호알칼리성 Bacillus sp. No. 4의 Cyclodextrin Glycosyltransferase에 의한 Glycosyl Sucrose의 생산과 저충치성 당으로서의 응용

손천배,유미경,김명희,문숙경 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Action of a cyclodextrin glycosyltransferase (CGTase) produce from alkalophilic Bacillus sp. No. 4 was studied in a solution containing starch and sucrose to prepare glycosyl sucrose syrup with good sweetness and antidecaying properties of teeth. In the initial stage of the reaction the CGTase produced cyclodextrin, however, the cyclodextrin disappeared and glycosyl sucrose was formed with the lapse of reaction time. The best proportion of sucrose to starch for prodution of glycosyl sucrose was about 1 : 1. The optimum pH and temperature of the coupling reaction was pH 6.0 and 60℃, respectively. Main composition of glycosyl sucrose syrup prepared with 20% starch and 20% sucrose was sucrose 18%, glucosyl sucrose (G_2F) 15.3% and amltosyl sucorse (G_3F) 11.3%. And glucose, maltose and maltotriose were produced very little. Smaller amounts of acid and insoluble glucan were formed in the syrup by Streptococcus mtans OMZ176 than in the sucrose. Therefore, the prepared glycosyl sucrose sucrose syrup is expected to prevent teeth from decaying.

黑麴菌의 變異에 있어서 生澱粉糖化 酵素活性의 變動

孫天培 충남대학교 자연과학연구소 1987 忠南科學硏究誌 Vol.14 No.1

This study was to induce mutant irradiated with UV rays from Soju brewing black-Koji molds-Aspergillus awamori that produces highlv raw starch saccharyfying enzymes, and the obtained mutant was investigated for fungal characters, condition of enzyme production, characteristics of purified enzyme. The mutant strain showed morphological characteristics such as smaller conidiospore, vesicle, conidial head, and lighter conidia compared with the parent strain. Also when it was cultivated in wheat bran media, the enzyme productivity was beat 2-3 days at 30℃. The raw starch saccharifying enzyme activity was increased approxymately two times and acid protease activity was increased seven times. By the enzyme purification, two glucoamylases were separated. The peak Ⅰ showed gelatinized starch saccharifying activity but the peak Ⅱ showed both raw and gelatinized starch saccharifing activity. The molecular weight, and the optimum pH of the raw starch saccarifying enzyme were 94,000, 3.5, and it was stable at pH 2-9. The amount of sugars in the carbohydrate - protein link - age was 17% and they were identified as mannose, glucose, and galactosamine. When various kinds of raw starches were saccharified by using enzyme which had same gelatinized starch saccharifing activity, it was found that the mutant strain saccharified faster than the parent strain. The saccharifying speed of the parent enzyme was in the order of corn>wheat>cassava>sweet potato>potato starch, but in the mutant strain enzyme, cassava starch saccharified faster than wheat starch. The acid protease produced from the mutant strain did not modify with raw starch saccharifying enzyme and it did not affect enzyme activity.

Aeromonas caviae No. S-76이 생산하는 Pullulanase의 정제, 특성 및 Maltosyl-β-Cyclodixtrin의 합성

손천배,김명희,이명자 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

The crude enzyme solution obtained by shaking culture of Aeromonas caviae No. S-76 isolated from soil as pullulanase producing bacterium was purified by 50 folds with 21% yield by salting out with ammonium sulfate and column chromatography using DEAE-Sephadex A-50 and Sephadex G-150. The purified pullulanase had a molecular weight of 118,000 approximately by SDS-polyacrylamide slab gel electrophoresis and pI of 4.3 by isoelectric focusing. And optimum reaction temperature and pH for pullulanase were 50℃ and 8.0, respectively. The purified enzyme was relatively stable at pH 6.0∼9.0 and below 45℃. This enzyme synthesized maltosyl-β-cyclodextrin from mixture of β-cyclodextrin and maltose.

Pullulanase를 생산하는 Aeromonas caviae No. S-76의 특성과 배양조건

손천배,김명희,이명자 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

A bacterial strain No. S-76 which produced pullulanase powerfully was isolated from soil. The isolated bacterium was 0.4∼0.6×0.8∼1.4 ㎛ in size, gram negative, rods, motile and was identified as Aeromonas caviae by Bergey's manual of determinative bacteriology with various characteristics investigated. The highest yield of pullulanase of the strain was obtained by using the following medium: 1% pullulan, soluble starch or corn starch as a carbon sources and 0.5% yeast extract, peptone as nitrogen sources with an initial pH of 9.0. The optimal culture conditions for production of pullulanase were at 32℃ for 2 days.

Γ-Cyclodextrin Glycosyltransferase 생산균주의 분리, 동정 및 효소 생산조건

김명희,손천배,임영희,배경숙,오태광 대전대학교 이과대학 기초과학연구소 1997 自然科學 Vol.- No.-

Cyclodextrin glycosyltransferase 생산균주를 토양으로부터 분리하여 형태학적, 생화학적 그리고 균주의 세포벽 지방산 조성분석에 의해 Bacillus brevis로 동정하였고, Bacillus brevis CD162로 명명하였다. 또한 배지조성에 따른 cyclodextrin glycosyltransferase의 최적생산조건을 검토한 결과, 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% KHPO₄, 0.05% MgSO₄·7H₂O, 1.5% Na₂CO₃ (pH 10.2)의 배지 조건에서 30℃에서 96시간 배양하였을 때 가장 높은 효소생산인 0.9 unit/ml을 얻을 수 있었다.(1997년 7월 10일 접수, 1997년 11월 21일 수리) A cyclodextrin glycosyltransferase-producing bacterium was newly isolated from soil using alkaline pH medium containg 1% Na₂CO₃, The isolated strain was identified as Bacillus brevis by morphological and biochemical characteristics, and designated Bacillus brevis CD162. The strain showed the best enzyme production of 0.9 unit/ml after 96 hrs of culture at 30℃ in a medium of 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% K₂HPO₄, 0.005% MgSO₄·7H₂O and 1.5% Na₂Co₃at initial pH 10.2

Bacillus brevis CD162 Cyclodextrin Glycosyltransferase의 정제 및 특성

김명희,손천배,임영희,오태광 대전대학교 이과대학 기초과학연구소 1997 自然科學 Vol.- No.-

Bacillus brevis CD162가 생산하는 cyclodextrin glycosyltransferase (CGTase)를 ammonium sulfate 침전, DEAE-Sephadex CL-6B 및 Sephadex G-150 컬럼 크로마토그래피를 수행하여 분리정제하였다. 정제된 CGTase는 분자량이 약74.000, 등전점은 약 6.3인 단백질이었고, 정제된 단백질을 SDS-PAGE한 후 변성된 단백질을 재활성시켜 zymogram을 수행한 결과 cyclodextrin glycosyltransferase임을 확인할 수 있었다. 이 효소의 최적활성 pH와 온도는 각각 8.0과 55℃이었으며, pH 5.5~9.0과 50℃까지 안정한 활성을 보였다. 또한, CGTase의 NH₂-말단 부위의 아미노산서열은 Ser-Val-Thr-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln 이었으며, 전분으로부터 cyclodextrin으로의 전환률을 분석한 결과, α-cyclodextrin은 1.3%, β-cyclodextrin은 33.9%, γ-cyclodextrin은 9.7% 이었다(1997년 7월 10일 접수, 1997년 9월 25일 수리) The cyclodextrin glycosyltrasferase (CGTase, EC 3.2.1.19) from Bacillus brevis CD162 was purified by precipitating with ammonium sulfate, DEAE-Sepharose CL-6B column chromatography and Sephadex G-150 column chromatography. The molecular mass and pI of the purified enzyme were estimated to be 74,000 and 6.3 by SDS-PAGe and isoelectric focusing, respectively. The purified enzyme was clearly identified as the CGTase by zymogram after SDS-PAGE. The optimum pH and temperature for the enzyme activity were 8.0 and 55℃, respectively. The enzyme was stable at the range of pH 5.5~9.0, and up to 50℃. The amino acid sequence from the NH₂-terminal of the purified CGTase was Ser-Val-Thr-Asn-Lys-Val-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln. The yields of the products from starch as the substrate were 1.3% for α-,33.9% for β-, and 9.7% for γ-cyclodextrin.

몇가지 酵母의 酸 및 알콜生成에 미치는 醱酵條件의 影響

朴允仲,孫天培 충남대학교 농업과학연구소 1977 農業技術硏究報告 Vol.4 No.2

These experiments were conducted to investigate the formation of organic acid and ethanol during fermentation by three yeast strains. One of these was industrial strain (No. 7) from Japan, and the others were wild types (No. 47, No. 239) isolated from Takju-mash and Strawbery, respectively. Conditions of fermentation were varied by differing the supply of oxygen (air), and by using different fermentation media The results obtained were as follows: 1) All the yeast strains produced higher amount of total organic acid and ethanol under the conditions which were aerobic, i.e. the flasks were opened during fermentation, than in case of using the flasks with fermentation bung. 2) Organic acid and ethanol were produced rapidly in the mash medium than in the semi-synthetic medium, i.e. total amount of organic acid and ethanol was maximized in a short time in the mash medium. 3) On the mash medium, the highest amount of organic acid was obtained by the strain No. 239, the next by No. 7 and the lowest by No. 47. Ethanol was produced on this medium with decreacing order of No. 47, No. 239, and No. 7. 4) The strain No. 239 was proved to be a powerful organic acid producer, yielding higher amount of organic acid especially under the aerobic conditions. 5) Above results suggests that the strain No. 239 could be of useful in alcoholic drink industry, due to its powerful ethanol-producing characteristic accompaning with high yielding of organic acids.

우수 사과酒酵母의 分離와 利用에 關한 硏究

朴允仲,金燦祚,李錫健,吳萬鎭,孫天培 충남대학교 농업과학연구소 1978 農業技術硏究報告 Vol.5 No.1

Extensive selection works on wild yeasts of fruits were carried cut to obtain strains which are applicable to apple wine making. Among the total number of 1,358 yeast strains which were isolated from various fruit samples collected from the vicinity of Daejeon and other regions of Korea, the strains SH-49, SH-129 and SH-338 were found to be useful. Then experiments on their morphological and physiological characteristics, and on the aspects of practical use in apple wine making were proceeded. The results obtained were as follows: 1. The strains SH-49 and SH-129, particularly SH-49, were appeared to have good fermentation ability, tolerance to sulfur dioxide and to produce fine quality of apple wine. 2. Apple wines made by using the strain, SH-49 and SH-129 contained less amount of total acids than those by other strains. 3. Apple wines of SH-49 and SH-129 were clarified rapidly during the primary fermentation period, and their absorbancy at 430 nm after 45 days of storage were approximately half of others. 4. Apple wine of SH-338 contained higher amount of residual sugar and its quality was superior to others. It is considered that this strain could be used in the production of apple wine of a characteristic quality. 5. The strains SH-49 and SH-338 were identified as a Saccharomyces cerevisiae according to Taxanomic Study of Yeasts by Lodder, however, classification of SH-129 was suspended for further study.

β-Amylase System Capable of Hydrolyzing Raw Starch Granules from Bacillus polymyxa No. 26 and Bacterial Identification

Sohn, Cheon Bae,Kim, Myung Hee,Bae, Jung Surl,Kim, Cheorl Ho 한국미생물 · 생명공학회 1992 Journal of microbiology and biotechnology Vol.2 No.3

A soil bacterium which produces raw starch-digesting β-amylase in culture medium, has been screened from soils. One strain, isolated and identified as Bacillus polymyxa No. 26, was selected as a β-amylase producing bacterium. Morphological and biological characteristics of the strain were found to be similar to those of a strain belonging to B. polymyxa. The electron microscopic observations of the bacterial vegetative cells and sporulated cells were extensively done to know the corelation between the enzyme synthesis and sporulation. When the bacterium was cultured on the appropriate media (3% dextrin, 0.3% beef extract,0.5% polypeptone, 1% yeast extract and 0.3% NaCl at pH 7.0 for 4 days) raw starch-digestible β-amylase was produced extracellularly. This strain produced 130 units of β-amylase per ml in a culture medium containing 3% dextrin at 30℃. This value is compared to those of other β-amylase-producing strains. The optimum pH and temperature for crude enzymes were pH 6.5 to 7.0 and 50℃, respectively. The enzymes were stable between pH 5.5 and 9.0 for 30 min at 45℃.

Biochemical Properties of Starch Granule Non-Digestive Enzyme(SGNA) of Bacillus polymyxa No.26

Sohn, Cheon Bae,Kim, Myung Hee,Bae, Jung Surl,Kim, Cheorl Ho 한국미생물 · 생명공학회 1992 Journal of microbiology and biotechnology Vol.2 No.3

A α-1,4-D-glucan maltohydrolase (β-amylase), secreted by the mesophilic aerobic bacterium Bacillus polymyxa No.26, was purified and characterized. The enzyme production was increased after a logarithmic phase of bacterial growth and paralleled with the onset of bacterial sporulation. By applying anion exchange chromatography and gel filtration the enzyme was purified 16.7-fold and had a specific activity of 285.7 units/㎎. Two enzyme activities were eluted on a column of DEAE-Sephadex chromatography, and they were designated as E-I for a major enzyme peak and E-II for a minor peak. Of them, E-I enzyme peak was further purified by using gel chromatography. The molecular mass of this enzyme was determined to be 64,000 daltons and consisted of a single subunit, showing an isoelectric point of 8.9. The enzyme was able to attack specifically the α-1,4-glycosidic linkages in soluble starch and caused its complete hydrolysis to maltose and β-limited dextrin. This amylolytic enzyme displayed a temperature optimum at 45℃ and a pH optimum at 7.0. The amino acid composition of the purified enzyme was quite similar to the other bacterial β-amylases reported. Surprisingly, the purified enzyme from this aerobe only exhibited hydrolytic activity on soluble starch, not on starch granules. The degradation of raw starch by β-amylase was greatly stimulated by pullulanase addition. These results differentiated from other β-amylases reported. Based on a previous result that showed the enzyme system involves in effective degradation of raw starch granules, this result strongly suggested that the purified enzyme (E-I) can be a synergistic part of starch granule-digestion and E-II plays a crucial role in digestion of starch granules.