted in more soluble interferon-${\alpha}$ expression caused by the reduction of the protein synthesis rate.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1664-1672
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• 2014

In this study, a fibrous bed bioreactor (FBB) was used for $\small{D}$-lactic acid ($\small{D}$-LA) production by Sporolactobacillus inulinus Y2-8. Corn flour hydrolyzed with ${\alpha}$-amylase and saccharifying enzyme was used as a cost-efficient and nutrient-rich substrate for $\small{D}$-LA production. A maximal starch conversion rate of 93.78% was obtained. The optimum pH for $\small{D}$-LA production was determined to be 6.5. Ammonia water was determined to be an ideal neutralizing agent, which improved the $\small{D}$-LA production and purification processes. Batch fermentation and fed-batch fermentation, with both free cells and immobilized cells, were compared to highlight the advantages of FBB fermentation. In batch mode, the $\small{D}$-LA production rate of FBB fermentation was 1.62 g/l/h, which was 37.29% higher than that of free-cell fermentation, and the $\small{D}$-LA optical purities of the two fermentation methods were above 99.00%. In fe$\small{D}$-batch mode, the maximum $\small{D}$-LA concentration attained by FBB fermentation was 218.8 g/l, which was 37.67% higher than that of free-cell fermentation. Repeate$\small{D}$-batch fermentation was performed to determine the long-term performance of the FBB system, and the data indicated that the average $\small{D}$-LA production rate was 1.62 g/l/h and the average yield was 0.98 g/g. Thus, hydrolyzed corn flour fermented by S. inulinus Y2-8 in a FBB may be used for improving $\small{D}$-LA fermentation by using ammonia water as the neutralizing agent.