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吳秉夏,黃殷成,李炯周,李啓瑚,朴官和,張海東,徐鉉昌 서울大學校農科大學 1984 서울대농학연구지 Vol.9 No.1
澱粉으로 부터의 alcohol 醱酵能을 增進시키기 爲하여 澱粉糖化性 菌株인 Saccharomyces diastaticus와 優秀한 alcohol 醱酵性 菌株인 Saccharomyces uvarum을 母菌株로 하여 이들간의 同屬異種間 原形質融合(interspecific protoplast fusion)을 通한 優秀한 澱粉醱酵 性 alcohol 生産性 菌株를 새로이 開發할 目的에서 다음과 같은 一漣의 實驗結果를 얻었다. S. diastaticus의 醱酵液과 S. diastaticus+S. uvarum 混合醱酵液의 風味特性등을 確認하였다. 風味成分 抽出은 methylene chloride와 diethylether를 가지고 neutral flavor fraction과 acidic flavor fraction으로 나누었고 gas chromatography를 通하여 同定 및 定量하였다. Neutral flavor fraction의 경우 S. diastaticus+S. uvarum 混合醱酵液이 S. diastaticus 醱酵液보다, ester成分中에서는 ethyl acetate와 ethyl undecanoate가 더 많았고, alcohol 成分中에서는 n-propanol과 n-butanol이 더 많았다. Acidic flavor fraction의 경우 C??~C?? fatty acid가 同定 및 定量되었는데 S. diastaticus+S. uvarum 混合醱酵液이 S. diastaticus 醱酵液보다 lauric acid, caprylic acid, capric acid 含量이 두드러지게 많았다. S. diastaticus의 glucoamylase 生産性, glucoamylase의 分離 精製, 酵素力價 그리고 酵素學的 特性에서 optimum pH는 5.0, optimum temperature는 55℃ 이었다. S. diastaticus와 S. uvarum을 母菌株로 이들 간의 protoplast fusion을 위한 基礎的인 硏究로서 두 菌株의 諸特性과 protoplast調製의 最適條件을 決定하고 protoplast의 regeneration 條件의 確立을 도모하였다.두 菌의 生育曲線에서 모두 培養開始 7~8 時間만에 對數期 中期에 到達되었으므로 protoplast 調製는 이 時期의 細胞를 쓰기로 하였다. Generation time은 S. diastaticus가 1.04, S. uvarum이 1.38 時間이었다. 細胞의 크기는 S. diastaticus 44.10?㎛³, S. uvarum 99.67㎛³로 S. uvarum이 2倍나 컸다. DNA 含量은 細胞 當 S. diastaticus 44.3fg, S. uvarum 37.6fg이었다. 30% glucose 및 soluble starch에 대한 두 菌株의 ethanol 醱酵能은 glucose에 對하여 S. uvarum 11.4%, S. diastaticus 8.9% 이었고 soluble starch에 對하여는 S. diastaticus 만이 6.9%이었다. 두 菌株는 generation time, 細胞크기 및 DNA 含量 等으로 보아 diploid strain임을 알 수 있었고, 融合株 選拔을 위한 marker 로는 Sacch. uvarum의 melibiose 資化能의 차이를 利用할 수 있음을 밝혔다. Protoplast의 調製에는 β-glucuronidase와 Zymoyase를 使用하였는데 두 酵素 反應最適條件은 β-glucuronidase는 pH 8.0에서 10% 濃度의 溶液으로, Zymolyase는 pH7.5에서 20㎛/ml의 濃度의 溶液으로 하여 모두 70分間 處理하는 것으로 決定하였으나 이 정도의 處理時間에서는 protoplast가 극히 不安定하게 되어 regeneration frequency가 떨어지는 것을 確認하였으며, 特히 Zymolyase 處理로 얻어진 protoplast의 regeneration率이 낮은 것은 Zymolyase中에 不純物로 微量 混在한 protease가 protoplast의 노출된 membrane-bound protein을 分解함으로써 protoplast를 破壞시키기 때문인 것으로 추측되었다. 融合實驗에 利用할 수 있을 정도의 regeneration frequency를 얻기 위해서는 Zymolyase를 45分間 處理하여 얻은 protoplast를 1.5%의 polyvinylpyrrolicone이 加해진 OYPD培地에서 重層法으로 展開하여 regeneration시키는 것이 좋은 것으로 판명되었다. As preliminary steps of protoplast fusion between Saccharomyces diastaticus and S. uvarum to develop a fusant of higher ethanol production from starch, characteristics of the two presumptive parent strains, optimal conditions for protoplast preparation and conditions for highrer regeneration frequency were investigated. To determine flavor characteristics of the parent strains, neutral and acidic flavor fractions were extracted from liquids fermented by S. diastaticus and S. diastaticus + S. uvarum with methylene chloride and diethly ether. The liquid by the mixed culture produced more ethly acetate, ethyl undecanoate, n-propanol, n-butanol, lauric acid, caprylic acid and capric acid than that by S. diastaticus. Glucoamylase from S. diastaticus was purified and activity, productivity, and characteristics were determined. Optimum conditions for the enzyme were pH 5.0 and 55℃. The two strains reached logarithmic phase in 7-8h during growth and the generation time was 1.04 in S. diastaticus and 1.38 in S. uvarum. Cell size and DNA content per cell of S. diastaticus were 44.10㎛³and 44.3 fg, and for S. uvarum, 99.67㎛³and 37.6fg. Ethanol productivities of S. diastaticus were 8.9% from 30% glucose and 6.9% from 30% starch and 11.4% from glucose with S. uvarum. Through determination of generation time, cell size, and DNA content per cell, both strains appeared as diploids, and differences in assimilability of melibiose and soluble starch of the two strains were selected as markers to determine the fusant. The optimal condition for protoplast formation was treatment of both strains with 10% ß-glucuronidase at pH 8.0 or 20㎍/ml Zymolyase at pH 7.5 for 70 min. While the regeneration frequencies were very low at 70min exposure to Zymolyase because of the instability of protoplasts, the yeasts treated for 45min were better for regeneration. The regeneration frequencies were also enhanced by 3-6 times when the regeration was carried out with 1.5% polyvinylpyrrolidone which stabilized protoplasts.
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朴官和,張判植 서울大學校 農科大學 1985 서울대농학연구지 Vol.10 No.1
Fusarium moniliforme으로부터 酵母細胞壁 分解酵素의 생산조건을 검토하고 分離,精製하여 효소특성을 硏究하였다. 효소생산은 배양기간 中 pH 7∼8을 유지시켜 주면서 Baker's yeast 10%와 Ammonium nitrate 0.2%의 첨가로 높은 活性이 있는 효소액을 얻을 수 있었고, 7日間 진탕배양으로 最高에 달하였다. (NH₄)₂SO₄로 분획한 후 Sephadex G-100 column chromatography를 行하여 세 개의 peak를 얻었으며, 첫 번째 peak는 proteose 및 lytic enzyme, 세번째 peak는 lytic enzyme임을 알았다. F.moniliform 효모세포벽 분해효소의 protoplast 제조 최적조건은 pH7.5및 37℃이었고 효모세포벽 분해율은 99.17% 정도였다. Cultural conditions of Fusarium moniliforme for lytic enzyme production have been investigated and the lytic enzyme was purified by (NH₄)₂SO₄fractionation, gel column chromatography and tannic acid precipitation. The lytic enzyme production in the culture of Fusarium moniliforme was greatly affected by pH change of the culture medium. A strong lytic activity on viable yeast cells could be obtained in a medium containing baker's yast (10%) and ammonium nitrate(0.2%) by keeping the pH 7-8 during cultivation. Three protein peaks were obtained from G-100 Chromatography of the crude enzyme. The third peak on chromatogram lysed viable yeast cells and the first had both proteolytic and lytic activity. The optimum pH for producing protoplast of yeast cell was pH 7.5 and the optimum temperature was 37℃.
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Horseradish Peroxidase 의 열불활성화에 Lecithin 과 지방산이 미치는 영향
박관화 한국농화학회 1979 Applied Biological Chemistry (Appl Biol Chem) Vol.22 No.1
A study was carried out about the effect of free fatty acids and lecithin on the thermal inactivation of horseradish peroxidase. In presence of lecithin peroxidase was inactivated very rapidly at 70℃ and pH 7.0, and showed also a rapid inactivation curve at 0℃ and pH 4.0. Linoleic acid was more effective in an O₂-stream than in a N₂-stream, but oleic acid showed a similar tendency in the presence of O₂-and N₂ stream. From the results of the experiment we suggested that the opening of the heme crevis of peroxidase in the presence of lecithin and the produced lipidperoxide from linoleic acid may accelerate the thermal inactivation of horseradish peroxidase respectively.
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Geotrichum candidum Lipase 의 열불활성(熱不活性)에 관하여
박관화 한국농화학회 1977 Applied Biological Chemistry (Appl Biol Chem) Vol.20 No.1
Lipase from Geotrichum candidum was heat inactivated in 0.1M phosphate buffer solution. The thermal inactivation followed first order kinetics for the range of temperatures 50°-80℃ except at 50℃. The changes in enthalpy, entropy and Gibbs free energy at 60℃ were 120.4 kJ/㏖, 73.0 J/㏖·K and 96.9 kJ/㏖ respectively a value of 19℃(Geotrichum candidum lipase) is greater than that of lipases from milk and pancreas. The effect of detergents, lecithin and linoleic acid or the thermal inactivation of lipase was found to be negligible.
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홍승서,김인철,윤종원,박관화,고영환 서울大學校 農科大學 1986 서울대농학연구지 Vol.11 No.1
A thermostable α-amylase were partially purified from Bacillus licheniformis ATCC 27811 by using ammonium sulfate fractionation. DEAE-cellulose CM-cellulose chromatography and Sephadex G-100 gel filtration chromatography. The general properties thermostability and effect of metal ions on the thermostability of the enzyme were characterized and compared with those of Termamyl® The partially purified α-amylase from B. licheniformis ATCC 27811 had optimum temperature of 75℃ and optimum pH between 6 and 9 In the presence of Ca ion the optimum temperature was enhanced to 90℃, and heat treatment of purified α-amylase at 80℃ for 20 min resulted in a loss of 12% of activity. Addition of Ca?? ion provided a marked increase in thermostability of Termamyl®
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