• Title/Summary/Keyword: 분리당화

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Cloning of $\beta$-Glucosidase Gene from Streptomyces coelicolor A3(2) and Characterization of the Recombinant $\beta$-Glucosidase Expressed in Escherichia coli (Streptomyces coelicolor A3(2)로 부터 $\beta$-Glucosidase 유전자 클로닝 및 재조합 효소의 특성)

  • Kim, Jae-Young;Kim, Bong-Kyu;Yi, Yong-Sub;Kang, Chang-Soo;Ahn, Joong-Hoon;Lim, Yoong-Ho
    • Microbiology and Biotechnology Letters
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    • v.37 no.2
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    • pp.99-104
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    • 2009
  • The $\beta$-glucosidase gene from Streptomyces coelicolor A3(2) was cloned and expressed in Escherichia coli. The ORF consisted of 1377 nucleotides encoding 51 kDa in a predicted molecular weight. Effects of pH indicated that the $\beta$-glucosidase showed similar activity using $\alpha$-pNPG($\rho$-nitrophenyl-$\alpha$-D-glucopyranoside), $\beta$-pNPG($\rho$-nitrophenyl-$\beta$-D-glucopyranoside), and $\beta$-pNPF($\rho$-nitrophenyl-$\beta$-D-fucopyranoside) at range of pH 3 to 10, and high activity using $\beta$-pNPGA ($\rho$-nitrophenyl-$\beta$-D-galactopyranoside) from pH 5 to 10, especially, 3.3 times higher activity at pH 9. Effects of temperature indicated that the $\beta$-glucosidase showed low activity using $\alpha$-pNPG, $\beta$-pNPG, and $\beta$-pNPF from $20^{\circ}C$ to $70^{\circ}C$, and increased activity using $\beta$-pNPGA from $30^{\circ}C$ to $50^{\circ}C$, 1.8 times higher activity at $50^{\circ}C$ than at $30^{\circ}C$. According to activity determination of other substrates, the enzyme was active on daidzin, genistin, and glycitin, inactive on esculin and apigenin-7-glucose. The EDTA and DTT as reducing agents inhibited $\beta$-glucosidase activity, but SDS and mercaptoethanol did not inhibit. Monovalent or divalent metal ions such as $MnSO_4$, $CaCl_2$, KCl, and $MgSO_4$ did not inhibited $\beta$-glucosidase activity. $CuSO_4$ and NaCl showed low inhibition, and $ZnSO_4$ inhibited 3.3 times higher than control.

Cellulosic Ethanol Production (셀룰로식 (Cellulosic) 에탄올 생산)

  • Chung, Chang-Ho
    • KSBB Journal
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    • v.23 no.1
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    • pp.1-7
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    • 2008
  • The world demand of ethanol as an alternative fuel for gasoline is increasing rapidly because of high oil price and global climate change. Most of ethanol is currently produced from corn grain or sugars in sugarcane and sugar beet. Because these sources compete with foods and animal feed and are not expected to be enough for future demand of ethanol. Thus, cellulosic ethanol from agricultural residues or wood has to be commercialized in near future. Typical cellulosic ethanol production consists of pretreatment, enzyme hydrolysis, fermentation and product separation. This paper reviews the principles and status of each step and discusses issues for cellulosic ethanol production.

Development of Black Garlic Yakju and Its Antioxidant Activity (흑마늘 발효주 개발 및 항산화 활성)

  • Lee, Hyo-Hyung;Kim, Ig-Jo;Kang, Sang-Tae;Kim, Yeong-Hoon;Lee, Jeong-Ok;Ryu, Chung-Ho
    • Korean Journal of Food Science and Technology
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    • v.42 no.1
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    • pp.69-74
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    • 2010
  • Black garlic has recently received significant attention due to its various health functional properties, and there has been an increase in demand for its use as a functional food. This study was performed to determine the optimum concentration for the fermentation of black garlic yakju. In addition, the antioxidant activity of the fermented black garlic yakju was examined. The alcohol content in the black garlic yakju significantly increased for 6 days and the pH gradually increased as the concentration of black garlic increased. The reducing sugar content at each black garlic concentration was maximal when it was fermented for 24 hours, and then rapidly decreased at longer fermentation periods. The main organic acids were lactic, citric, malic and oxalic acid. Also, the lactic acid content increased as the concentration of the black garlic increased where as the content of other organic acids decreased. The total polyphenol content, ferric ion reducing antioxidant power (FRAP) activity and DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging activity of black garlic yakju increased as the concentration of black garlic increased. The sensory characteristics of fermented black garlic yakju were evaluated in terms of color, flavor, taste and overall acceptability, and the highest overall acceptability value was obtained for yakju containing a black garlic concentration of 1-3%. Therefore, the optimum concentration of black garlic was determined to be 1% for the production of high quality black garlic yakju.

Studies on the Brewing of Kochuzang (Red Pepper Paste) by the Addition of Yeasts (효모첨가(酵母添加)에 의(依)한 고추장의 양조(釀造)에 관(關)한 연구(硏究))

  • Lee, Taik-Soo
    • Applied Biological Chemistry
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    • v.22 no.2
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    • pp.65-90
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    • 1979
  • This study was conducted to establish the brewing method which would be useful for the production of Kochuzang. Kojis, which were made from various materials and microorganisms under a covered condition, were investigated and compared. Yeasts (Saccharomyces rouxii and Torulopsis versatilis) were added to Kochuzang, and the enzyme activity, microflora, chemical composition, nitrogen content, alcohol content and free sugars of Kochuzang were investigated and analysed. The results obtained are as follows: 1. Koji making (1) Glutinous rice-soybean group was superior to glutinous rice group in the saccharogenic and liquefying amylase activities of three day-Koji. (2) Protease activity (acid, neutral and alkaline) of glutinous rice-soybean Koji, which was inoculated with Aspergillus oryzae A, was increased till the 5th day, while other groups showed maximum activity after the 3rd day. (3) The maximum cellulose activity of Aspergillus oryzae B-Koji and A-Koji was observed after the 2nd day and the 3rd day, respectively. High cellulose activity of Aspergillus oryzae B-Koji and A-Koji was respectively shown in glutinous rice group and glutinous rice-soybean group at maximum. (4) Compared with glutinous rice Koji, glutinous rice-soybean Koji gave larger number of yeast and aerobic bacteria. 2. Kochuzang Fermentation (1) Each Kochuzang group shoved different liquefying and saccharogenic amylase activities. The highest activities were generally shown in 10 to 40 days after mashing and remarkably reduced in the last stage of aging. (2) Protease activities of each group were strong in order of acid, neutral and alkaline protease. Especially acid protease showed highest activity at the 40th to 50th day Kochuzang. (3) Each group showed maximum cellulase activity in the 40th and 50th day-Kochuzang and then decreased. (4) Osmophilic yeast of yeast-added Kochuzang after one-month aging was distinctively outnumbered compared with non-yeast-added Kochuzang, but two groups were similar after two months. (5) Yeast-added group and non-added group gave almost the same number of halophilic lactic acid bacteria in Kochuzang, but the non-added group gave slightly larger number of aerobic bacteria than the yeast-added group. (6) Amino nitrogen contents in all test group were increased rapidly till the 60th day of Kochuzang aged. After that the contents were increased slowly. (7) Ethyl alcohol contents of 20day-fermented Kochuzang were high in order of Saccharomyces rouxii-added group, Torulopsis versatilis-added group, Saccharomyces rouxii and Torulopsis versatilis mixed group and non-yeast-added group. But all test group showed about 2% in ethyl alcohol content after 40days of aging. (8) Alcohol content in the 7 month-aged Kochuzang of all test groups was high in order of ethyl alcohol, n-butyl alcohol, n-propyl alcohol and iso-propyl alcohol. Torulopsis versatilis-added group had the highest value of ethyl alcohol, n-propyl alcohol and n-butyl alcohol. (9) Reducing sugar in Kochuzang was increased after 20 days of aging compared with the 10days-ferment. The reducing sugar content in Saccharomyces rouxii-added group was distinctively small compared with that of other groups, decreasing after 30days of aging. (10) Rhamnose, fructose, glucose and maltose were isolated from the 10 day fermented Kochuzang. Raffinose was also found after 300 days-aged group, and fructose content was high in the 300days-aged Kochuzang. However, glucose content was smaller than that of 10days-fermented Kochuzang. (11) For the organoleptic tests of Kochuzang, taste, flavour and color of yeast-added group were superior to the non yeast-added group. Especially the complex yeast group among the yeast added groups were the best of all. Yeast-added group after 300 days of aging took higher paint in flavour test than that of non-added group. Therefore, brewing method like complex yeast added group seems to be advantageous for short time brewing Kochuzang.

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Microbiological and Enzymological Studies on Takju Brewing (탁주(濁酒) 양조(釀造)에 관(關)한 미생물학적(微生物學的) 및 효소학적(酵素學的) 연구(硏究))

  • Kim, Chan-Jo
    • Applied Biological Chemistry
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    • v.10
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    • pp.69-100
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    • 1968
  • 1. In order to investigate on the microflora and enzyme activity of mold wheat 'Nuruk' , the major source of microorganisms for the brewing of Takju (a Korean Sake), two samples of Nuruk, one prepared at the College of Agriculture, Chung Nam University (S) and the other perchased at a market (T), were taken for the study. The molds, aerobic bacteria, lactic acid bacteria, and yeasts were examined and counted. The yeasts were classified by the treatment with TTC (2, 3, 5 triphenyltetrazolium chloride) agar that yields a varied shade of color. The amylase and protease activities of Nuruk were measured. The results were as the followings. a) In the Nuruk S found were: Aspergillus oryzae group, $204{\times}10^5$; Black Aspergilli, $163{\times}10^5$; Rhizogus, $20{\times}10^5$; Penicillia, $134{\times}10^5$; Areobic bacteria, $9{\times}10^6-2{\times}10^7$; Lactic acid bacteria, $3{\times}10^4$ In the Nuruk T found were: Aspergillus oryzae group, $836{\times}10^5$; Black Aspergilli, $286{\times}10^5$; Rhizopus, $623{\times}10^5$; Penicillia, $264{\times}10^5$; Aerobic bacteria, $5{\times}10^6-9{\times}10^6$; Lactic acid bacteria, $3{\times}10^4$ b) Eighty to ninety percent of the aerobic bacteria in Nuruk S appeared to belong to Bacillus subtilis while about 70% of those in Nuruk T seemed to be spherical bacteria. In both Nuruks about 80% of lactic acid bacteria were observed as spherical ones. c) The population of yeasts in 1g. of Nuruk S was about $6{\times}10^5$, 56.5% of which were TTC pink yeasts, 16% of which were TTC red pink yeasts, 8% of which were TTC red yeasts, 19.5% of which were TTC white yeasts. In Nuruk T(1g) the number of yeasts accounted for $14{\times}10^4$ and constituted of 42% TTC pink. 21% TTC red pink 28% TTC red and 9% TTC white. d) The enzyme activity of 1g Nuruk S was: Liquefying type Amylase, $D^{40}/_{30},=256$ W.V. Saccharifying type Amylase, 43.32 A.U. Acid protease, 181 C.F.U. Alkaline protease, 240C.F.U. The enzyme activity of 1g Nuruk T was: Liquefying type Amylase $D^{40}/_{30},=32$ W.V. Saccharifying type amylase $^{30}34.92$ A.U. Acid protease, 138 C.F.U. Alkaline protease 31 C.F.U. 2. During the fermentation of 'Takju' employing the Nuruks S and T the microflora and enzyme activity throughout the brewing were observed in 12 hour intervals. TTC pink and red yeasts considered to be the major yeasts were isolated and cultured. The strains ($1{\times}10^6/ml$) were added to the mashes S and T in which pH was adjusted to 4.2 and the change of microflora was examined during the fermentation. The results were: a) The molds disappeared from each sample plot since 2 to 3 days after mashing while the population of aerobic bacteria was found to be $10{\times}10^7-35{\times}10^7/ml$ inS plots and $8.2{\times}10^7-12{\times}10^7$ in plots. Among them the coccus propagated substantially until some 30 hours elasped in the S and T plots treated with lactic acid but decreased abruptly thereafter. In the plots of SP. SR. TP. and TR the coccus had not appeared from the beginning while the bacillus showed up and down changes in number and diminished by 1/5-1/10 the original at the end stage. b) The lactic acid bacteria observed in the S plot were about $7.4{\times}10^7$ in number per ml of the mash in 24 hours and increased up to around $2{\times}10^8$ until 3-4 days since. After this period the population decreased rapidly and reached about $4{\times}10^5$ at the end, In the plot T the lactic acid becteria found were about $3{\times}10^8$ at the period of 24 fours, about $3{\times}10$ in 3 days and about $2{\times}10^5$ at the end in number. In the plots SP. SR. TP, and TR the lactic acid bacteria observed were as less as $4{\times}10^5$ at the stage of 24 hours and after this period the organisms either remained unchanged in population or ceased to exist. c) The maiority of lactic acid bacteria found in each mash were spherical and the change in number displayed a tendency in accordance with the amount of lactic acid and alcohol produced in the mash. d) The yeasts had showed a marked propagation since the period of 24 hours when the number was about $2{\times}10^8$ ㎖ mash in the plot S. $4{\times}10^8$ in 48 hours and $5-7{\times}10^8$ in the end period were observed. In the plot T the number was $4{\times}10^8$ in 24 hours and thereafter changed up and down maintaining $2-5{\times}10^8$ in the range. e) Over 90% of the yeasts found in the mashes of S and T plots were TTC pink type while both TTC red pink and TTC red types held range of $2{\times}10-3{\times}10^7$ throughout the entire fermentation. f) The population of TTC pink yeasts in the plot SP was as $5{\times}10^8$ much as that is, twice of that of S plot at the period of 24 hours. The predominance in number continued until the middle and later stages but the order of number became about the same at the end. g) Total number of the yeasts observed in the plot SR showed little difference from that of the plot SP. The TTC red yeasts added appeared considerably in the early stage but days after the change in number was about the same as that of the plot S. In the plot TR the population of TTC red yeasts was predominant over the T plot in the early stage which there was no difference between two plots there after. For this reason even in the plot w hers TTC red yeasts were added TTC pink yeasts were predominant. TTC red yeasts observed in the present experiment showed continuing growth until the later stage but the rate was low. h) In the plot TP TTC pink yeasts were found to be about $5{\times}10^8$ in number at the period of 2 days and inclined to decrease thereafter. Compared with the plot T the number of TTC pink yeasts in the plot TP was predominant until the middle stage but became at the later stage. i) The productivity of alcohol in the mash was measured. The plot where TTC pink yeasts were added showed somewhat better yield in the earely stage but at and after the middle stage the difference between the yeast-added and the intact mashes was not recognizable. And the production of alcohol was not proportional to the total number of yeasts present. j) Activity of the liquefying amylase was the highest until 12 hours after mashing, somewhat lowered once after that, and again increased around 36-48 hours after mashing. Then the activity had decreased continuously. Activity of saccharifying amylase also decreased at the period of 24 hours and then increased until 48 hours when it reached the maximum. Since, the activity had gradually decreased until 72 hours and rapidly so did thereafter. k) Activity of alkaline protease during the fermentation of mash showed a tendency to decrease continusously although somewhat irregular. Activity of acid protease increased until hours at the maximum, then decreased rapidly, and again increased, the vigor of acid protease showed better shape than that of alkaline protease throughout. 3. TTC pink yeasts that were predominant in number, two strains of TTC red pink yeasts that appeared throughout the brewing, and TTC red yeasts were identified and the physiological characters examined. The results were as described below. a) TTC pinkyeasts (B-50P) and two strains of TTC red pink yeasts (B-54 RP & B-60 RP) w ere identified as the type of Saccharomyces cerevisiae and TTC pink red yeasts CB-53 R) were as the type of Hansenula subpelliculosa. b) The fermentability of four strains above mentioned were measured as follows. Two strains of TTC red pink yeasts were the highest, TTC pink yeasts were the lowest in the fermantability. The former three strains were active in the early stage of fermentation and found to be suitable for manufacturing 'Takju' TTC red yeasts were found to play an important role in Takju brewing due to its strong ability to produce esters although its fermentability was low. c) The tolerance against nitrous acid of strains of yeast was marked. That against lactic acid was only 3% in Koji extract, and TTC red yeasts showed somewhat stronger resistance. The tolerance against alcohol of TTC pink and red pink yeasts in the Hayduck solution was 7% while that in the malt extract was 13%. However, that of TTC red yeasts was much weaker than others. Liguefying activity of gelatin by those four strains of yeast was not recognized even in 40 days. 4. Fermentability during Takju brewing was shown in the first two days as much as 70-80% of total fermentation and around 90% of fermentation proceeded in 3-4 days. The main fermentation appeared to be completed during :his period. Productivity of alcohol during Takju brewing was found to be apporximately 65% of the total amount of starch put in mashing. 5. The reason that Saccharomyces coreanuss found be Saito in the mash of Takju was not detected in the present experiment is considered due to the facts that Aspergillus oryzae has been inoculated in the mold wheat (Nuruk) since around 1930 and also that Koji has been used in Takju brewing, consequently causing they complete change in microflora in the Takju brewing. This consideration will be supported by the fact that the original flavor and taste have now been remarkably changed.

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