纖維素 分解酵素에 關한 硏究 : 第3報 强力 纖維素 分解菌의 分離 및 同定 Ⅲ.Isolation and identification of mold producing high cellulolytic activity

成洛癸 진주농과대학 1969 진주농과대학 연구논문집 Vol.- No.8

自然界에 分布되어 있는 絲狀菌 133株를 分離하여, 濾紙巖壞活性 C.M.C. 糖化活性을 調査하였다. Mucor屬의 大部分은 兩活性이 弱하였으며, Pennicillium屬의 大部分은 C.M.C 糖化活性이 弱한 反面, 或種의 菌株는 濾紙巖壞活性이 良好하였고, Rhizopus屬의 大部分은 이의 反對現象을 나타내었다. Aspergillus屬 가운데는 濾紙巖壞活性이 良好한 것이 있는가하면, C.M.C. 分解力이 强한 것도 있었다. 半腐朽한 아까시나무(Robinia Pseudacacia Linne)에서 分離한 1菌株는 兩活性이 强하였음으로, 이 菌株에 對해서 그 性狀을 調査하였던바 Trichoderma viride의 性狀과 一致하였음으로 Trichoderma viride로 同定하였다. The molds of 133 strains were isolated from the nature and investigated the enzymic activities-the sacch-arifying activity on C.M.C. (Carboxyl Methyl Cellulose) and the disintegrating activity on filter paper. Most of Penicillium genus had a weak saccharifying activity on C.M.C., however, some of them showed high disintegrating activity on filter paper. The strains of Rhizopus showed high activity on C.M.C. and weak activity on filter paper. Both cell ulolytic activities of Mucor genus ies of were weak. Aspergillus genus had high activity on C.M.C. and a few of them had high disintegrating activity on filter paper. Both celluloytic activities Mucor genus were weak. Since one strain of mold isolated from Locust acasia (Robinia Pseudacasia Linne) showed high activity on C.M.C. and filter paper, its microbiological properties were studied. This strain of mold was identified as Trichoderma viride.

호알칼리성 Bacillus sp.F204와 Bacillus sp.K17의 원형질체 융합

성낙계,노종수,박석규,정영철 순천대학교 기초과학연구소 1990 基礎科學硏究誌 Vol.1 No.-

To develop cellulase and xylanase-producing strain by protoplast fusion, alkalophilic Bacillus sp. F204 and K17 were treated with NTG(N-methyl-N'-nitro-N-nitrosoguanidine) and isolated antibiotics resistant strains of S20 (Km', Cm') and G70(Str'). The frequency of protoplast formation was about 95% when cells of mid-log phase were treated with 200㎍/㎖ lysozyme at 37℃ for 30-45 minutes. Under addition of 0.4-0.5M sodium succinate, 0.5% casamino acid, 1.5% polyvinylpyrrolidone, 25mM MgCl_2 and 50mM CaCl_2 to the regeneration medium, the regeneration frequency of Bacillus sp. F204 and K17 was 24.9% and 26.2%, respectively. the fusion frequency was 6.6×10^-6 in the presence of 30% polyethylene glycol 6000 containing 50mM Ca^++ at 45℃ for 5 minutes. Cellulase complex and xylanase activities of fusant were compared with parental strains.

纖維素 分解酵素에 關한 硏究 : 第Ⅱ報 Aspergillus 屬이 生産하는 Cellulase의 性質에 對하여 Ⅱ On the character of cellulase by Aspergillus strain

成洛癸 진주농과대학 1968 진주농과대학 연구논문집 Vol.- No.7

Aspergillus屬 18株를 分離하여 그中 Cellulose 分解力이 强한 1菌株(Asp-A15)를 選擇하여 이 菌株가 生産하는 Cellulase를 粗精製하여 基質을 分解할때 그 性質에 對한 實驗을 하여 다음과 같은 結果를 얻었다. 1) Aspergillus屬 中에서는 黑麴菌類가 比較的 酵素活性이 强하였고, 可溶性 Cellolose 分解力이 强한 것이 반드시 天然 Cellolose 分解力도 强하지 않다는 事實을 알았다. 2) 本酵素의 最適活性 pH는 3.5∼4.5이며 水素 Ion에 對한 安定度는 pH 3∼7로서 그 範圍가 넓다. 3) 本酵素의 作用最適溫度는 40∼45℃이며 70℃에서는 20分만에 約 90%가 失活되였다. 4) 金屬 Ion에 對한 影響으로서는 阻害作用이 가장 甚한것이 Hg++>Ag++>Ba++>Pb++ 等이며 Ca++ K+ Na+ 等은 活性을 Control때 보다 增加시켰다. One strain of Aspergillus, Producing Powerful cellulase(β-1.4 glucan 4 glucano hydrolase)was Scree ned from 18 strains of Aspergillus which isolated from the nature and some enzymetic characteristics were studied with crude purified state. The results obtained were as follows: 1) Of the Aspergillus, black Aspergillus had relatively high cellulase activity. A strain having high activity on the water soluble cellulase was not always coincidenced with hydrolysing activity on the natural cellulose. 2) The cellulase of last screened strain had optimum pH 3.5-4.5 and wide range of pH stability from pH 3 to pH 7. 3) Optimum temperatures of this enzyme action was 40-45℃ and the activity was decreased about 90% at 70℃ for 20 min treating. 4) This cellulase activity was inhibited by heavy metalic ions as following order ?? and activated or protected of its activity by ??

Trichoderma viride가 生成하는 纖維素 分解酵素의 分別 및 酵素學的 性質에 關한 硏究

成洛癸 진주농과대학 1971 진주농과대학 연구논문집 Vol.- No.10

One strain of mold producing powerful cellulolytic enzyme complexes was screened from various strains of molds which were isolated from nature, and its microbiological properties and cellulolytic enzymes were studied. The results obtained were summarized as follows: 1) One strain of mold isolated from Locust acasia (Lobinia pseudacasia linne) showed remarkably high cellulolytic activity on the various substrates. This strain, by its microbiological properties, was identified as Trichoderma viride. 2) It was found that there were five different kinds of cellulolytic enzymes in the cellulase derived from Trzchoderma viride. Their physical and enzymological properties were as follows: i) The optimum pH of each enzyme was 5.0 and the range of stability with respect to pH was generally from 4.0 to 6.0. ii) The optimum temperature of CMCase, β-glucosidase and filter paper saccharifying enzyme was 60℃, but that of filter paper disintegrating enzyme was 50℃. The heat stabilities of β-glucosidase, filter paper disintegrating enzyme, CMCase and filter paper saccharifying enzyme were 30, 40, 50, and 55℃, respectively. iii) When the concentration of metal ions was 2×10³M. ,the activities of CMCase and filter paper disintegrating enzyme were found to be inhibited by ?? and ??. At the same concentration, filter paper saccharifying enzyme was inhibited by ??, EDTA, ??, and ??, while β-glucosidase was inhitibited by all the above metal ions. 3) The purification and fractionation of the cellulolytic enzyme complexes by organic solvents, and column chromatography with ion exchange resin, were as follows: i) In the purification, β-glucosidase could be collected completely using a 60% solution of acetone and ethanol. In the case of filter paper disintegrating enzyme, the best result was obtained by the use of an 80% acetone solution. ii) A 100% yield of these enzymes was obtained by the use of saturated aqueous ammonium sulfate solution(60%). iii) The cellulolytic enzyme complexes could be separated into a few fractions by chromatography with cellulose powder. Most of the filter paper disintegrating enzyme was absorbed, but the others were eluted. iv) The cellulolytic enzyme complexes could be separated into filter paper disintegrating enzyme and filter paper saccharifying enzyme by gauze column chromatography. v) The doted fraction could be separated into CMCase, β-glucosidase and filter paper saccharifying enzyme by column chromatography with DEAE-sephadex A-50, Amberite XE-64 and Sephadex G-200. vi) The filter paper disintegrating enzyme present in the absorbed fraction was resolved by column chromatography with DEAE-sephadcx A-50. 4) The characteristics of the separated fractions were as follows: i) The ?? value of CMCase was 0.125% and that of β-glucosidase was 0.045%. ii) The filter paper saccharifying enzyme and filter paper disintegrating enzyme differed with respect to the distance moved during electrophoresis, and in ?? values obtained in paper chromatography. iii) The filter paper disintegrating enzyme was completely inactivated by heating at 70℃ for 10 minutes, and filter paper saccharifying enzyme activity reduced by about 80 percent. iv) The decomposing pattern of filter paper saccharifying enzyme was mainly an exo-type and that of filter paper disintegrating enzyme was an endo-type. 5) It has been previously reported that filter paper disintegrating enzyme is indentical with filter paper saccharifying enzyme: however, the present study indicates that these two enzymes differ in physical and enzymological properties. Therefore it is suggested that there are five cellulolytic components in Trichoderma viride cellulase.

組織素 分解酵素를 生成하는 高溫性 곰팡이의 分離 및 特性

성낙계,강인수,박채규,정영철,김두현 慶尙大學校 1990 論文集 Vol.29 No.1

고온에서 잘 생육하고 cellulose 및 xylan 분해력 이 우수한 곰팡이를 분리할 목적으로 pH5.0, 50℃, Czapek-Dox pulp 배지에서 celluase complex와 xylanase생산이 우수한 곰팡이 H-702균주를 최종선정하여 균학적 성상을 조사한 결과, Aspergillus속과 거의 일치하였으며, 본 공시균주가 효소 생산에 미치는 영향과 효소학적 성질의 결과를 요약하면 다음과 같다. 생육 및 효소생성의 최적온도는 50℃이고, 균체증식 최적 pH는 5.0, cellulase 생산 최적 pH 는 4.5 xyanases는 4.0으로 나타났으며, 효소최대 생성 배양 4일째에, 균체증식은 약 3일경에 최대에 도달하였다.또한 α-1, 4-linkage를 가진 섬유성 물질 왕성하였으며 glucose 와 같은 저분자물질에서는 효소생합성이 현저히 저해되었는데 이런 결과로 볼때 이균주는 carbon catabolite repression을 받고 있었다. 효소생산에 적합한 탄소원으로는 α-cellulose,avicel, filter paper 등 이였으며, 질소원으로는 암모니아가 효과적이었으며 무기염류는 Ca??, Mg??, Mn??, Co??에서 효소생합성이 좋은것으로 나타났다. 계면활성제는 0.15% Tween 80이 좋았다. 개선된 KD배지에서는 각종 효소활성도가 1.2-1.6배 증가하였으며 또한 KD배지에서 각종탄소원을 지질로하여 효소 생합성을 조사한 결과 3% a-cellulose 에서 효소활성도가 좋았고 값싼 기질인폐신문지에서도 효소활성이 높게 나타났다. 조효소의 최적 온도는 cellulas complex 는 70℃, xylanase는 60℃로 나타났으며 열안정성은 cellulas complex 70℃까지는 30분간 , 80℃에서는 잔존활성이 약 93%였고 xylanase는 70℃에서도 거의 안정하였다.최적 pH는 cellulas complex는 5.0, xylanase는 5.5였으며, pH안정성은 3.0-8.0까지 안정하였다.조효소의 미치는 금속이온의 영향은 Ca??,Mg?? Mn??에의해 활성화 되었으며 Pb??, Cu??, Mg?? 에 의해 현저히 저해 현상을 나타냈다. In order to isolate a fungus which grow well in the high temperature and has the excellent decomposing power of cellulose and xylose a fungus. H-702 which produced potently cellulase complex(CMCase,avicelase and β-glucosidase) and xylanase in Czapek-Dox pulp medium (pH 5.0, 50℃) was finally selected. Then as a result of its bacteriological properties investigated it was almost the same properties as Apergillus sp.. The effect influencing to the enzyme production of the isolated strain and enzymological properties are followed. The optimal temperature and pH for cell growth and enzyme production were 50℃, 5.0, 4.5 and 4.0.respectively . The highest production of enzyme was shown when the culture time was three days and the greatest growth of cell was shown about after two days. In the cellulosic materials(CMC.avicel and α-cellulose etc.) containing α-1.4-linking the vigorous production if enzyme was shown and in the small molecular substance such as glucose was remarkably repressed. According to these results, this stain was under the control of carbon catabolite repression.α-cellulose , avicel and filter paper were suitable for carbon source in the enzyme production and NH? for nitrogen source and Ca??, Mg??, Mn?? and Co?? for inorganic salt. 0.15% Tween 80 was a good surface active agent. In improved KD medium each enzyme activity was increased 1.2 to 1.4 times. As a result of investigating enzyme biosynthesis, which each carbon source was used as substrate in the KD medium, it was shown a good enzyme activity. The optimal temperature of crude enzyme in cellulase complex and xylanase were 70 ℃and 60℃, respectively. Thermal stability was shown for 30 minutes until 70℃in cellulase complex and its residual activity at 80℃ was about 93%. It almost was stable at 70℃in xylanase. The optimal pH for cellulase complex and xylanase were 5.0 and 5.5.respectively and they were stable in the range of pH 3.0 TO 8.0. The crude enzyme was activated by Ca?? , Mg??and Mn?? but inhibited by Pb??, Cu?? and Ag?? remarkably.

Cellulase의 利用에 關한 硏究 : 第 1 報 大豆蛋白의 抽出에 對하여 Ⅰ. On the extraction of the soybean protein

金明燦,成洛癸,奇宇京 진주농과대학 1969 진주농과대학 연구논문집 Vol.- No.8

絲狀菌 Cellulase의 食品加工에의 利用의 一環으로 大豆와 비지에 對하여 Protein의 抽出試驗을 하였으며 實地 豆腐製造에 利用하여 다음과 같은 結果를 얻었다. 1) 本實驗室에서 製造한 Trichoderma 劑 酵素는 大豆와 비지에 對하여 Protein 抽出率이 높았다. 2) Trichoderma 酵素에 混在하는 大部分의 Protease는 熱處理(60℃30分)에 依하여 Cellulase component의 많은 失活없이 除去되었다. 3) 熱處理한 同 Trichoderma Cellulase를 使用하여 大豆와 비지로 부터 豆腐를 만들어 顯著한 收量 增加를 얻었다. As the part of the utilization of celluloytic enzyme derived from Trichoderma viride to the food processing, the extractability of the protein from soy bean and soy bean refuse was studied and some practical point in the preparoion of the soy bean curd was investigated. The results obtained were summerized as follows: 1) The crude enzyme from Tricoderma viride prepared in our laboratory had high extractability of protein from both soy bean and soy bean refuse. 2) Most of protease from Trichoderma viride was eliminated with little loss of celluloytic activity by heat treatment. 3) The increased yield of soy bean curd from soy bean refuse or soy bean was obtained using heat treated crude enzyme.

섬유질 가수분해물로부터 효율적인 Ethanol 생산균주의 분리

고학룡,문종상,심기환,성낙계 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.4

섬유성 가수분해물로부터 효율적으로 ethanol을 생산하는 세 가지 균주를 밀기울 당화액에서의 집적 배양에 의해 토양에서 분리하였다. 효모인 KM-09와 KM-402 및 세균인 HG-225의 생리학적 및 생화학적 특성은 각각 Candida sp. 및 Klebsiella sp.과 거의 유사하였다. KM-09와 HG-225 균주는 발효당으로 xylose와 cellobiose를 이용하였고 HG-225는 발효시 넓은 당 이용성을 가졌다. KM-09, KM-402 및 HG-225의 최적 생육 pH 및 온도는 각각 5.8, 5.6 및 6.8 그리고 32℃, 30℃ 및 38℃였다. 분리균에 의한 밀기울 당화액에서 ethanol 발효 동안 최대 ethanol 생산온도는 최적생육온도보다 다소 높았으며, 0.2% MgSO_4의 첨가는 ethanol 생산성을 향상시켰다. 세 가지 균주 중 KM-09의 ethanol 함량이 약 2.3%(v/v)로서 가장 높았고 HG-225의 ethanol 생산속도가 가장 빨랐으며 이때 최대 생산성은 배양 4일 후였다. KM-09와 HG-225는 4%(w/v) xylose로부터 각각 1.42 및 1.05%(v/v)의 ethanol을 생산하였으나 균 증식속도는 glucose에 비해 매우 느린 것으로 나타났다. 한편, KM-402는 glucose에서는 가장 ethanol 생산성이 좋았으나 xylose 및 cellobiose에서는 생산하지 못하였다. There strains able to efficiently produce ethanol from cellulosic hydrolysates were isolated from soil samples by enrichment culture in liquid saccharified wheat bran medium. The profiles of physiological and biochemical properties of two yeasts KM-09 and KM-402 and a bacterium Hg-225 were almost identical from those of Candida sp. and Klebsiella sp., respectively. Strains KM-09 and HG-225 used xylose and cellobiose as fermentable sugars, and HG-225 had a wide range of sugar utilization for ethanol fermentation. The optimal pH and temperature for growth of KM-09, KM-402 and HG-225 were 5.8, 5.6 and 6.8 and 32℃, 30℃ and 38℃, respectively. During the ethanol fermentation in saccharified wheat bran by the isolated strains, optimal temperature for ethanol production was more or less higher than those for growth, and addition of 0.2%(w/v) MgSO_4 into the medium enhanced ethanol productivity. Of the three strains ethanol content of KM-09 was the highest with about 2.3%(v/v), and ethanol production rate of HG-225 was faster than the others and maximum productivity was after 4 days. KM-09 (1.42%, v/v) and HG-225 (1.05%, v/v) produced ethanol from 4%(w/v) xylose but growth rate was slower than on glucose. Otherwise KM-402 showed the highest ethanol productivity on glucose, but no ethanol was detected on xylose and cellobiose.

Streptomyces griseoplanus SL20209에 의한 Aminopeptidase M 저해제의 생산 조건

고학룡,전효곤,성낙계,고영희 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.3

생산용 배지(GSS) 100 ㎖을 포함하는 500㎖ 삼각 플라스크에서 Streptomyces griseoplanus SL20209를 28℃, 3일간 배양했을 때 aminopeptidase M(AP-M) 저해제 생산이 최대에 도달하였으며, 그 이후 pH가 알카리성으로 변하면서 다소 감소하였다. Arabinose, xylose, mannose 및 soluble starch가 저해제 생산에 좋은 탄소원인 반면, glucose는 균생육에는 좋으나 저해제 생산은 강하게 저해하였다. Soybean meal, fish meal, gluten meal 및 peanut powder와 같은 천연 유기질소원이 저해제 생산에 좋은 질소원이었으며, soytone이나 peptone 및 NH_4Cl과 NH_4NO_3와 같은 무기질소원이 함유된 배지에서는 저해제 생산이 빈약하였다. 배지내에 각각 0.5%(w/v)의 yeast extract난 0.05%의 K_2HPO_4의 첨가는 균생육을 촉진시켜 저해제 생산을 증가시켰으며, CaCo_3(0.3%)와 zeolite(0.5%)도 저해제 생산 증가의 효과를 나타내었다. 저해제 생산을 위한 최적 배양온도 및 초기 pH 범위는 각각 28℃와 6.0~7.0이었다. Arabinose(ASY 배지) 또는 soluble starch(SSY 배지) 3%, SBM 2.5%, yeast extract 0.5%, K_2HPO_4 0.05%, CaCO_3 0.1% 및 zeolite 0.3%(pH 6.8)의 조성의 두 개선된 배지에서 초기의 GSS 배지에서 보다 저해제 생산이 1.8배 증가되었다. Maximum amount of the aminopeptidase M inhibitors produced by Streptomyces griseoplanus SL20209 in 500 ml-Erlenmeyer flask was accumulated after cultivation for 3 days at 28℃, thereafter the amount of inhibitors decreased slowly with a pH change to alkaline. Arabinose, xylose, mannose and soluble starch were good carbon sources for the production of the inhibitors. On the other hand, glucose was only good for the cell growth but potently inhibited the production of inhibitors. Natural organic nitrogen sources such as soybean meal, fish, meal, gluten meal and peanut powder were good for the production of inhibitors, however, soytone, peptone and inorganic nitrogens such as NH_4Cl and NH_4NO_3 were poor. Inclusion of yeast extract (0.5%, w/v) or K_2HPO_4 (0.05%) into the production medium increased the production of inhibitors by accelerating cell growth. The production of inhibitors was slightly increased on the medium containing CaCo_3(0.3%) and zeolite (0.5%), respectively. Optimal temperature and initial pH range for the production of inhibitors were determined to be 28℃ and 6.0~7.0, respectively. Employing two improved production media consisting of 3% arabinose or soluble starch, 2.5% soybean meal, 0.5% yeast extract, 0.05% K_HPO_4 0.1% CaCO_3 and 0.3% zeolite (pH 6.8), 1,8-fold increase in the amount of inhibitors was achieved, comparing with the basal medium used.

纖維素 分解酵素에 關한 硏究(第6報) : 培地組成이 Trichoderma viride(??)의 酵素生産에 미치는 影響 Ⅵ.Effect of Composition of medium on cellulase Productivity of Trichoderma viride(??)

趙武濟,奇宇京,成洛癸 진주농과대학 1971 진주농과대학 연구논문집 Vol.- No.10

1) Trichoderma viride(??)의 Cellulase 生産에 최적 固休培地로서 수세밀기울과 여지분을 9:1로 혼합하여 질소원으로서 ammonium sulfate를 2∼3%, 효소 생성 촉진제로서 citric acid를 2∼2.5% 첨가한 것이 가장 양호하였다. 2) 배양기간은 30℃에서 4∼5일간 배양이 가장 효과적이었다. Various carbon sources, nitrogen sources and inducers were tested in order to increase celluase productivity of Trichoderma viride(O₂-1) in the solid media. It was most favorable conditions for cellulase production when 10% of filter paper powder as carbon source, 2-3% of ammonium sulfate as nitrogen source and 2% of citric acid as inducer were added to basic media of washed wheat bran. According to cultivation period, cultivation for 4-5 days in 30℃ showed maximum activity.

Pectolytic Enzyme에 關한 硏究 (第一報) : 分離細菌 ??가 生産하는 Macerating Enzyme와 Pectolytic Enzymes에 關하여 Ⅰ. Macerating Enzyme and Pectolytic Enzymes Produced by Isolated Bacteria (??)

李千洙,金鍾奎,奇宇京,成洛癸 慶尙大學校 1972 論文集 Vol.11 No.-

고구마 軟腐病所에서 分離한 細菌 ??가 生産하는 酵素中에서 macerating acton을 하는 酵素를 究明하기 위한 資料로 쓰기 위해 macerating enzyme의 生成에 미치는 pectin의 影響과 生成한 macerating enzyme와 pectolytic enzymes의 一般전 性質을 조사한 결과는 다음과 같다. 1. 細菌 ??는 酵素生成培地(wheat bran media)에 pectin을 加하므로써 macerating enzyme을 效果的으로 生産했다. 2. pectic acid liquefying activity, pectin liquefying activity, pectin esterase,와 macerating enzyme은 各各 1) 最適作用 pH가 7.2∼7.8, 7.2, 6.0, 6.5∼7.5 2) 最適作用溫度는 50℃, 50℃, 60℃, 50℃ 3) pH 安定性은 3.0∼8.0, 5.0∼8.0, 3.0∼8.0, 및 3.0∼8.0이었다. The influence of pectin on production of macerating enzyme by isolated bacterial ?? and the enzymological properties of its macerating enzyme produced and pectolytic enzymes were studed. The results obtained were summarized as follows: 1. Macerating enzyme was effectively produced in the media adding pectin. 2. The optimum pH of pectic acid liquefying activity, pectin liquefying activity, pectin esterase and macerating enzyme were 7.2∼7.8, 7.2, 6.0 and 6.5∼7.5 respectively. 3. The optimum temperature of pectic acid liquefying activity, pectin liquefying activity and macerating enzyme were 50℃., and that of pectin esterase was 60℃. 4. The pH stability of pectic acid liquefying activity, pectin esterase and macerating enzyme were 3.0∼8.0, and that of pectin liquefying activity was 5.0∼8.0.