• 한국미생물·생명공학회지
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• 제22권4호
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• pp.401-406
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• 1994

The medium compositions of Alcaligenes eutrophus were optimized for increasing PHB productivity. It is very important to optimize the concentrations of inorganic salts and trace eleme- nts as well as carbon and nitrogen sources to maximize cell growth rate and productivity. The fed-batch culture of Alcaligenes eutrophus by dual feeding of ammonia water and glucose under optimized initial medium concentrations was carried out. Glucose was fed manually according to glucose consumption rate and ammonia water by pH-stat. The final cell concentrations and PHB content in 30 hours were 122 g/l and 65% of dry cell weight(yielding 79 g of PHB/l), respectively and 2.64 g/l/hr of PHB production rate was obtained.

• KSBB Journal
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• 제15권2호
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• pp.174-180
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• 2000

본 연구에서는 다른 host cell 에 비하여 여러 가지 장점을 가지고 있는 Methylotrophic yeast 중 Pichia pastoris와 Hans-enula polymorpha의 fed batch 실험을 통하여 유전자 재조합 단백질 발현최적조건을 구하여 각 균주의 유전자 재조합 albumin 발현 최적화 연구를 수행하였다. 글리세롤이 두 균주의 promoter의 AOX 1과 MOX promoter repression에 미치는 영향을 확인한 바 H. polymorpha가 P. pastoris보다 promoter repression이 심함을 알 수 있었다. 두 균주의 promoter를 induction시키는 최적 메탄올 농도는 P. pastoris의 경우 메탄올 8g/L, H. polymorpha의 경우는 13 g/L임을 알 수 있었다. 또한 메탄올에 의한 induction시기는 두 균주 모두 O.D. 4 정도되는 exponential growth stage에서 메탄올을 첨가하는 경우가 초기 세포 성장단계에 메탄올을 첨가한 경우에 비해 약 20% 정도 높아짐을 확인하였다. 두 균주의 재조합 albumin 발현에 미치는 pH의 영향을 조사하였는데, p. pastoris의 경우 pH 5에서 가장 높은 albumin 생산성을 보여 약 300mg/L albumin을 발현하였고, H. polymorpha 의 경우 pH 5와 6에서 최대 약 180 mg/L의 albumin을 발현하였지만 pH 8에서는 이의 절반 수준에 그쳤다. 두 균주의 최적 fed-batch 방법을 확인하는 실험을 수행하였는데 P. pastoris의 경우의 최적 fed-batch 방법은 mixed feeding은 바람직하지 않고 글리세롤 배지를 공급하여 세포를 성장시킨 후 글리세롤 공급을 멈추고 바로 메탄올로 전환하는 방법을 효과적이며, H. polymorpha의 경우 비성장속도 제어를 통한 글리세롤 공급으로부터 메탄올 공급으로의 단계적 전환방법이 균주의 albumin 발현에 큰 영향을 준다는 것을 알 수 있었다. 이 방법을 통하여 두 균주의 고농도 배양 실험을 수행한 결과 P. pastoris의 경우는 O.D. 300에서 약 4.7g albumin/L를 발현하였다. 이와같은 결과를 바탕으로 산업체에서 methlotrophic yeast를 이용한 상업화를 계획함에 있어서 host로서의 균주를 선택할 수 있는 기본 자료를 제공함과 아울러 균주가 선택된 후에 그 균주를 이용한 재조합 단백질 최적화 방법을 제공하여 줄 것으로 생각된다.

• 한국미생물·생명공학회지
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• 제28권6호
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• pp.341-348
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• 2000

Selection of high producer strain, optimization of production medium and cultivation in bioreactor system were carried out in order to produce an antifungal substance, PAFS in large amounts which sources and 41 kinds of nitrogen sources, a synthetic medium consisting of fructose(70 g/1) and ammonium sulfate (10g/l) and a complex medium including galactose(30g/l), fructose(20g/l) and cottonseed flour(35g/l) were determined as opti-mized media for PAFS production. In bioreactor studies examining physiological characteristics of the pro- ducer microorganism with the complex medium, typical pattern of diauxic growth was observed as demonstrated by the result that fructose was not used before almost exhaustion on readily utilizable carbon source, galactose. When galactose was supplemented additionally during the fermentation period. PAFS pro-ductivity did no increases any more, indicating that large portion of the added galactose was used for cell growth instead of biosynthesis of the secondary metabolite. It was deduced that PAFS production could be enhananced by employing fed-batch operation in order to overcome the apparent phenomenon of catabolite repression and /or inhibition.

• KSBB Journal
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• 제5권1호
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• pp.25-35
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• 1990

In the Xanthan gum fermentation by Xanthomonas campestris there are problems of the large energy consumption by long fermentation time, the mass transfer of oxygen and nutrients by high viscous fermentation broth. In this study, the media optimization and the fed batch fermentation were carried out to decrease fermentation time and increase Xanthan gum yield. The $O_2$ uptake rate (OUR) and $CO_2$ evolution rate(CER) which were obtained from the analysis of fermentation exit gas using a gas chromatograph were investigated. As a result, the fermentation time decreased at optimal assimilable nitrogen concentration but increased at poor or rich assimilable nitrogen concentration, the Xanthan gum biosynthesis was stimulated under the limited condition of assimilable nitrogen source and the optimum fermentation medium was obtained as follow; Glucose=30g / l, Peptone=8.0g / l, $K_2HPO_4=2.0g/l$, $MgS0_47H_2O=10g/l$, Sodium acetate=20g/l, Sodium pyruvate=0.5g/1. As the agitation speed and nitrogen concentration increased, the $O_2$ uptake rate and $CO_2$ evolution rate increased. The OUR and CER were 37.3mmol $O_2/\;l$ hr and 20.2 mmol $CO_2/\;L$ hr at peptone 11g / l and agitation speed 990RPM, respectively. In fed batch fermentation, the final concentration of Xanthan gum was enhanced up to 29g / l.

• 한국미생물·생명공학회지
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• 제31권1호
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• pp.46-50
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• 2003

젓갈에서 분리된 Lactococcus lactis SA72균주로부터 유용 박테리오신인 lacticin SA72의 생산을 최적화하기 위해 배양용 배지 및 배양조건을 검토하였다. 유산균의 대표적인 배양 배지인 BHI, MRS, Ml7배지에 대해서 검토한 결과, 이중 MRS배지가 최적 배지로 결정되었다. 이 MRS배지에 0.5% glucose를 첨가한 경우, 더 높은 활성을 얻을 수 있었다. pH의 영향을 검토하였을 때는, 초기 pH에서는 pH 7이었고, 발효조에서의 배양기간 동안 pH 6으로 조절할 때가 높은 활성을 보였다. 배양온도는 $32^{\circ}C$로 유지할 때가 활성이 우수하였다. 회분식 배양에서는 최대 3,200AU/m1을 얻을 수 있었으며, 유가식 배양으로 10g/1 glucose를 주기적으로 공급하였을 때 , 약 6,400 AU/ml로 가장 높은 활성을 얻을 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1178-1185
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• 2022

Steroids are a class of compounds with cyclopentane polyhydrophenanthrene as the parent nucleus, and they usually have unique biological and pharmacological activities. Most of the biosynthesis of steroids is completed by a series of enzymatic reactions starting from cholesterol. Synthetic biology can be used to synthesize cholesterol in engineered microorganisms, but the production of cholesterol is too low to further produce other high-value steroids from cholesterol as the raw material and precursor. In this work, combinational strategies were established to increase the production of cholesterol in engineered Saccharomyces cerevisiae RH6829. The basic medium for high cholesterol production was selected by screening 8 kinds of culture media. Single-factor optimization of the carbon and nitrogen sources of the culture medium, and the addition of calcium ions, zinc ions and citric acid, further increased the cholesterol production to 192.53 mg/l. In the 5-L bioreactor, through the establishment of strategies for glucose and citric acid feeding and dissolved oxygen regulation, the cholesterol production was further increased to 339.87 mg/l, which was 734% higher than that in the original medium. This is the highest titer of cholesterol produced by microorganisms currently reported. The fermentation program has also been conducted in a 50-L bioreactor to prove its stability and feasibility.

• 한국미생물·생명공학회지
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• 제28권6호
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• pp.355-360
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• 2000

Candida rugosa IFO0750의 UV-변이주를 제조하여 butyric acid를 D-$\beta$-hydroxybutyrin acid(이하 D-$\beta$-HBA)로 전환하는 데 이용하였다. 후보 변이주 중 활성이 가장 높은 Candida rugosa CM42를 이용하여 발효를 수행한 후 NMR 분석, polarimeter 분석 등을 통하여 생성된 물질이 D-$\beta$-HBA 임을 확인하였다. Chemostar 배양을 이용하여 D-$\beta$-HBA 발효 생산의 주요 영향인자를 분석하였으며, 균체의 활성을 나타내는 비생산성의 최대치는 균체의 비증식 속도를 0.06, 발효조 내의 glucose와 butyric acid의 농도를 각각 10g/L와 8.7 g/L로 각각 유지 할 때 얻어졌다. 회분식 배양 중에 glucose와 butyric acid를 공급하여 발효조 내의 glucose 및 butyric acid 농도를 최적조건으로 유지하는 fed-batch 발효를 수행하였다. 배양 180 시간 후에 D-$\beta$-HBA 농도가 약 12.4 g/L에 도달하였으며 회분식 발효에 비하여 4.7배 증가하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권1호
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• pp.43-51
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• 2002

The cloning and expression of $\beta-glucosidase$ II, encoded by the gene ${\beta}glu2$, from thermotolerant yeast Pichia etchellsii into Escherichia coli is described. Cloning of the 7.3 kb BamHI/SalI yeast insert containing ${\beta}glu2$ in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid into E. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At $50^{\circ}C$, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of $0.14\;h^{-l}$. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

• 산업식품공학
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• 제14권3호
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• pp.217-221
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• 2010

플라스크 실험과 fermentor 발효실험으로 분리균주인 Acetobacter aceti B20 균주의 초산발효를 위한 몇가지 조건을 최적화하였다. 통기교반 조건이 제한된 flask 실험에서 B20 균주의 생육은 에탄올 농도에 민감하게 반응하여 4%의 에탄올 농도에서는 거의 생육이 되지 않았으며, 초산 생성량도 미미하였다. Flask 배양에서 B20 균주의 생육은 포도당 농도가 3%일 때 가장 좋았으나 농도가 증가할 수록 생육이 저해되었다. ${27^{\circ}C}$와 ${30^{\circ}C}$의 온도에서 A. aceti B20 균주의 생육과 초산생성은 유사하였으며, 이보다 낮거나 높은 온도에서는 생육과 초산 생성이 모두 저하되었다. B20 균주의 최적 발효온도는 $27{\pm}3^{\circ}C$ 범위로 생각된다. Fermentor의 교반속도가 높아질수록 B20 균주의 생육도와 초산생성량이 증가하여 500 rpm일 때 초산농도 5.34%, 발효수율은 57.2%이었다. Batch식 발효에서 초기 에탄올 농도가 7%일 때 발효 120시간째 산도가 5.34%로 가장 높았으며, 이 때의 발효수율은 56.1%로 가장 양호하였다. Fedbatch식 발효에서 초산농도는 2회 feeding할 때 144시간째 8.76%로 최고에 도달하였으며, 이 때 발효수율은 50.6%로 feeding 횟수가 증가할수록 낮게 나타났다.

• Journal of Microbiology and Biotechnology
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• 제34권3호
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• pp.710-724
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• 2024

Flavobacterium can synthesize xanthophyll, particularly the pigment zeaxanthin, which has significant economic value in nutrition and pharmaceuticals. Recently, the use of carotenoid biosynthesis by bacteria and yeast fermentation technology has shown to be very efficient and offers significant advantages in large-scale production, cost-effectiveness, and safety. In the present study, JSWR-1 strain capable of producing xanthophyll pigment was isolated from a freshwater reservoir in Wanju-gun, Republic of Korea. Based on the morphological, physiological, and molecular characteristics, JSWR-1 classified as belonging to the Flavobacterium species. The bacterium is strictly aerobic, Gram-negative, rod-shaped, and psychrophilic. The completed genome sequence of the strain Flavobacterium sp. JSWR-1 is predicted to be a single circular 3,425,829-bp chromosome with a G+C content of 35.2% and 2,941 protein-coding genes. The optimization of carotenoid production was achieved by small-scale cultivation, resulting in zeaxanthin being identified as the predominant carotenoid pigment. The enhancement of zeaxanthin biosynthesis by applying different light-irradiation, variations in pH and temperature, and adding carbon and nitrogen supplies to the growth medium. A significant increase in intracellular zeaxanthin concentrations was also recorded during fed-batch fermentation achieving a maximum of 16.69 ± 0.71 mg/l, corresponding to a product yield of 4.05 ± 0.15 mg zeaxanthin per gram cell dry weight. Batch and fed-batch culture extracts exhibit significant antioxidant activity. The results demonstrated that the JSWR-1 strain can potentially serve as a source for zeaxanthin biosynthesis.