• Journal of Plant Biotechnology
• /
• 제36권4호
• /
• pp.309-319
• /
• 2009

Transgenic plant cell cultures for the production of biopharmaceuticals including monoclonal antibodies, recombinant proteins have been regarded as an alternative platform in addition to traditional microbial fermentation and mammalian cell cultures. Plant-made pharmaceuticals (PMPs) have several advantages such as safety, cost-effectiveness, scalability and possibility of complex post-translational modifications. Increasing demand for the quantity and diversity of pharmaceutical proteins may accelerate the industrialization of PMP technology. Up to date, there is no plant-made recombinant protein approved by USFDA (Food and Drug Administration) for human therapeutic uses due to the technological bottlenecks of low expression level and slight differences in glycosylation. Regarding expression levels, it is possible to improve the productivity by using stronger promoter and optimizing culture processes. In terms of glycosylation, humanization has been attempted in many ways to reduce immune responses and to enhance the efficacy as well as stability. In this review article, all these respects of transgenic plant cell cultures were summarized. In addition, we also discuss the general characteristics of plant cell suspension cultures related with bioreactor design and operation to achieve high productivity in large scale which could be a key to successful commercialization of PMPs.

• Journal of Microbiology and Biotechnology
• /
• 제2권2호
• /
• pp.92-97
• /
• 1992

Microbial transformation of (-)kaur-l6-en-19-oic acid and (-)pimara-9(1l), 15-dien-19-oic acid from A. koreanum was investigated. Throughout the screening of the capability of metabolizing these bioactive diterpenoids, two microorganisms have chosen among various fungi and streptomycetes tested. Scale-up fermentation with Fusarium oxysporum KCTC 6051 produced two metabolites related to the precursor diterpenoids. The two metabolites were isolated by column chromatography and identified by chemical and spectroscopic methods as $2\beta$, $16\alpha$-dihydroxy kauran-19-oic acid and $16\alpha$-hydroxy kauran-19-oic acid. However any microorganisms capable to transform (-) pimara-9(11), 15-dien-19-oic acid was not screened in this condition.

• Journal of Microbiology and Biotechnology
• /
• 제27권5호
• /
• pp.965-974
• /
• 2017

The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and response surface methodology, under which the yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature, and particle diameter size were 1.01E-07 M, $55.2{\pm}0.3^{\circ}C$, and 9.388 nm for the scFv expressed by B. choshinensis, and 4.53E-07 M, $52.5{\pm}0.3^{\circ}C$, and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
• /
• pp.60-61
• /
• 2004

Metabolic engineering is now a well established discipline, used extensively to determine and execute rational strategies of strain development to improve the performance of micro-organisms employed in industrial fermentations. The basic principle of this approach is that performance of the microbial catalyst should be adequately characterised metabolically so as to clearlyidentify the metabolic network constraints, thereby identifying the most probable targets for genetic engineering and the extent to which improvements can be realistically achieved. In order to harness correctly this potential, it is clear that the physiological analysis of each strain studied needs to be undertaken under conditions as close as possible to the physico-chemical environment in which the strain evolves within the full-scale process. Furthermore, this analysis needs to be undertaken throughoutthe entire fermentation so as to take into account the changing environment in an essentially dynamic situation in which metabolic stress is accentuated by the microbial activity itself, leading to increasingly important stress response at a metabolic level. All too often these industrial fermentation constraints are overlooked, leading to identification of targets whose validity within the industrial context is at best limited. Thus the conceptual error is linked to experimental design rather than inadequate methodology. New tools are becoming available which open up new possibilities in metabolic engineering and the characterisation of complex metabolic networks. Traditionally metabolic analysis was targeted towards pre-identified genes and their corresponding enzymatic activities within pre-selected metabolic pathways. Those pathways not included at the onset were intrinsically removed from the network giving a fundamentally localised vision of pathway functionality. New tools from genome research extend this reductive approach so as to include the global characteristics of a given biological model which can now be seen as an integrated functional unit rather than a specific sub-group of biochemical reactions, thereby facilitating the resolution of complexnetworks whose exact composition cannot be estimated at the onset. This global overview of whole cell physiology enables new targets to be identified which would classically not have been suspected previously. Of course, as with all powerful analytical tools, post-genomic technology must be used carefully so as to avoid expensive errors. This is not always the case and the data obtained need to be examined carefully to avoid embarking on the study of artefacts due to poor understanding of cell biology. These basic developments and the underlying concepts will be illustrated with examples from the author's laboratory concerning the industrial production of commodity chemicals using a number of industrially important bacteria. The different levels of possibleinvestigation and the extent to which the data can be extrapolated will be highlighted together with the extent to which realistic yield targets can be attained. Genetic engineering strategies and the performance of the resulting strains will be examined within the context of the prevailing experimental conditions encountered in the industrial fermentor. Examples used will include the production of amino acids, vitamins and polysaccharides. In each case metabolic constraints can be identified and the extent to which performance can be enhanced predicted

• 한국미생물·생명공학회지
• /
• 제10권1호
• /
• pp.9-14
• /
• 1982

유산균의 동결보존에 있어서 활성변화를 시험하고, 유산균을 종균으로 사용하는 생산공장등 에서 종균의 활성이 감소되지 않고 장기간 보존할 수 있는 방법을 검토하였다. 10% 환원탈지유 10$m\ell$에 전배양한 시험균액을 1 백금이 접종하여 냉장고의 동결고에 4개월간 저장하면서 starter로서의 활성을 측정하기 위하여 융해후 1대와 융해후 2대로 구분하였다. 양결란주를 적온에서 48시간 배양한 것을 융해후 1대로 하고 이것을 10% 멸균환원탈지유 10$m\ell$에 계대배양한 것을 융해후 2대로 하였다. 본실험의 결과 다음과 같은 결과를 얻었다. 1. L. casei는 동결보존에 대하여 매우 안정하며 동결 120일간의 생균수와 산도에 있어서, 각각 99.88%, 98.93%의 활성을 유지하였다. 2. L. casei의 융해후 2대의 동결난수를 발효유의 mother starter로 사용하여 culture starer, seed starter를 제조한 결과 동결 120일째의 것도 충분한 활성도를 나타내었다. 3. L. bulgaricus는 동결기간의 경과에 따라 생균수보다 산생성력의 감소현상이 크게 나타났으나 융해후 2차에는 회복되어, 120일째의 산도가 96.20%를 유지하였다. 4. Str. lactis의 융해후 2대는 동결 120일째에 생균수와 산도가 대조균에 비하여 99.50%, 96.63%를 나타내었다.

• Journal of Ginseng Research
• /
• 제42권2호
• /
• pp.199-207
• /
• 2018

Background: Ginseng is a well-known traditional Chinese medicine that has been widely used in a range of therapeutic and healthcare applications in East Asian countries. Microbial transformation is regarded as an effective and useful technology in modification of nature products for finding new chemical derivatives with potent bioactivities. In this study, three minor derivatives of ginsenoside compound K were isolated and the inducing effects in the Wingless-type MMTV integration site (Wnt) signaling pathway were also investigated. Methods: New compounds were purified from scale-up fermentation of ginsenoside Rb1 by Paecilomyces bainier sp. 229 through repeated silica gel column chromatography and high pressure liquid chromatography. Their structures were determined based on spectral data and X-ray diffraction. The inductive activities of these compounds on the Wnt signaling pathway were conducted on MC3T3-E1 cells by quantitative real-time polymerase chain reaction analysis. Results: The structures of a known 3-keto derivative and two new dehydrogenated metabolites were elucidated. The crystal structure of the 3-keto derivative was reported for the first time and its conformation was compared with that of ginsenoside compound K. The inductive effects of these compounds on osteogenic differentiation by activating the Wnt/b-catenin signaling pathway were explained for the first time. Conclusion: This study may provide a new insight into the metabolic pathway of ginsenoside by microbial transformation. In addition, the results might provide a reasonable explanation for the activity of ginseng in treating osteoporosis and supply good monomer ginsenoside resources for nutraceutical or pharmaceutical development.

• 한국유기농업학회지
• /
• 제25권1호
• /
• pp.135-148
• /
• 2017

본 연구를 통해 논, 시설재배 밭 토양, 쓰레기장, 하천 및 호수의 퇴적 토양 등 22개소에서 분리한 총 6종의 광합성세균 중 호기 암 배양이 가능한 Rhodobacter sphaeroides PS-24를 분리하였다. 형태학적 특징으로는 그람음성의 막대모양으로, 운동성이 있었다. 분리균주의 16S rRNA 염기서열을 분석한 결과 Rhodobacter sphaeroides ATH2.4.1과 99%의 상동성을 나타내었으며, 본 연구에서 Rhodobacter spharoides PS-24로 명명하여 연구를 수행하였다. 선별균주를 modifed Van niel's yeast 배지에서 배양 후 생성된 carotenoid를 추출한 결과 $12.03{\pm}0.15mg/L$의 함량이 측정되었으며, 반응표면분석법 중 Plackett burman 분석방법과 Box-Behnken 분석방법을 통해 carotenoid 생산에 영향을 미치는 요인을 분석하고 농도를 최적화하였다. 분석결과 각각의 독립변수 yeast extract -0.4144 (1.23 g/L), $Na_2CO_3$ 0.8541 (3.71 g/L)와 $MgSO_4$ 1.00 (1.00 g/L)의 농도를 선정하였으며, 이를 바탕으로 배지 조성을 최적화한 결과 yeast extract 1.23 g, sodium acetate 1 g, $NH_4Cl$ 1.75 g, NaCl 2.5 g, $K_2HPO_4$ 2 g, $MgSO_4$ 1.0 g, mono-sodium glutamate 7.5 g, $Na_2CO_3$ 3.71 g, $NH_4Cl$ 3.5 g, $CaCl_2$ 0.01 g/ liter로 선정하였다. 최적배지를 대상으로 5 L, 50 L, 500 L scale-up을 진행한 결과 최종 carotenoid는 각각 17.98 mg/L, 18.03 mg/L, 18.11 mg/L로 조사되었다. 최적배지의 경우 modified Van niel's yeast 배지보다 약 1.5배 많은 carotenoid를 생산하였으며, 대량배양을 통한 scale-up 과정 시 carotenoid의 생산량은 크게 변화하지 않는 것으로 조사되었다. 따라서 본 연구를 바탕으로 산업적으로 다양하게 사용되고 있는 carotenoid를 생산하는 광합성세균 Rhodobacter spharoides PS-24를 개발하였으며, 본 연구를 바탕으로 유기농축산에 사용이 가능한 기능성 미생물제제를 개발하고자 한다.

• Korean Chemical Engineering Research
• /
• 제46권3호
• /
• pp.451-464
• /
• 2008

삼상 역 유동층은 유동하거나 부유하는 입자의 크기가 매우 작은 경우나 유동입자의 밀도가 액체보다 작은 담체나 접촉매체 또는 촉매전달물질인 경우에 생물반응기, 발효공정, 폐수처리공정, 흡착, 흡수공정 등에 매우 효과적으로 사용될 수 있어서 그 적용성은 날로 증대되고 있다. 그러나, 삼상 역 유동층에 대해서는 많은 연구가 진행되지 않아 왔으며 수력학적 특성에 대한 연구조차도 미흡한 실정이다. 삼상 역 유동층을 이용한 많은 종류의 반응기와 공정들의 운전과 설계 그리고 scale-up을 위해서는 삼상 역 유동층에서 수력학적 특성과 열전달과 물질전달과 같은 이동현상에 대한 정보는 필수적이라는 것은 자명한 사실이다. 따라서, 본 총설에서는 삼상 역 유동층에 대한 정보들을 공학적 측면에서 요약하고 재정리하여서 이 분야의 현장에서 필요한 지식들을 제안하고자 하였다. 본 논문은 수력학적 특성, 열전달 특성 그리고 물질전달 특성의 세 부분으로 이루어져있다. 즉, 수력학적 특성 부분에서는 운전변수가 상 체류량, 기포의 특성 그리고 유동입자의 분산에 미치는 영향을 검토하였으며, 열전달 특성 부분에서는 삼상 역 유동층에서의 운전변수가 열전달 계수에 미치는 영향을 고찰하였고, 열전달 모델에 대한 정리를 하였으며, 물질전달 특성 부분에서는 운전변수가 연속액상의 축방향 분산계수 및 액상 부피물질전달계수에 미치는 영향에 대해 고찰하였다. 또한, 각 절에서 유동입자의 최소유동화속도, 상 체류량, 기포특성, 유동입자의 요동빈도수 및 유동입자의 분산 등과 같은 수력학적 특성과 열전달 계수 그리고 연속액상의 축방향 확산계수와 물질전달계수 등을 예측할 수 있는 상관식들을 제안하였다. 본 총설의 마지막 절에서는 삼상 역 유동층의 공업적 응용을 위해 앞으로 더 연구해야하는 내용에 대해 제안을 하였다.

• The Plant Pathology Journal
• /
• 제33권3호
• /
• pp.337-344
• /
• 2017

To develop a commercial product using the mycoparasitic fungus Simplicillium lamellicola BCP, the scale-up of conidia production from a 5-l jar to a 5,000-l pilot bioreactor, optimization of the freeze-drying of the fermentation broth, and preparation of a wettable powder-type formulation were performed. Then, its disease control efficacy was evaluated against gray mold diseases of tomato and ginseng plants in field conditions. The final conidial yields of S. lamellicola BCP were $3.3{\times}10^9conidia/ml$ for a 5-l jar, $3.5{\times}10^9conidia/ml$ for a 500-l pilot vessel, and $3.1{\times}10^9conidia/ml$ for a 5,000-l pilot bioreactor. The conidial yield in the 5,000-l pilot bioreactor was comparable to that in the 5-l jar and 500-l pilot vessel. On the other hand, the highest conidial viability of 86% was obtained by the freeze-drying method using an additive combination of lactose, trehalose, soybean meal, and glycerin. Using the freeze-dried sample, a wettable powder-type formulation (active ingredient 10%; BCP-WP10) was prepared. A conidial viability of more than 50% was maintained in BCP-WP10 until 22 weeks for storage at $40^{\circ}C$. BCP-WP10 effectively suppressed the development of gray mold disease on tomato with control efficacies of 64.7% and 82.6% at 500- and 250-fold dilutions, respectively. It also reduced the incidence of gray mold on ginseng by 65.6% and 81.3% at 500- and 250-fold dilutions, respectively. The results indicated that the new microbial fungicide BCP-WP10 can be used widely to control gray mold diseases of various crops including tomato and ginseng.

• 한국축산식품학회지
• /
• 제31권6호
• /
• pp.928-934
• /
• 2011

본 연구에서는 파일럿 스케일의 에멘탈치즈를 제조하였으며, 치즈제조 과정에서 PAB를 첨가하지 않은 치즈를 대조군으로 사용하여 숙성 중 lactic acid의 대사와 지방의 가수분해에 의한 화학적 변화를 연구하였다. 실험결과 에멘탈치즈의 숙성 중 lactic acid는 최초 9.39 g/kg에서 숙성종료(90일)까지 2.56 g/kg으로 감소하였으며, 1.48 g/kg의 acetic acid와 6.11 g/kg의 propionic acid를 생성하였다. 반면 대조군은 숙성 종료일(90일)에 lactic acid의 함량이 15.96 g/kg까지 증가하였으며, acetic acid와 propionic acid는 각각 0.25 g/kg과 0.09 g/kg이 생성됨에 따라 에멘탈치즈가 숙성 중 PAB에 의한 propionic acid 발효특성을 확인하였다. 숙성 중 유리지방산 분석 결과 숙성 종료일(90일)에 에멘탈치즈의 총 유리지방산 함량은 6,628.2 mg/kg이었으며, 대조군의 총 유리지방산 함량은 1,605.4 mg/kg으로서 에멘탈치즈에 사용된 PAB가 높은 지방분해력을 보였다. 또한 에멘탈치즈에서 유리된 지방산의 조성은 숙성 중 LCFFA(C14:0-C18:2)가 높은 비율을 차지 하였으며, 이 중 palmitate(C16:0), stearate(C18:0) 및 oleate(C18:1)가 주요 지방산이었다.