• 한국미생물·생명공학회지
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• 제37권2호
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• pp.140-146
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• 2009

Cyclosporin A 분자의 구성 아미노산 가운데 분지 아미노산인 valine, leucine, isoleucine, threonine 등에 대한 첨가 효과를 확인한 결과, L-valine 첨가구는 비첨가구보다 cyclosporin A 역가는 3.7배, cyclosporin A 구성비율은 약 40%로 가장 크게 증가하였다. 그리고 L-valine의 최적 농도는 0.75%로서 비첨가구에 비하여 cyclosporin A 역가는 3배, cyclosporin A 구성비율은 30% 증가하였다. 그러나 L-valine 1% 단독 첨가구보다 L-valine 0.5%, L-leucine 0.5% 혼합구가 cyclosporin A 역가에서는 약 16% 우수하였고, cyclosporin A 구성비율에서는 약 5% 증가하였다. 또한 다양한 유기질소원의 첨가 효과를 확인한 결과, meat peptone인 Bacto-peptone 첨가구는 유기질소원 비첨가구보다 cyclosporin A의 역가는 약 2배, cyclosporin A 구성비율은 약 13% 증가하였고, Bacto-peptone의 최적농도는 1%인 것으로 확인되었다. 그러나 Casiton, Tryptone, NZ-amine 등 우유 유래 유기질소원 첨가구의 경우, 비첨가구보다 cyclosporin A 역가가 오히려 약 10-20% 감소하는 현상을 보여 cyclosporin A 생합성을 억제하는 것으로 확인되었다. 본 연구에서 최적화된 배지 조건에서, 모균주인 T. inflatum ATCC 34921와 UV 조사로 얻어진 4종의 cyclosporin A 고생산 변이주 4종의 생산성을 비교한 결과, 변이주 YHC-004의 cyclosporin A 역가는 3,430 mg/L로 모균주인 ATCC 34921보다 약 7.1배로 증가하였지만, 모균주와 변이주 4종의 cyclosporin A 구성비율은 약 93-94% 범위에서 비슷한 값을 보였다.

• Journal of Microbiology and Biotechnology
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• 제19권5호
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• pp.462-467
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• 2009

Cyclosporin A (CyA) produced by Tolypocladium inflatum is a promising drug owing to its immunosuppressive and antifungal activities. From an industrial point of view, the necessity to obtain a suitable and economic medium for higher production of CyA was the aim of this work. The present study evaluated the effect of different fermentation parameters in solid state fermentation, such as selection of solid substrate, hydrolysis of substrates, initial moisture content, supplementation of salts, additional carbon, and nitrogen sources, as well as the inoculum age and size, on production of CyA by Tolypocladium inflatum MTCC 557. The fermentation was carried out at $25{\pm}2^{\circ}C$ for 9 days. A combination of hydrolyzed wheat bran flour and coconut oil cake (1:1) at 70% initial moisture content supported a maximum production of $3,872{\pm}156\;mg$ CyA/kg substrate as compared with $792{\pm}33\;mg/kg$ substrate before optimization. Furthermore, supplementation of salts, glycerol (1% w/w), and ammonium sulfate (1% w/w) increased the production of CyA to $5,454{\pm}75\;mg/kg$ substrate. Inoculation of 5 g of solid substrate with 6 ml of 72-h-old seed culture resulted in a maximum production of $6,480{\pm}95\;mg$ CyA/kg substrate.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1055-1060
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• 2001

Experimental data for the on-line estimation of cell concentration and growth rate are presented. For this purpose, we utilized the on-line calculation of the oxygen uptake rate (OUR), which was derived from a liquid phase dynamic mass balance for the oxygen during the active growth phase in cyclosporin A (CyA) fermentation. The cell yield coefficient, based on the oxygen $(Y_{x/o})$for both suspended and immobilized cells of Tolypocladium inflatum, was estimated as $1.9 gDCW/gO_2$ from a very good linear correlation between the cell mass produced and the total oxygen consumed. The calculated yield showed a good agreement with the value of $(Y_{x/o})$ generated from the correlation between the cell growth rate and the oxygen uptake rate. In addition, further experimental data are given, which were also applied to determine the specific oxygen uptake rate of T. inflatum cells during the exponential phase of CyA fermentation. A theoretical basis for the analysis of these fermentation parameters is also provided.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.1930-1936
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• 2007

A sequential optimization strategy based on statistical experimental designs was employed to enhance the production of cyclosporin A (CyA) by Tolypocladium inflatum DSMZ 915 in a submerged culture. A 2-level Plackett-Burman design was used to screen the bioprocess parameters significantly influencing CyA production. Among the 11 variables tested, sucrose, ammonium sulfate, and soluble starch were selected, owing to their significant positive effect on CyA production. A response surface methodology (RSM) involving a 3-level Box-Behnken design was adopted to acquire the best process conditions. Thus, a polynomial model was created to correlate the relationship between the three variables and the CyA yield, and the optimal combination of the major media constituents for cyclosporin A production, evaluated using the nonlinear optimization algorithm of EXCEL-Solver, was as follows (g/l): sucrose, 20; starch, 20; and ammonium sulfate, 10. The predicted optimum CyA yield was 113 mg/l, which was 2-fold the amount obtained with the basal medium. Experimental verification of the predicted model resulted in a CyA yield of 110 mg/l, representing 97% of the theoretically calculated yield.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.128-134
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• 2008

The kinetics of cell growth and Cyclosporin A (Cyc A) production by Tolypocladium inflatum were studied in shake flasks and bioreactors under controlled and uncontrolled pH conditions. In the case of the shake flask, the production time was extended to 226 h and the maximal antibiotic concentration was 76 mg/l. When scaling up the cultivation process to a bioreactor level, the production time was reduced to only 70h with a significant increase in both the cell growth and the antibiotic production. The maximal dry cell weights in the case of the controlled pH and uncontrolled pH cultures in the bioreactor were 22.4g/l and 14.2g/l, respectively. The corresponding maximal dry cell weight values did not exceed 7.25g/l with the shake flask cultures. The maximal values for Cyc A production were 144.72 and 131.4 mg/l for the controlled and uncontrolled pH cultures, respectively. It is also worth noting that a significant reduction was observed in both the dry cell mass and the antibiotic concentration after the Cyc A production phase, whereas the highest rate of antibiotic degradation was observed in the stirred tank bioreactor with an uncontrolled pH. Morphological characterization of the micromorphological cell growth (mycelial/pellet forms) was also performed during cultivation in the bioreactor.

• Journal of Microbiology and Biotechnology
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• 제20권7호
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• pp.1086-1091
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• 2010

This study explored the use of gellan gum as an immobilization matrix for the production of cyclosporin A (CyA) by immobilized spores and mycelia of Tolypocladium inflatum MTCC 557. Different carriers, such as gellan gum, sodium alginate, celite beads, and silica, were tested as immobilization carriers, along with the role of the carrier concentration, biomass weight, number of spore-inoculated beads, and repeated utilization of the immobilized fungus. The maximum CyA production was 274 mg/l when using gellan gum [1% (w/v)], and a mycelial weight of 7.5% (w/v) supported the maximum production of CyA. Additionally, the addition of a combination of $_L$-valine (6 g/l) and $_L$-leucine (5 g/l) after 48 h of fermentation produced 1,338 mg/l of CyA when using gellan gum. The immobilized mycelia beads were found to remain stable for four repetitive cycles, indicating their potential for semicontinuous CyA production.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1385-1392
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• 2009

This work evaluates the effect of different amino acids on production of Cyclosporin (CyA) production in solid-state fermentation that was previously optimized for different fermentation parameters by one factor at-a-time for the maximum production of CyA by Tolypocladium inflatum MTCC557. Based on the Plackett-Burman design, glycerol, ammonium sulfate, $FeCl_3$, and inoculum size were selected for further optimization by response surface methodology (RSM). After identifying effective nutrients, RSM was used to develop mathematical model equations, study responses, and establish the optimum concentrations of the key nutrients for higher CyA production. It was observed that supplementation of medium containing (% w/w) glycerol, 1.53; ammonium sulfate, 0.95; $FeCl_3$, 0.18; and inoculum size 6.4 ml/5g yielded a maximum of 7,106 mg/kg as compared with 6,480 mg CyA/kg substrate using one factor at-a-time. In the second step, the effect of amino acids on the production of CyA was studied. Addition of $_L$-valine and $_L$-leucine in combination after 20 h of fermentation resulted in maximum production of 8,166 mg/kg.

• KSBB Journal
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• 제11권5호
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• pp.613-621
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• 1996

Wild-type 곰팡이인 Tolypoc/adium inflatum의 고정상배양을 통해 탄소원, 질소원, 아미노산 등이 peptide 항생제 계통 의 면역억제제인 cyclosporin A(CyA) 생합성에 미치는 영향을 조사하였다. 질소 원으로서 ammonium sulfate가 첨가된 경우에, f fructose 또는maltose 등을 탄소원으로 이용하는 배 지가 glucose가 첨가된 배지에 비해 월등히 높은 C CyA 생산성을 보였으나. ammonium sulfate의 부 재시에는 탄소원들의 종류에 관계없이 CyA 생산성 이 매우 낮게 나타났다. 질소원의 경우는 무기 질소 원인 ammonium sulfate가 CyA 생산에 가장 훌륭 한 성분으로 작용했으며, 그밖에 ammonium phos p phate, ammonium citrate 역시 CyA 생산을 어느 정도 증가시키는 효과를 보였다. 최척 ammonIum s sulfate의 농도는 109/L로 밝혀 졌으며 배양초기 에 a ammonium sulfate릎 첨가해 주는 경우가 배양중간 에 첨가하는 경우보다 CyA 생산에 더 효율적인 것 으로 나타났다. 아미노산의 경우 L-valine에 의한 C CyA 생합성 증진 효과가 가장 투렷하게 나타났다. 최적 L-valine 농도는 109/L이였으며 L-valine이 배양 초반부터 배지 중에 존재하는 것이 CyA 생합 성에 가장 유리한 것으로 나타났다.

• KSBB Journal
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• 제9권2호
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• pp.200-210
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• 1994

Cyclosporin A(Cy A) 생산을 위한 회분식 생물 반응기 실험에서, 고정상세포를 이용함으로써 액상 배양과 비교할 때 생물공정 개선의 가능성이 있음을 제시하였다. 고농도 배지를 생산균주가 지수기 생장단계인 발효개시 후 139시간에 첨가하였을 때, 고정상배양과 액상배양 모두에서, 균주의 재활성 및 재생장으로 인해 CyA의 생산기간이 연장되어, 발효개시 후 250시간까지 최대 CyA 농도를 유지하였다. 반면에 배지의 첨가가 없는 단순 회분식 배양의 경우, 두 경우 모두 정체생장 단계에서 CyA의 생산성이 빠른 속도로 감소하였다. 주목할 점은 고정상 세포의 경우 CyA수율($Y_{p/x}$)이 고농도 배지를 첨가한 후에도 지수기때의 수율의 80%에 이르는 높은 값을 계속 유지할 수 있었으나, 이와는 대조적으로 액상 세포는 단지 58%만을 유지할 수 있었다. 그 결과 고정상배양의 최대 CyA생산성 이 액상배양과 비교하여 약 2배 정도 증가하였다.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.188-191
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• 2005

Abstract Cyclosporins are a family of clinically-important immunosuppressive cyclic peptides produced by Tolypocladium inflatum. The structural modification of cyclosporins via hydroxylation at various positions of N-methyl leucines in cyclosporin A leads to a dramatic change of their bioactive spectra. Among over 100 soil actinomycetes screened, two actinomycetes species, Sebekia benihana and Pseudonocardia autotrophica, were identified to contain superior cyclosporin A hydroxylation activities. A HPLC-based cyclosporin A hydroxylation assay revealed that each strain possesses distinctive hydroxylation specificity and regiospecificity; mono-hydroxylation at the 4th N-methyl leucine of cyclosporin A by S. benihana, and di-hydroxylations at both 4th and 9th N-methyl leucines of cyclosporin A by P. autotrophica. The conversion yields for cyclosporin A hydroxylation by both S. benihana and P. autotrophica were significantly improved from less than 10% and 18% up to 58% and 45%, respectively, in the optimized culture containing molybdenum with 0.05 g/l of cyclosporin A concentration. An ancymidol-specific inhibition of cyclosporin hydroxylation also suggested that the regiospecific cyclosporin hydroxylation might be catalyzed by a putative cytochrome P450 mono-oxygenase enzyme.