Aspergillus awamori와 Zymomonas mobilis로 구성된 혼합고정화 배양계의 최적 조건

이상원(Sang-Won Lee),박석규(Seok-Kyu Park),손봉수(Bong-Soo Shon),최수철(Soo-Cheul Choi),서권일(Kwon-Il Seo),성낙계(Nack-Kie Sung),김홍출(Hong-Chul Kim) 한국식품영양과학회 1995 한국식품영양과학회지 Vol.24 No.5

Gel bead의 효율적인 이용을 목적으로 산소요구성이 전혀 다른 두 균주로 구성된 혼합고정화 배양계의 개발을 시도하였다. 호기성 균으로서 A. awamori, 혐기성균으로서 Z. mobilis를 사용하여 생전분으로 부터 에탄올 생산을 행한 결과는 다음과 같다. 두 균주의 최적 혼합비율은 A. awamori 1.25×10^9 spore/L-gel, Z. mobilis 0.5g cell/L-gel이었다. 배양이 완료된 후의 gel bead 내의 균주 분포는 각각 혐기부와 호기부, 즉 gel bead의 표면부와 중심부로 생육장소가 구분되어 있었다. A-Z계의 배양에서는 에탄올의 수율이 낮았고, 균사가 gel bead로 부터 노출되었다. 배양 개시 후 36시간째에 배양기의 면전을 특수 제조한 check valve가 부착된 실리콘 plug로 교환하여 혐기적 배양을 행한 A-Z 36계에서는 gel bead로 부터 균사의 누출이 상당히 억제되었고, pH가 4.3을 유지하면서, 에탄올의 수율이 대조구 보다 약 2배 높게 나타났다. Co-immobilized mixed culture system(A-Z system) composed of two different oxygen-demanding strains, aerobic(Aspergillus awamori) and anaerobic(Zymomonas mobilis) strains, in a Ca-alginate gel beads was developed to increase ethanol production from raw starch as a carbon source. Optimal mixture ratio of A. awamori and Z. mobilis was 1.25×10^9 pores/L-gel and 0.5g cells/L-gel, respectively. After 120 hours of cultivation, gel beads distinguished oxygen-rich surface for A. awamorifrom oxygen-deficient central part for Z. mobilis. At A-Z culture system, yield of ethanol on glucose, Y_(p/s)=0.18, was very low and there was high leakage of cells from surface of gel beads. At A-Z 36 culture system with changing silicon check valve for cotton plug at 36 hours in A-Z culture system, there was no cell leakage from gel beads, pH was maintained at around 4.3 during cultivation, and yield of ethanol on glucose, Y_(p/s)=0.36, showed 2 times higher than that of control culture system (cotton plug culture).

Aspergillus awamori와 Zymomonas mobilis로 구성된 혼합고정화 배양계의 에탄올 생산에 미치는 Neupectin-L의 영향

이상원,조용운,김홍출,박석규,성낙계 ( Sang Won Lee,Yong Un Cho,Hong Chul Kim,Seok Kyu Park,Nack Kie Sung ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.2

In order to reduce energy input in direct ethanol production from raw starch by co-immobilized Aspergillus awamori(A) and Zymomonas mobilis(Z), A-Z 36 culture system which was changed to anaerobic after 36 h of aerobic fermentation without sterilization was investigated. This immobilized cell system can not be carried out under unsterile conditions because of growth of microbial contaminants from original medium. Among some food additives such as sorbic acid, benzoic acid, dehydroacetic acid, p-hydroxybenzoic acid, Vantocil IB and Neupectin-L, Vantocil IB and Neupectin-L were a potent antibacterial agent in A-Z 36 culture cell system and were not affected in hydrolysis of substrate as compared with the case of control. Ethanol yield(6.9 g/ℓ) in system of addition of 0.1% Neupectin-L was slightly higher than that in control(6.4 g/ℓ). When 2% starch was fed five times in fed-batch culture with 0.1% Neupectin-L, ethanol yield and productivity were 34 g/ℓ and 2.0 g/ℓ/day, respectively.

Actinomycetes GF 155-2 에 의한 pepsin 저해물질의 생산 및 정제

박석규(Seok - Kyu Park),성낙계(Nack - Kie Sung),이상원(Sang - Won Lee) 한국미생물생명공학회 1989 한국미생물·생명공학회지 Vol.17 No.2

Rhizopus japonicus와 Zymomonas mobilis의 혼합고정화 배양계에 의한 생전분으로부터 에탄올 생산

최수철(Soo-Cheol Choi),이상원(Sang-Won Lee),박석규(Seok-Kyu Park),성찬기(Chan-Ki Sung),손봉수(Bong-Soo Shan),성낙계(Nack-Kie Sung) 한국식품영양과학회 1996 한국식품영양과학회지 Vol.25 No.4

호기성의 Rhizopus japonicus와 협기성 Zymomonas mobilis로 구성된 혼합고정화 배양계(R-Z계)를 제조하고, 생전분으로부터 에탄을 생산에 응용하였다. R. japonicus를 고정화배양하므로서 액체배양에서 보다 2배 높은 glucose량을 얻었다. R-Z계의 에탄을 생산량은 1.67g/L(Y_(P/S), 0.094)이었지만, 배양 24시간째부터 산소공급을 억제한 R-Z 24계에서는 6.54g/L(Y_(P/S), 0.38)의 에탄올을 얻어 대조구의 약 4배를 향상시켰다. 회분배양에서는 5%의 기질 농도가 적당하였으며, 생산된 에탄올은 15.02g/L(Y_(P/S), 0.36)이었다. 2% 기질을 5회 첨가한 유가배양에서는 2%기질의 회분배양과 동등한 수율인 0.38을 얻었다. Ethanol production from raw starch was performed using the co-immobilized culture system of Rhizopus japonicus and Zymomonas mobilis(R-Z). Glucose production in immobilized R. japonicus culture was 2-fold higher than that in free cell culture. Ethanol production was 1.67g/L(Y_(P/S), 0.094) and 6.54g/L(Y_(P/S). 0.38) in R-Z and R-Z 24 culture system, respectively. R-Z system was modified and designated as R-Z 24 system by replacing cotton plug with silicon check valve after 24h fermentation with R-Z system. Optimal substrate concentration for ethanol production in batch culture was 5%(w/v) and ethanol concentration produced was 15.02g/L(Y_(P/S), 0.36). Ethanol yield(Y_(P/S), 0.38) in fed-batch culture of 5 times with 2%(w/v) substrate was equal to that in batch culture of 2% (w/v) substrate.

섬유소 분해효소를 생성하는 Aspergillus wentii 와 Aspergillus nidulans의 원형질체 융합

성낙계,이상원,정영철,강신권,노종수 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.5

Asp. wentii와 Asp. nidulans의 원형질체 재생은 2-DG가 30㎍/㎖ 첨가된 포자현탁액을 4시간 전배양할 때 적당하였고 ergosterol, myoinositol, casamino acid, BSA가 함유된 CBE 재생용 배지에서 효과적이었으며, 30% 이상 재생률을 나타내었다. 원형질체 융합은 10mM CaCl_2가 함유된 pH7.5의 30% PEG 4000으로 37℃에서 10분간 처리했을 때 가장 양호하였으며, 융합빈도는 8.2×10 exp(-4)을 나타내었다. 가장 우수한 융합주인 FWN-56은 CMCase, avicelase, β-glucosidase 및 xylanase를 동시에 분비하였으며 친주에 비하여 활성이 2.3배, 1.5배, 1.8배, 2.5배 각각 증가하였고, 또한 MM에 4중 이상 보관 후의 segregant율이 1% 이내였으므로 유전적 안정성은 높았으며, 분생포자 DNA 함량은 1.4∼1.6배였다. 또한 핵의 크기도 친주에 비하여 큰 것으로 보아 융합주임을 확인하였다. Regeneration of protoplast was effective by preincubating spore suspension containing 30㎍/㎖ of 2-DG for 4 hours, and CBE medium containing casamino acid, bovine serum albumin, ergosterol and myoinositol was found to be more efficient than any other regeneration medium tested in this experiment. The regeneration frequency was about 30%. Optimal conditions for conidial protoplast fusion were obtained by treatment of protoplasts with 10 mM CaCl_2 and 30% polyethylene glycol 4000(pH 7.5) as fusogenic agent at 37℃ for 10 minutes. The fusion frequency was 8.2×10 exp(-4). The higher productivity of enzyme of fusant FWN-56 was achived: 2.3-fold for CMCase, 1.5-fold for avicelase, 1.8-fold for β-glucosidase and 2.5-fold for xylanase compared to that obtained in two parental strains. The genetic stability of fusant after maintenance on minimal medium for more than 4 weeks was high because segregant rate was below 1%. The conidial DNA content of fusant was 1.4-1.6 times higher than that of the parental strains. The nucleus size of fusants were also higher than that of each parental strains.

Xanthomonas sp. EPS-1이 생산하는 다당류의 리올로지 특성

손봉수,박석규,강신권,이상원,성낙계 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.3

Xanthomonas sp. EPS-1 균주의 배양액으로부터 분리한 다당류 EPS-1의 분자량은 2.1×10exp(6) dalton으로 추정되며, 구성성분을 HPLC, IR 및 NMR을 이용하여 분석한 결과 mannose, glucose, galactose 및 glucosamine으로 이루어져 있다. 다당류 EPS-1 용액은 Power-law model을 따르는 비뉴우톤체의 의가소성 특성을 나타내었고, 1.0% 농도에서 점조도 지수(consistency index)와 유동지수(flow behavior index)는 각각 10.8352 poise·sec^(-n)과 0.4419였다. 또한 이 용액은 pH 5∼13까지의 범위에 대하여 안정하였으며, 저농도에서도 높은 점성을 나타내었고, NaCl에 대해서도 매우 안정하였다. For the screening of a new functional exopolysaccharide, sugar composition and rheologiccal properties of exopolysaccharide produced from Xanthomonas sp. EPS-1 were investigated. The average molecular weight of exopolysaccharide was determined to be approximately 2.1×10exp(6) dalton. The new exopolysaccharide EPS-1 was composed of mannose, glucose, galactose and glucosamine. IR analysis showed that the exopolysaccharide EPS-1 was assumed to be polymer with carbohydrates. NMR analysis showed that exopolysaccharide EPS-1 was presumed to be 4 units of sugar and trace of CH_3 group. Exopolysaccharide EPS-1 solution showed a characteristic of non-Newtonian fluid properties. At the concentration of 1.0%, the consistency index and the flow behavior index were shown at 10.8352 poise·sec “ and 0.4419, respectively. All dispersions were pseudoplastic fluids described accurately by Power-law model. Exopolysaccharide EPS-1 was highly viscous at low concentration, with good stability over a wide range of pH 5 to 13. The excellent compatibility of exopolysaccharide EPS-1 was represented with salts such as sodium chloride.

다당류를 생산하는 미생물의 분리와 배양특성

손봉수,박석규,강신권,이상원,성치남,성낙계 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.3

토양으로부터 다당류를 생산하는 미생물을 분리하고 생성능이 우수한 한 균주를 최종 선정하여 균학적 성상을 조사한 결과 Xanthomonas 속과 거의 일치하였다. 최적배지 조성은 sucrose 50 g, peptone 1.5 g, KH_2PO_4 2 g, MgSO_4·7H_2O 2 g, NaCl 3 g, CaCO_3 0.05 g, FeSO_4·7H_2O 0.07 g, MnSO_4·7H_2O 0.05 g, 증류수 1리터였다. 최적 플라스크 배양에서 다당류 14.6 g/가 생산되었으며, 이때 수율은 29.8%였고 생산된 다당류는 분산효과와 겔 형성능이 우수하였다. A screening was performed to isolate exoploysaccharide-producing microorganisms, which synthesized specific exopolysaccharide for the substitutive of commercial polysaccharides, from natural sources. Soil bacterium, one of 378 mucoid isolates, was finally selected as potential producer of polysaccharides which made the culture broth very viscous and thus examined in detail for optimal medium composition. Isolated strain was identified as Xanthomonas sp. EPS-1 from the results of morphological and biochemical characteristics. The composition of optimal medium for exopolysaccharide production was as follows: 50 g sucrose, 1.5 g peptone, 2 g KH_2PO_4, 2 g MgSO_4·7H_2O, 3 g NaCl, 0.05 g CaCO_3, 0.07 g FeSO_4·7H_2O and 0.05 g MnSO_4·7H_2O in 1 liter of distilled water, From the experiments of temperature and pH dependence, the optimal conditions for exopolysaccharide biosynthesis seemed to be 30℃’and 8.0, respectively. About 14.9 gram of maximum exopolysaccharide per liter was obtained at the initial pH 8.0. 30℃ and 250 rpm in a flask culture. The exopolysaccharide EPS-1 had such potential as an emulsifying agent and a gelling agent in comparision with commercial exopolysaccharide.