• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.327-332
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• 1994

Optimum conditions for the production of acetoin and ammonia as the precursors of tetramethylpyrazine(TMP) were determined using Lactococcus lactis subsp. lactis biovar. diacetylactis FC1 in a modified Lactose-citrate broth containing galactose, citrate, and arginine. The cell growth and the productivity of acetoin and ammonia were remarkably increased in an aerobic culture with 10 $\mu M$ of hematin. For the optimum conditions of acetoin and ammonia production, the concentration of citrate and arginine were adjusted to 156 mM and 50 mM after 18 hr cultivation, and citrate and galactose to 156 mM and 50 mM after 36 hr cultivation, respectively. In these conditions, acetoin and ammonia were produced to the final concentration of 127 mM and 195 mM, which were the highest concentations, respectively. The optimum conditions of the TMP production were also determined as follows; 4 hours at 121, pH 8.3, and the maximal yield of TMP under these conditions was 0.81 g/l.

• Journal of Microbiology and Biotechnology
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• 제22권3호
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• pp.371-377
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• 2012

The present study describes medium-chain-length polyhydroxyalkanoates (mcl-PHAs) production by the Pseudomonas Gl01 strain isolated from mixed microbial communities utilized for PHAs synthesis. A two-step fed-batch fermentation was conducted with glucose and waste rapeseed oil as the main carbon source for obtaining cell growth and mcl-PHAs accumulation, respectively. The results show that the Pseudomonas Gl01 strain is capable of growing and accumulating mcl-PHAs using a waste oily carbon source. The biomass value reached 3.0 g/l of CDW with 20% of PHAs content within 48 h of cultivation. The polymer was purified from lyophilized cells and analyzed by gas chromatography (GC). The results revealed that the monomeric composition of the obtained polyesters depended on the available substrate. When glucose was used in the growth phase, 3-hydroxyundecanoate and 3-hydroxydodecanoate were found in the polymer composition, whereas in the PHAs-accumulating stage, the Pseudomonas Gl01 strain synthesized mcl-PHAs consisting mainly of 3-hydroxyoctanoate and 3-hydroxydecanoate. The transcriptional analysis using reverse-transcription real-time PCR reaction revealed that the phaC1 gene could be transcribed simultaneously to the phaZ gene.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1938-1944
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• 2008

The procpb gene encoding human procarboxypeptidase B (proCPB, GeneBank access code AJ224866) was cloned and its Pichia expression plasmid, $pPIC9{\alpha}$/hproCPB (9.2 kb), was constructed, in which procpb was under the control of the AOXl promoter and connected to the downstream of the mating factor ${\alpha}$-1 ($MF{\alpha}1$) signal sequence. The plasmid was linearized by digestion with Sacl, and integrated into the genome of P. pastoris strain GS115. By culturing of Pichia transformant on methanol medium, the human proCPB was successfully expressed and secreted into the culture supernatant. Moreover, Western blot analysis of the extracellular proteins showed proCPB bands clearly at a molecular mass of 45 kDa, confirming the expression of proCPB with its right size. The CPB activity reached about 3.5 U/ml and 12.7 U/ml in the flask and fermentor batch cultures of Pichia transformant, respectively. No CPB enzyme activity was found in the intracellular fraction. When the fed-batch cultivation was performed with methanol and glycerol mixture as a feeding medium, the extracellular CPB activity was increased to 42.0 U/ml, which corresponds to a 3.3-fold higher level of CPB activity than that of batch culture. The $K_m$ and $k_{cat}$ values of recombinant human CPB enzyme for hippuryl-$_L$-Arg as a substrate were estimated to be 0.16 mM and $11.93\;sec^{-1}$, respectively.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권3호
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• pp.138-149
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• 2002

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stage etc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.308-311
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• 2006

In an attempt to increase production of LK8, an 86-amino-acid kringle fragment of human apolipoprotein(a) with three disulfide linkages, protein disulfide isomerase (PDI) was coexpressed in recombinant Saccharomyces cerevisiae harboring the LK8 gene in the chromosome. Whereas overexpression of the LK8 gene without coexpressing PDI was detrimental to both host cell growth and LK8 production, coexpression of PDI increased the LK8 production level by 2.5-fold in batch cultivation and 5.0-fold in fed-batch cultivation compared with the control strain carrying only the genomic PDI gene.

• 한국미생물·생명공학회지
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• 제30권1호
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• pp.68-72
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• 2002

RNA 함량이 증가되고, 증식속도가 더 빠른 효모 변이주를 선별하기 위해, 모균주 Saccharomyces cerevisiae MTY62 세포에 화학적 돌연변이제인 ethylmethane sulfonate를 처리하여, YPD 배지에서는 잘 자라고 KCl 함유 배지에서는 자라지 않는 변이주들을 선별하였다. 이 변이주들 중 시험관 및 플라스크 배양을 통해 균체농도와 RNA 함량이 모균주 MTY62에 비해 각각 1.5배, 1.3배 증가한 M40-10 변이주를 최종적으로 선별하였다. 변이주 M40-l0을 발효조 회분배양한 결과, 최대비증식속도는 $0.38 h^{-1}$ , RNA 농도는 3210 mg-RNA/1, RNA 함량은 183mg-RNA/g-DCW 값을 보여, 모균주에 비해 각각 23%, 15%, 12%씩 증가하였다. M40-10 변이주를 간헐적 유가배양한 결과, 최대 균체농도는 35.6 g-DCW/1을, 최대 RNA 농도는 5677mg-RNA/l을, RNA함량은 160 mg-RNA/g-DCW을 나타내어 모균주보다 우수하였다. 일정속도의 유가배양에서도 M40-10 변이주의 최대 균체농도는 46.4g-DCW/1, RNA 농도는 6270mg-RNA/1, RNA 함량은 135mg-RNA/g-DCW을 보였다. 이들 유가배양에서 배양 중반기인 20시간 전후에서는 모균주에 비해 변이주의 세포농도는 30%, RNA 농도는 10% 정도 증가되었다. 또한 유가배양 말기까지도 RNA 분해는 거의 일어나지 않아, M40-10 변이주는 산성 RNase 활성이 크게 감소한 변이주임을 알 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.719-722
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• 2001

Silk proteins from Nephila clavipes are fibrous proteins containing repetitive sequences with both crystalline and amorphous domains. In order to obtain high-level production of silk protein, the synthetic genes had 16 contiguous units of the consensus repeat sequence of the silk protein were expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter. For production of recombinant silk protein in large amounts, pH-stat fed-batch cultures were carried out. The recombinant silk protein was produced as soluble forms in E. coli, and the recombinant silk protein content was as high as 11% of the total protein. When cells were induced at $OD_{600}$ of 60, the amount of silk protein produced was 6.49 g/L. After simple purification steps, 9.2 mg of silk protein that was more than 80% pure was obtained from a 50 mL culture, and the recovery yield was 26.3%.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.641-646
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• 2003

An accurate and efficient method for measuring the mass of hairy roots using conductometry is established. A conductivity equation expressed in terms of the concentration of the ion species in the medium is suggested. By using this equation, the effect of the individual ions on the total conductivity can be quantitatively analyzed. An equation for the in situ estimation of the cell growth coefficient for determining the mass of hairy roots is established based on measurements of the nitrogen concentration and conductivity during cultivation. The proposed equation does not require preliminary experiments to determine the cell growth coefficient. Instead, the physiological characteristics of the plant species are reflected by introducing the cellular nitrogen content. Since the cell growth coefficient is determined by measuring the major ionic nutrient concentrations, it is more effective to express the dynamics of an actual culture system. This improved method for determining the mass of hairy roots was successfully utilized in a fed-batch culture system.

• Journal of Microbiology and Biotechnology
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• 제1권2호
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• pp.126-129
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• 1991

In order to produce ${\gamma}-linolenic$ acid by Mucor sp. KCTC 8405P. the fungus was cultivated in fed-batch culture with two phases. i.e., growth in yeast-like form and induction to hyphal growth by pH shift of the culture medium during cultivation. The synchronous growth of the fungus into the appropriate sizes was important for the high density cell culture of this dimorphic fungus. Dissolved oxygen concentration in the medium did not affect degree of unsaturation of fatty acids and ${\gamma}-linolenic$ acid content. Under the culture conditions applied in this experiment. the fungus was found to produce 100 g/l dry mycelia containing 40% of the lipids, where ${\gamma}-linolenic$ acid comprised about 9% of the total extractable fatty acids.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권5호
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• pp.303-305
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• 2003

Scale-up of hirudin production from Saccharomyces cerevisiae from bench-scale to pilot-scale was carried out based on constant volumetric oxygen transfer coefficient (K$\sub$L/a). Fed-batch mode of cultivation using step-wise feeding strategy of galactose was employed for the production of hirudin in a 30-L and a 300-L pilot-scale fermentor. The final hirudin concentrations were achieved 390 mg/L and 286.1 mg/L, and the volumetric productivities were 80.4% and 90.7% with the 30-L and 300-L fermentors, respectively, compared to the productivity of the 5-L bench-scale fermentor.