대두 ${\alpha}-galactosidase$의 정제 및 성질

금종화,오만진,김성렬,Keum, Jong-Hwa,Oh, Man-Jin,Kim, Seong-Yeol 한국응용생명화학회 1991 Applied Biological Chemistry (Appl Biol Chem) Vol.34 No.3

To elucidate enzymatic properties of ${\alpha}-galactosidase$ (EC 3, 2, 1, 22) from germinated soybean, changes in the enzyme activities and oligosaccharide contents during germination of soybean were determined. ${\alpha}-Galactosidase$ from germinated soybean was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. Their chemical and enzymatic properties was investigated. ${\alpha}-galactosidase$ activity of sobeam was maximized when it was germinated at $25^{\circ}C$ for 120 hour. Raffinose and stachyose in soybean were decomposed completely after 96 hours and 120 hours of germination, respectively. Soybean ${\alpha}-galactosidase$ was purified by 6.6 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Cellulose and Sephadex A-50, and gel filtration on Sephadex G-150. Its specific activity was 825 Units/mg protein and the yield was 2.5% of the total activity of crude extracts. The purified ${\alpha}-galactosidase$ of soybean was found to be homogeneous by polyacrylamide gel electrophoresis and by HPLC. Isoelectric point of soybean ${\alpha}-galactosidase$ was determined analytical isoelectric focusing to be pH 4.8. The soybean ${\alpha}-galactosidase$ was monomeric and its molecular weight was estimated to be 30,000 by SDS-PAGE. The optimal temperature and pH for the soybeam ${\alpha}-galactosidase$ activity were $40^{\circ}C$ and pH 6.0 and 75% of its activity was lost by heating at $60^{\circ}C$ for 10 min. The enzyme was appeared to have higher affinity to raffinose than to stachyose. The Km value of soybean enzyme was 5.3 mM for ${\rho}-nitrophenyl-{\alpha}-D-galactopyranoside$ and the activation energy on PNPG was calculated to be 13.02 Kcal per mole. 대두 발아 과정 중의 ${\alpha}-galactosidase$를 추출하여 염석, 이온교환 크로마토그래피 및 겔 여과 등의 방법으로 정제한 후 정제효소의 효소학적 성질을 검토하였다. 대두 ${\alpha}-galactosidase$의 활성은 $25^{\circ}C$에서 120시간 발아시켰을 때 가장 높았으며, 대두 중의 raffinose는 96시간, stachyose는 120시간 발아시켰을 때 완전히 분해되었다. 대두 ${\alpha}-galactosidase$는 황산암모늄염석, DEAE-Cellulose 및 DEAE-Sephadex A-50 이온교환 크로마토그래피, Sephadex G-150 겔 여과 등에 의하여 비활성은 825U/mg protein으로써 6.6배까지 정제되었으며 수율은 2.5%이었고 HPLC와 PAGE에 의하여 순도를 확인하였다. 정제효소의 등전점은 pH 4.8이었고, 분자량은 30,000인 monomer이었으며 정제효소의 최적작용 PH는 6.0, 최적작용온도는 $40^{\circ}C$ 이었고, $60^{\circ}C$에서 10분 처리시 25%의 잔존 활성을 나타내었다. 정제효소는 stachyose보다 raffinose를 쉽게 분해하였으며 PNPG에 대한 Km값은 5.3 mM, 활성화 에너지는 13.02 cal/mole이었다.

흑마늘을 첨가하여 조제한 김치 양념소의 품질특성

유광원,황종현,금종화,이경행,Yu, Kwang-Won,Hwang, Jong-Hyun,Keum, Jong-Hwa,Lee, Kyung-Haeng 한국식품영양학회 2016 韓國食品營養學會誌 Vol.29 No.5

To enhance the physiological activities and reduce the off-flavor of garlic in kimchi, we manufactured kimchi seasoning replaced with 25~75% black garlic, we evaluated microbiological, physico-chemical and sensory evaluation. The changes of total aerobic and lactic acid bacteria were similar between the control and the kimchi seasoning replaced with black garlic during storage periods. The lightness of the kimchi seasoning replaced with black garlic was lower than that of the control and decrease of lightness was proportional to the concentration of black garlic. The redness and yellowness of the kimchi seasoning replaced with black garlic were decreased to the concentration of added black garlic and changes of the redness in all the samples were slightly increased during storage periods. But the yellowness did not change during storage periods. The changes of pH and acidity did not differ between the control and the samples of replaced with black garlic during storage periods. The sensory parameters including taste and flavor did not differ among treatments during storage periods. But color and overall acceptance of the control and sample replaced with 25% black garlic were higher than those of the samples replaced with 50 and 75% black garlic.

몇종의 세균과 Saccharomyces cerevisiae에 대한 식품첨가물의 향균 특성

이종수,오준세,김나미,금종화,이석건,Lee, Jong-Soo,Oh, Jun-Sei,Kim, Na-Mi,Keum, Jong-Hwa,Lee, Suk-Kun 배재대학교 자연과학연구소 1996 自然科學論文集 Vol.8 No.2

현재 식품첨가물로 많이 사용되고 있는 유기산과 안정제 및 색소 등의 각종 세균과 Sacch. cerevisiae 에 대한 향균 특성을 조사하였다. 산도 조절용으로 사용되고 있는 젖산, 사과산, 호박산 및 주석산은 L. acidophilus와 Sacch. cerevisiae에 대하여 향균성이 없었으나 B. subtilis등의 세균에 대하여는 향균성이 있었고 특히 사과산은 P. aeruginosa에 대하여 강한 향균력이 있었다.(최소생육저지농도 : 0.05%). 안정제로서의 알긴산과 펙틴은 B. subtilis, E. coli, P. aeruginosa에 대하여 비교적 강한 향균성을 보였고 L. acidophilus에 대하여는 향균성이 없었다. 황색 색소(홍화엘로우)와 적색 색소(Red powder-N)는 향균성이 없었고 표백제인 $NaHSO_3$의 세균에 대한 최소생육저지농도는 0.05%, Sacch. cerevisiae에 대하여는 0.5%로 향균성이 있었다. In order to survey the safety of some food additives, antimicrobial activity of acidulants, stabilizers, antioxidants, natural coloring materials and bleaching agents against 5 strains of bacteria and Sacch. cerevisiae were investigated by dilution method and minimal inhibitory concentration(MIC) method. Malic acid as acidulants displayed the effective antimicrobial activity in vitro against P. aeruginosa and its MIC is 0.05%. Alginic acid and pectin as stabilizer also displayed strong antimicrobial activity against B. subtilis, E. coli and P. aeruginosa, and tannin(antioxidants) and $NaHSO_3$ displayed antimicrobial activity against all bacteria tested. However gums(Arabia, Xanthan, Gua) and natural coloring materials(Hongwha Yellow, Red powder-N) were not affected to growth of bacteria and Sacch. cerevisiae.

大豆 및 Aspergillus niger α-galactosidase의 酵素學的 硏究

琴鍾和,吳萬鎭 충남대학교 농업과학연구소 1991 농업과학연구 Vol.18 No.1

To elucidate enzymatic properties of α-galactosidases (EC3, 2. 1. 22) from germinated soybean and Aspergillus niger changes in the enzyme activities and oligosaccharide contents during germination of soybean were determined and α-galactosidases from germinated soybean and wheat bran culture of Aspergillus niger were purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. Their chemical and enzymatic properties were investigated and the results obtained were summarized as follows : 1. α-Galactosidase activity of soybean was maximized when it was germinated at 25℃ for 120 hours. And raffinose and stachyose in soybean were decomposed completely after 96 hours and 120 hours of germination. respectively. 2. The highest level of α-Galactosidase activity was obtained when Aspergillus niger was grown on wheat bran medium at 30℃ for 96 hours. 3. Soybean α-galactosidase was purified by6.6 fold by ammonium slufate fractionation, ion exchange chromatography on DEAE-Cellulose and Sephadex A-50., and gel filtration on Sephadex G-150. Its specific activity was 825 units/㎎ protein and the yield was 2.5% of the total activity of crude extracts. 4. Aspergillus niger α-galactosidase was purified by 23.7 fold. Its specific activity was 1,929 units/㎎ protein and the yield was 14% of the total activity of wheat bran culture. 5. The purified α-galactosidases of soybean and. Aspergillus niger were found to be homogeneous by polyacrylamide gel electrophoresis and by HPLC. 6. Chemical properties of the purified α-galactosidases were : 1) The soybean α-galactosidase was monomeric and its molecular weight was estimated to be 30.000 by SDS-PAGE whereas the Aspergillus niger α-galactosidase was a tetrameric glycoprotein which consisted of identical subunits with molecular weight of 28,000 each. 2) Isoelectric points of α-galactosidases were determined analytical isoelectric focusing to be : pH 4.8 for the soybean enzyme; and pH 4.6 for the Aspergillus niger enzyme, respectively. 3) Among the amino acids in active sites of both soybean and Aspergillus niger α-galactosidases, histidine was identified by chemical modification of diethyl pyrocarbonate. 1 4) The activity of soybean α-galatosidase was inhibited by 2-mercaptoethanol and L-cysteine. 7. Enzymatic properties of the purified α-galatosidases were : 1) The optimal temperature and pH for the α-galactosidase activity were : 40℃ and pH 6.0 for the soybean enzyme : and 40℃ and pH 6.5 for the Aspergillus niger enzyme. 2) The soybean α-galactosidase was stable at the range of pH 5.5 to 6.5 and at temperature below 45℃, however 75% of its activity was lost by heating at 60℃ for 10 min. The Aspergillus niger α-galactosidase was stable at the range of pH 6.0 to 7.0 and at temperature below 45℃, but 55% of its activity was lost under the same condition. 3) No significant difference was found in substrate specificity between (he bean and enzymes and both appeared to have higher affinity to raffinose than to stachyose. The enzymes were inhibited by galactose. 4) The Km values of soybean and Aspergillus niger enzymes were 5.3mM and 5.0mM for p-nitrophenyl-α-D-galactopyranoside, 50.0mM and 37.0mM for raffinose. and 55.5mM and 55.5mM for stachyose, respectively. 5) The activation energy and Q_10 value on p-nitrophenyl-α-D-galactopyranoside were calculated to be : 13.024 ㎉ per mole and 2.0 for the soybean enzyme; and 8.515 Kcal per mole and 1.38 for the Aspergillus niger enzyme.

마늘 식초 및 호박 식초에 관한 연구

금종화 한국식품영양학회 1999 韓國食品營養學會誌 Vol.12 No.5

5% 에탄을 용액에 마늘을 파쇄하여 10% 가하고 Acetobacter aceti 3281로 35℃에서 26일간 발효시켜서 마늘식초를 제조하였다. 호박식초는 호박술을 기질로 하여 Acetobacter aceti 3281로 35℃에서 26일간 발효시켜서 제조하였다. 마늘식초와 호박식초의 총당은 0.04 ㎎/ml 및 1.53 ㎎/m1, 환원당은 0.122 ㎎/ml 및 0.406 ㎎/ml, 비중은 1.001 및 1.004, 에탄올은 모두 0.06% 및 0.02%, 균체수는 8.52 및 8.48 CFU/ml, pH는 3.06 및 3.20, 산도는 4.98 및 5.02를 나타냈다. 기호도는 마늘식초는 2.7. 호박식초는 3.9를 나타냈다. The garlic vinegar brewed with 5% ethanol solution added 10% crushed garlic was fermented by Acetobactor aceti 3281 at 30℃ for 26 days. Pumpkin wine vinegar was made from acetic acid fermentation of pumpkin wine at 35℃ for 26 days. The garlic vinegar and pumkin wine vinegar contained 0.04㎎/ml and 1.53㎎/ml of total sugar, 0.122/㎎/ml and 0.406㎎/ml of reducing sugar, and 0.06 and 0.02% of ethanol. Specific gravity of garlic vinegar and pumkin wine vinegar was 1.0012 and 1.0015, repectively. Viable cell count of garlic vinegar and pumkin wine vinegar was 8.53 and 8.48CFU/ml, respectively. pH of garlic vinegar and pumkin vinegar was 3.06 and 3.20, respectively. Acidity of garlic vinegar and pumkin wine vinegar was 4.98 and 5.02, respectively. Sensory evaluation garlic of vinegar and pumkin wine vinegar was 2.7 and 3.9, respectively.

Aspergillus niger α-Galactosidase의 정제 및 성질

금종화,오만진,김찬조 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.5

Aspergillus niger가 생산하는 α-glactosidase의 효소학적 성질을 조사하기 위하여 시험균주를 밀기울 배양한 후 생성된 α-glactosidase를 염석, 이온교환 크로마토그래피 및 겔 여과 등의 방법으로 정제한 후 정제효소의 효소학적 성질을 검토하였다. Asp. niger를 밀기울 배지에서 30℃, 4일 배양했을 때 효소활성이 가장 높았으며 α-glactosidase는 황산암모늄 염석, DEAE-cellulose 및 DEAE-Sephadex A-50 이온교환 크로마토그래피, Sephadex G-150 겔 여과 등에 의하여 23.7배까지 정제되었으며 비활성이 1,220 U/㎎·protein, 수율 14%이었고 HPLC와 PAGE에 의해 순도가 확인되었다. 정제효소는 당 단백질로서 등전점은 4.6이었고 분자량이 28,000인 monomer 4개로 구성된 tetramer이었다. 정제효소의 최적작용 pH는 6.5, 최적작용온도는 40℃이었고 60℃에서 10분 처리시 46%의 잔존활성을 나타내었다. 정제효소는 stachyose보다 raffinose를 쉽게 분해하였고 PMPG에 대한 K_m값은 5.0mM, 활성화에너지는 8.515Cal/mole이었다. To elucidate enzymatic properties of α-glactosidase(EC 3.2.1.22) from Asp. niger, α-glactosidase from wheat bran culture was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. And then its enzymatic propeties were investigated. The highest level of α-glactosidase activity was obtained when Asp. niger was grown on wheat bran medium at 30℃ for 96 hours. The α-glactosidase was purified by 23.7 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Celluose and Sephadex A-50, and gel filtration on Sephadex G-150 and its specific activity was 1,229 Units/㎎ protein and the yield was 14% of the total activity of wheat bran culture. The purified α-glactosidase was found to be homogeneous by polyacrylamide gel electrophoresis and HPLC. The α-glactosidase was a tetrameric glycoprotein which consisted of identical subunits with molecular weight of 28,000 each by SDS-PAGE and isoelectric point was determined analytical isoelectric focusing to be pH 4.6. The optimal temperature and pH for the α-glactosidase activity were 40℃ and pH 6.5, respectively, and 54% of its activity was lost by heating at 60℃ for 10 mins. It was appeared to have higher affinty to raffinose than to stachyose. The K_m value and activation energy of α-glactosidase were 5.0 mM and 8.515㎉ per mole for p-nitrophenyl-α-D-galactopyranoside, respectively.

과요오드산 산화당에 의한 효소의 안정성

금종화 한국식품영양학회 2001 韓國食品營養學會誌 Vol.14 No.3

NaIO_4-산화 전분당을 Bzcillus licheniformisα-아밀라아제와 반응시켜서 시프염기 형성으로 당단백질로 변형시켜서 안정성을 확인하였다. 100℃에서의 열안정성은 10분 뒤에, pH 9.7에서 변형한 효소>pH 8.0에서 변형한 효소>비변형 효소의 순으로 높았다. 그러나 변형 및 안정성에 α-cyclodextrin(α-CD)을 사용한 결과 큰 차이는 나지 않았다. pH 8.0에서 α-CD존재 하에 변형한 효소는pH8∼11의 알칼리 쪽에서 가장 높은 안정성을 나타냈으나 pH5∼7 사이에서는 다른 효소보다 낮았다. pH 9.7에서 변형하지 않은 효소는pH 5부터 pH 13까지 서서히 증가하였고 pH 9.7에서 α-CD존재 하의 효소는pH 5부터 7까지 증가하다가 그후 pH 13까지 서서히 감소하였다. α-CD 존재 하의 비변형 효소는 pH 7과 10에서 피크를 나타낸 다음 pH 12 이후에는 급격히 낮아졌다. 변형한 효소는 HPLC의 유출시간이 빨라져서 변형하지 않은 효소보다 분자량이 큰 것으로 나타났다. 분자량 크기는 비변헝 효소 < pH 8,0에서 변형할 효소 < pH 9.7에서 변형한 효소의 순으로 컸다. NaIO_4-oxidized soluble starch was added to Bacillus licheniformis α-amylase by Schiff base reaction to make glycoprotein. Thermal stability at 100℃, in 10 minutes, proved high in the order of enzyme modified at pH 9.7, enzyme modified at pH 8.0 and native enzyme, respectively. But when α-cyclodextrin(α-CD) was used to modification and stabilization, the result showed no big difference. Modified enzyme under α-CD at pH 8.0 shows highest stability in pH 8∼11, while low in the pH 5∼7, compared to the other enzyme. Native enzyme at pH 9.7 gradually increased up to pH 13 from pH 5, enzyme under α-CD at pH 9.7 increased from pH 5 to 7 and little by little decreased up to pH 13. Native enzyme under α-CD peaked pH 7 and pH 10, and lowered sharply after pH 12. As molecular weight became larger than native enzyme, HPLC retention time of modified enzyme quickened. The molecular weight proved large in the order of modified enzyme at 9.7, enzyme modified at pH 8.0, and native enzyme.

大豆요구르트 製造過程中의 成分變化

琴鍾和,吳萬鎭 충남대학교 농업과학연구소 1984 農業技術硏究報告 Vol.11 No.1

This experiment was carried out to obtain the fundamental data for development of digestibility and quality enhanced product of soy yogurt. Soy yogurt was processed from raw materials of soybean, defatted soybean and sprouted soybean which inoculated with Lactobacillus acidophilus and Bifidobacterium bifidum as a starter. Changes of chemical compositions, viable cell count and saccharides during processing were investigated including acceptibility of manufactured products. The results were summarized as follows; 1 . Defatted soy milk fermented with Lactobacillus acidophilus was slowed the greatest initial acid productivity and sprouted soy milk was showed the greatest growth of lactobacillus acidophilus. 2. Acid production was accelerated when 2% glucose was used in soy milk. 3. Addition of reconstituted skim milk in soy milk and defatted soy milk increased acid production but was not showed the effect in the sprouted soy milk. 4. Sprouting soybean, the contents of raffinose and stachyose were decreased but those of glucose was increased. 5. When soy milk was fermented with Lactobacillus acidophilus, the contents of raffinose and stachyose were decreased. 6. As a result of panel test, sprouted soy yogurt which was produced by addition of reconstituted skim milk of 10% showed the greatest flavor and tastes.

水質汚染 指標種으로서의 淡水藻類에 關한 硏究

朴鐘聲,崔信錫,申昌男,琴鍾和 忠南大學校 環境問題硏究所 1984 環境硏究 Vol.2 No.2

In order to determine the fresh- water algae as indicators of water pollution, an attempt was conducted from February 28 to October 20, 1984 at the six stations of the Geum River. 1. The St. 1 and 2 showed farirly good water quality. The value of BOD at St. 3 was 6.4 ppm exceeding the permitted level of pollution (5 ppm). Those values at St. 4, 5, and 6 were 2.0- 5.0ppm below the permitted level. 2. DO, BOD, COD, C1-,and NH₃-N contents were significantly different from one another of the surveyed stations of the Geum River. 3. The algae collected at all surveyed stations were of 52 genera 119 species. The relative abundance of Nostoc planctonicum and Oscillatoria limosa were very high at St. 3 where the level of pollution was relatively high and at St. 6 where the water quality was affected by the sea-water under the tidal condition. 4. In the Geum River, Gomphonema olivaceum could be regarded as the most important indicator of the water pollution. The permitted levels of BOD, COD, C1-, and NH₃-N contents for its survival are 5.0, 7.9, 39.1, and 4.1 ppm, respectively.

대두 α - galactosidase 의 정제 및 성질

오만진,금종화,김성렬 한국농화학회 1991 Applied Biological Chemistry (Appl Biol Chem) Vol.34 No.3

To elucidate enzymatic properties of α-galactosidase (EC 3, 2, 1, 22) from germinated soybean, changes in the enzyme activities and oligosaccharide contents during germination of soybean were determined. α-Galactosidase from germinated soybean was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. Their chemical and enzymatic properties was investigated. α-galactosidase activity of sobeam was maximized when it was germinated at 25℃ for 120 hour. Raffinose and stachyose in soybean were decomposed completely after 96 hours and 120 hours of germination, respectively. Soybean α-galactosidase was purified by 6.6 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Cellulose and Sephadex A-50, and gel filtration on Sephadex G-150. Its specific activity was 825 Units/㎎ protein and the yield was 2.5% of the total activity of crude extracts. The purified α-galactosidase of soybean was found to be homogeneous by polyacrylamide gel electrophoresis and by HPLC. Isoelectric point of soybean α-galactosidase was determined analytical isoelectric focusing to be pH 4.8. The soybean α-galactosidase was monomeric and its molecular weight was estimated to be 30,000 by SDS-PAGE. The optimal temperature and pH for the soybeam α-galactosidase activity were 40℃ and pH 6.0 and 75% of its activity was lost by heating at 60℃ for 10 min. The enzyme was appeared to have higher affinity to raffinose than to stachyose. The Km value of soybean enzyme was 5.3 mM for ρ-nitrophenyl-α-D-galactopyranoside and the activation energy on PNPG was calculated to be 13.02 K㎈ per mole.