TRICHOSPORON CUTANEUM의 LIPASE에 關한 硏究 = Studies on the Lipase of Trichosporon cutaneum

국문 초록 (Abstract)

1. Lipase 生産菌의 分離 및 同定
(1) Olive oil을 唯一炭素源으로 하는 培地를 使用하여 空氣 및 土壤 中에서 300餘株의 Lipase 生産菌을 分離하였으며 이들의 Lipase 生産能을 檢討하여 有用菌株 1株를 選定하였다.
(2) 選定된 菌株의 形態學的 및 生理學的性質을 檢討한 바 Lodder 分類法의 Tricosporon cutaneum에 屬하는 것으로 認定되었다.
2. 培養條件
大豆粉 2%, soluble starch 2%, (NH₄)₂SO₄ 0.1%, K₂HPO₄ 0.5%, MGSO₄7H₂O 0.1%의 基本培地를 使用하여 選定菌株의 Lipase 生産條件을 檢討한 結果는 아래와 같다.
(1) Lipase 生産의 基本 最適條件으로서는 Initial pH 7.0~7.5, 培養溫度 30℃, 振탕速度 120 oscills/min. 內外 이었다.
(2) 窒素源으로서는 2%의 大豆粉과 0.1%의 (NH₄)₂SO₄를 混用하는 것이 가장 效果的이었으며 大豆粉을 單用한 경우에 比하여 Lipase이 生産이 8倍以上 增大 되었다.
(3) 大豆粉(2%)을 含有하는 基本培地에 培養하였을 때에는 炭素源添加의 影響이 거의 없었으며 xy-lose, arabinose, glucose, fructose, mannose를 添加하면 Lipase의 生産이 크게 阻害되었다.
(4) 基本培地에 0.5%의 olive oil을 添加하면 Lipase의 生産이 20% 以上 增大 되었다.
(5) 基本培地 中의 大豆粉을 脫脂大豆粉으로 代置할 경우에는 Lipase 生産이 1/2 以下로 低下되었으나 이것에 0.5~1.0%의 大豆油를 添加하면 基本培地의 경우보다 오히려 Lipase의 生産이 若干 增大되었다.
(6) 基本培地에 1.0%의 CaCO₃를 添加하면 Lipase의 生産이 30% 以上 增大 되었다.
(7) 基本培地中의 K₂HPO₄ 및 MgSO₄7H₂O의 濃度는 各各 0.6% 및 0.2% 時에 Lipase의 生産이 가장 良好 하였다.
(8) 最適培養條件下에서 培養하면 96時間까지 Lipase의 生産量이 增加되었다.
3. Lipase의 精製
選定菌株의 培養液을 遠沈하여 菌體 其他成分을 除去하고 硫安鹽析(0.3~0.5 飽和), Sephadex G-25에 依한 脫鹽, polyethylene glycol에 依한 濃縮, Acetone分別(40~60%), 透析, DEAE-Cellulose column chromatography들을 거쳐 specific activity 112.8u/㎎·protein, 原活性의 18.8倍, 收率 3.8%까지 精製히였다.
4. 精製酵素의 性質
(1) 本 lipase의 作用最適 pH는 8.0, 最適溫度는 37℃이었고 pH 4.0~9.0에서 安定하였으며, 15℃, 60分間處理時 5%, 50℃ 60分間 處理時, 約88%가 失活되었다.
(2) Sodium cholate는 essential activator로서 作用하였으며 0.1~0.4% 添加時에 15~18倍 以上 活性化 하였다.
(3) Ca^(2+) 및 Mg^(2+)도 activator로서 作用하였으며 Ca^(2+)보다 Mg^(2+)이 더 效果的이었다.
(4) Cu^(2+) 및 Hg^(2+)이 强한 阻害作用을 하였으며 Pb^(2+), Fe^(2+), Zn^(2+), Sn^(2+)등도 阻害 하였다.
(5) 植物油 中 sesame oil, perilla oil, soybeen oil, 및 olive oil를 特히 잘 分解하고 rape seed oil은 잘 分解하지 못했으며 tributyrin, triacetin, Tween, Span등은 分解하나 微弱하였다.

다국어 초록 (Multilingual Abstract)

1. Isolation and Identification of Lipolytic Microorganisms.
(1) A Potent lypolytic strain was selected by an extensive screening test of microorganisms isolated from air and various soils on the medium containing olive oil as sole source of carbon.
(3) Morphological and Physiological characteristics of the selected strain had been investigated, and it has been identified to "Trichosporon cutaneum" by the classification of LODDER^((23)).
2. Cutural Conditions
The selected strain was cultured with shaking at 30℃ for 24 hrs, or more, and the basal medium was composed of 2%(w/v) of soybean meal, 2% of soluble starch, 0.1% of (NH₄)₂SO₄, 0.5% of K₂HPO₄ and 0.1% of MgSO₄ 7H₂O. Cultural conditions for lipase production by the strain have been studied, and the results obtained were as follows.
(1) The optimum condition for lipase production were: initial pH 7.0 to 7.5, temperature 30℃ and the velocity of oscillation about 120 oscills./min.
(2) Among the various nitrogen compounds, soybean meal(2%) and (NH₄)₂SO₄(0.1%) mixture were the best nitrogen sources for the lipase prodution increasing more than 8 times the case of using soybean meal(2%) alone.
(3) When the strain was cultured in the basal medium containing 2% of soybean meal, the carbon sources had little effect on the lipase production, and xylose, arabinose, glucose and mannose reduced the lipase production sharply.
(4) The lipase production was increased as much as 20% and more by the addition of 0.5% of olive oil to the basal medium.
(5) When the soybean meal in basal medium was substitted for defatted soybean meal, the lipase production was decreased below a half, and the addition of soybean oil(0.5- 1.0%) to the latter increased the lipase production slightly more than in the case of using soybean meal basal medium.
(6) The lipase production was increased as much as 30% and more by the addition of 1.0% of CaCO₃ to the basal medium, compared with no addition.
(7) The most effective concentration of K₂HPO₄ and MgSO₄·7H₂O in the basal medium for the lipase production was 0.6% and 0.2% respectively.
(8) When the strain was cultured under the opitimum cultural conditions, the increase of the lipase production was continued up to 96 hrs.
3. Puification of Lipase.
The purificasion of lipase from the selected strain was successfully carried out by the following steps.
Cell-free extract was salted out with ammonium sulfate(0.-0.5 saturation), desalted by Sephadex G-25, concentrated by polyethylene glycol, fractionated with acetone(40-60%), dialyzed, and fractionated by DEAE-Cellulose column chromatography.
The specific acivity(112.8u/㎎·protein) of the purified enzyme was 18.8fold of the centrifuged culture broth, and the yield was 3.8% of the original activity.
4. Properties of Purified Enzyme.
(1) The optimum conditions for lipase activity were pH 8.0 and 37℃, and the enzyme was stable in the range of pH 4.0-9.0 and was inactivated 5% and more by treatment at 15℃ for 60min. and 88% at 50℃ for 60min.
(2) The purified enzyme required essentially sodium cholate and the lipase activity was increased 15 to 18 fold in the presence of 0.1 to 0.4% of sodium cholate, compared with no addition.
(3) The enzyme was activated by Ca^(2+) and Mg^(2+), and the degree of activation of Mg^(2+) was higher than Ca^(2+).
(4) The strong inhibition was observed with Cu^(2+) and Hg^(2+), although the enzyme action was also inhibited by Pb^(2+), Fe^(2+), Zn^(2+) and Sn^(2+).
(5) Among the natural fats and oils tested, sesame oil, Perilla oil, soybean oil and olive oil were also inhibited by Pb^(2+), Fe^(2+), Zn^(2+) and Sn^(2+).
The enzyme was also capable of hydrolyzing tributyrin, triacetin, tween-80 and span-20: however, the hydrolysis rate was much slower than the above oils except rape seed oil.