효소제이용에 의한 주정발효에 대하여

성낙계 한국농화학회 1967 Applied Biological Chemistry (Appl Biol Chem) Vol.8 No.1

Starch was liquefied by the bacteriological α-amylase and saccharified separately by four strains of mold as saccharogenic enzymes, then these was fermented for research the fermentation rates, the usefulness of application of four strains of mold and the S.P to need in proportion increasing of concentration of mixed-starches for brewing in this experiment. The obtained results were as follows: 1) In the ability of saccharificatien, fermentation and acid resistance. Aspergillus usamii mut shirousamii, which were used to produce the koji, were excellent, however, Rhizopus delemar had heavy pollution in a lot of production of koji, were-as its qualities as enzyme was good. 2) The necessary S.P in proportion to the concentration of mixed-starch for brewing was found in this experiment in which was fermented in the various concentration of the adding S.P and the mixed-starches for brewing. 3) In the experiment of almost manufacture scale, the S.P needed essentially in the saccharogenic formentation was found and the usefulness of the necessary S-P was observed, and in fact in the manufacture scale it. was safer to add the S.P more than 1.2% because it must be considered that the ability of enzymes is reduced by heat or acid. 4) The period of fermentation was shorten because the conditions for yeast propagation were excellent as a results of starches being liquefied by bacteriological amylase, and saccharified by molds as saccharogenic enzymes.

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.

고온·알칼리성 Bacillus sp.의 Catabolite Repression 저항성 변이주로부터 Cellulase 및 Xylanase의 생산

최수철,정영철,강갑석,심기환,성낙계 慶尙大學校 1990 論文集 Vol.29 No.2

고온·알칼리성 cellulase 및 hemicellulase 생성 미생물을 분리할 목적으로 pH 10.3의 PY-CMC배지에서 55℃로 3회 연속 직접배양하여 효소 생성능이 우수한 20여균주를 분리하였고, 그 중에서 2개균주를 최종 선정하여 균학적 성상을 조사한 결과 고온·알칼리성 Bacillus속과 거의 일치하였다. 선정, 동정된 고온·알칼리성 Bacillus sp. F204와 F1020은 α-1, 4-glucose 등에서는 효소 생합성이 왕성하였으나, glucose와 같은 단당류에서는 효소가 전혀 유도되지 않았는데, 이 두 균주는 carbon catabolote repression을 받고 있었다. 2-deoxy glucose는 야생주에서는 강력한 repressor로 작용하였고, 야생주의 catabolite repression을 해제 시키는데 antimetabolite로 작용한다는 것이 밝혀졌다. NTG(500㎍/㎖, 50℃, 20∼60분)와 UV(2분) 처리후 2-DG(20mM)에 직접배양하여 강력한 catabolite repression resistant strain인 M-211과 M-367을 분리하였다. M-211과 M-367은 2-DG를 energy원으로 이용하였으며, glucose에 대하여 CMCase는 1.5%, avicelase 및 xylanase는 약 1.0%까지 저항성을 보였다. 또한 두개의 변이주의 효소 활성은 CMCase 2배, avicelase 1.5배 및 xylanase3배의 증가를 보였으며, 각 효소생합성을 위한 gene expression은 cAMP-independent transcription을 하는 것으로 나타났다. Cellulase and xylanase synthesis of thermophilic alkalophilic Bacillus sp. F204 and F1020 isolated in our lab, were repressed by glucose and induced by compounds structurally related to α-1,4 linkage (avicel cellulose and xylan et. al.). Catabolite repression-resistant mutants were obtained through treatment of NTG(500㎍./㎖), UV(2 min) and enrichment culture in 2-deoxy glucose (20 mM) containing PY-glucose on the wild type strain. T재 catabolite repression resistant mutants were not repressed by glucose and produced cellulase complex and xylansase when in grown in the presence of 1.0% glucose and 30mM 2-DC, which were repressors in wild type. Enzyme production was greater with the mutants (CMCase of 2 times, avicelase of 1.5 times, xylanase of 3 times) than with the wild type strains under various test conditions. T재 mutants M-211 and M-367 were potential catabolite repression resistant mutants and their gene expression was cAMP independent transcription.

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.

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.

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 ??

사상균에 의한 구연산발효에 관한 연구 (제4보) 가용성전분 및 당밀에 의한 구연산발효

성낙계,김명찬,심기환,정덕화 ( Nack Kie Sung,Myung Chan Kim,Ki Hwan Shim,Duck Hoa Chung ) 한국미생물생명공학회 1980 한국미생물·생명공학회지 Vol.8 No.3

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.

속간 원형질체 융합에 의한 섬유질 기질로 부터 L - lysine 생산균주 개발 - 원형질체의 형성 및 재생 -

성낙계,정덕화,이무영,정영철 ( Nack - Kie Sung,Duck - Hwa Chung,Mu - Young Lee,Young - Chul Chung ) 한국미생물생명공학회 1988 한국미생물·생명공학회지 Vol.16 No.2

호알칼리성 Bacillus sp . F204 와 Bacillus sp . K17 의 원형질체 융합

성낙계,노종수,박석규,정영철 ( Nack - Kie Sung,Jong - Su Roh,Seok - Kyu Park,Young - Chul Chung ) 한국미생물생명공학회 1988 한국미생물·생명공학회지 Vol.16 No.4