• 한국식품영양학회지
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• 제7권4호
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• pp.259-266
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• 1994

The hyperthermophilic archaebacterium Thermococcus profundus Isolated from a deep-sea hydrothermal vent system, produced several amylolytic enzymes such as extracellular amylase and pullulanase, intracellular a-1,4-91ucosidase in respone to the presence of complex carbohydrates In the growth medium. This strain showed high activities on 0.5% maltose than on complex carbohydrates One of the amylases was partially purified by ammonium sulfate precipitation, DEAE-Toyopearl chromatography. The amylase exhibited maximal activity at pH 5.5 and 80$^{\circ}C$, and was stable in the range of pH 5.5 to 9.5 and up to 80$^{\circ}C$ for 30 min. The enzyme activity was no dependence on Ca2+ and not inhibited by detergents. The amylase hydrolyzed soluble starch, amylose, amylopectin and glycogen to produce maltose and maltotriose with trace amounts of glucose, but not pullulan and ${\alpha}$-, ${\beta}$-, ${\gamma}$-cyclodextrin. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were hydrolyzed in an endo fashion.

• 미생물학회지
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• 제46권1호
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• pp.80-85
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• 2010

보리 맥아로부터 발견된 서로 다른 알파아밀라제 동질효소(AMY1, AMY2)는 80%에 달하는 높은 아미노산 서열의 상동성을 보이지만, 두 효소의 특성은 서로 달라 AMY1 효소는 낮은 농도의 칼슘 조건에서 최대 활성을 보이는 반면, AMY2 효소는 높은 칼슘이온 농도에서 높은 활성을 나타낸다. 또한 BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) 단백질은 AMY2 효소만을 특이적으로 저해한다. 따라서 본 연구에서는 AMY1과 AMY2 효소의 유전자를 I, II, III의 세 부위로 나눈 후, 제한효소 처리에 의해 일부 부위를 상호 치환한 4종의 chimera 효소를 추가로 제조하고, Pichia pastoris 균주에서 대량 발현하였다. 이들 효소의 특성을 비교한 결과, 제 I 부위만이 상호치환된 AMY211 및 AMY122 효소의 경우, AMY1과 AMY2의 중간적 칼슘 의존성을 나타내었으며, BASI에 의한 저해효과는 AMY2의 제 I, II 부위를 포함하는 AMY221 효소에서만 관찰되었다. 따라서 보리 아밀라제의 제 I 부위 및 제 II 부위에 존재하는 아미노산 잔기들이 칼슘 의존성 및 BASI와의 결합에 중요한 역할을 담당하는 반면 제 III 부위는 이들 효소의 활성 차이에 영향을 미치지 않음을 확인하였다.

• 생명과학회지
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• 제12권4호
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• pp.483-489
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• 2002

S. cerevisiae에서 glucoamylase 유전자의 promoter와 signal sequence 그리고 MF$\alpha$1의 prosequence를 이용하여 합성 곤충 defensin를 발현하고 항균활성을 보유한 형태로 분비하는데 성공하였다. Defensin의 여러 생화학적인 특성을 조사한 결과 열 안정성이 높아 10$0^{\circ}C$에서 30분간 가열하여도 항균활성을 온전히 유지하였으며 조사한 pH 영역, 2.0-12.0에서 항균활성의 변화가 없었다. 또한 여러 단백질 분해효소를 처리하면 항균활성이 완전히 사라졌으나 전분질 분해효소, 섬유소분해효소 및 지질분해효소의 처리는 항균 활성에 전혀 영향이 없었다. 황산암모늄침전, SP-Sepharose column cormatography, RP-HPLC 등의 조작을 통해 defensin을 순수한 형태로 정제하였으며 Tricine-SDS-PAGE를 통해 분자량이 약 4.0 kDa임을 확인하였고 정제한 defensin은 항균활성을 보유하였다.

• 미생물학회지
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• 제26권2호
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• pp.73-81
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• 1988

$\alpha$-Amylase (EC.3.2.1.1) of Neurospora crassa (ATCC9279) was cloned in E. coli HB101 using shotgun method, and the enzymes isolated from both N. crassa and E. coli were compared. Chromosomal DNA isolated from the spores of N. crassa was partially digested with PstI restriction endonuclease and rejoined to pBR322 which had been digested with the same enzyme. The resulting recombinant DNA were introduced into E. coli HB101 which had competancy by treating with $CaCl_{2}$. As the result, about 8000 colonies which showed tetracycline resistance were selected and two of the colonies which had 13.5Kb recombinant plasmid exhibit starch degrading activity on starch-containing plate when treated with D-cycloserine. $\alpha$-Amylases from both N.crassa and E. coli were isolated by using ammonium sulfate precipitation, DEAE-cellulose ion exchange column chromatography and Bio-Gel P150 gel foltration column. As the result, about 81.3 fold and 5.6 fold purifications in specific activities were obtained respectively, and specific activities of the gel filtrates were 6.1u/mg and 85u/mg respectively. The properties of both enzymes were compared and they showed quite the similar patterns in optimal temperature, optimal pH and had same molecular weight about 100,000 daltons on gel filtration method. Optimal temperatures for both enzymes were $70^{\circ}C$ and optimal pH were about 6 and 10.

• 미생물학회지
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• 제17권2호
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• pp.81-93
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• 1979

The mycelium of Rhizopus nigricans was harvested at intervals during the sporulation periods, fractioned into various cell components and analyzed the con!eiits of various cell materials in order to clarify the optimum conditions of sporulation and some characteristics of the metabolism during tke sporulation periods. The changes in enzyme activities, such as amylase and protease, were also measured during the sporulation period,. 1. Mycelium in distilled water culture, as control, did not sporulate but mycelial mat cultured in Petridish without mutrient spourulated. Optimum temperature range for sporulation was $20{\sim}25^{\circ}C$. 2. During the sporulation and maturation periods, proteins, especially alkali-labile protein were decreased remarkably but free amino acid and ninhydrin reactive substances in acid soluble fraction were increased, compared with control. 3. Acid solable polyphosphate was decreased but acid insoluble polyphosphate was increased, during the sporulation. 4. Carbohydrate and hexosamine in acid soluble fraction were increased, while carbohydrate in alkali insoluble residual fraction was decreased during the sporulation periods. 5. Amounts of UV-absorbing material in deoxyribonucleic acid fraction was increased a little but those in ribonucleic acid fraction was decreased, compared with control. 6. Intracellular amylases and proteases activities insporulating mycelial mat were increased continuously during the sporulation and maturation periods.

• Journal of Microbiology and Biotechnology
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• 제20권9호
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• pp.1307-1313
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• 2010

An extracellular ${\alpha}$-amylase from Lactococcus lactis IBB500 was purified and characterized. The optimum conditions for the enzyme activity were a pH of 4.5, temperature of $35^{\circ}C$, and enzyme molecular mass of 121 kDa. The genome analysis and a plasmid curing experiment indicated that $amy^+$ genes were located in a plasmid of 30 kb. An analysis of the phylogenetic relationships strongly supported a hypothesis of horizontal gene transfer. A strong homology was found for the peptides with the sequence of ${\alpha}$-amylases from Ralstonia pikettii and Ralstonia solanacearum. The protein with ${\alpha}$-amylase activity purified in this study is the first one described for the Lactococcus lactis species, and this paper is the first report on a Lactococcus lactis strain belonging to the amylolytic lactic acid bacteria (ALAB).

• 한국식품영양과학회지
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• 제27권3호
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• pp.367-375
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• 1998

The physicochemical properties of the $\alpha$-amylase inhibitors from black bean and naked barley is Korea were investigated. Preincubation time for maximum inhibition was 30min and no activity change was seen after that time. Optimum pH of the $\alpha$-amylase inhibitors from the black bean and naked barley was pH 7.0 and the inhibitory activities were stable in the range of pH 6.0~8.0 in both phosphate and Tris-HCI buffer solutions. Both inhibitors maintained more than 50% of activity after incubation for 17 min at 7$0^{\circ}C$. The inhibitors from the black bean and naked barley maintained more than 50% of activities after treatment for 40 min and 30 min with pepsin, and 30 min and 50 min with trypsin, respectively. Both inhibitors functioned via a noncompetitive mechanism and were active against porcine pancreatic and human salivary $\alpha$-amylases. The activities of both inhibitors were linear for the ionic stength ranging from 0 to 0.9. The addition of 70 mM maltose to the reaction mixture caused a maximum increase in the relative activities of both inhibitors, but it did not affect the dissociation of the EI complex. The activities of both inhibitors were significantly enhanced by adding 1mM of K+ or Mg2+.

• 한국식품영양과학회지
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• 제16권2호
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• pp.128-135
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• 1987

고온성 cellulose 분해이용세균인 Herpetosiphon geysericola CUM 317 균주가 생성하는 ${\alpha}-amylase$, ${\beta}-amylase$ 및 glucoamylase를 황산암모늄 염석, DEAE-cellulose chromatography, CM-cellulose chromatography 방법으로 각각 부분정제하였다. 이들 ${\alpha}-amylase$, ${\beta}-amylase$ 및 glucoamylase의 감자 전분에 대한 Km치는 $2.31mg/m{\ell}$, $7.69mg/m{\ell}$ 및 $8.33mg/m{\ell}$였으며, 각 분자량은 84,000 dalton, 76,000 dalton 및 80,000 dalton의 크기로 나타났다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권3호
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• pp.230-234
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• 2006

In this study, we describe a one-step chemoenzymatic reaction for the production of natural blue pigments, in which the geniposide from Gardenia extracts is transformed by glycosidases to genipin. Genipin is then allowed to react with amino acids, thereby generating a natural blue pigment. The ${\beta}-glycosidases$, most notably Isolase (a variant of ${\beta}-glucanase$), recombinant ${\beta}-glycosidases$, Cellulase T, and amylases, were shown to hydrolyze geniposide to produce the desired pigments, whereas the ${\alpha}-glycosidases$ did not. Among the 20 tested amino acids, glycine and tyrosine were associated with the highest dye production yields. The optimal molar ratio of geniposide to glycine, two reactants relevant to pigment production, was unity The natural blue pigments produced in this study were used to dye cotton, silk, and wool. The color yields of the pigments were determined to be significantly higher than those of other natural dyes. Furthermore, the color fastness properties of these dyes were fairly good, even in the absence of mordant.

• Food Science and Biotechnology
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• 제15권2호
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• pp.298-302
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• 2006

Kochujang was fermented using hot red pepper, meju prepared with soybean and rice, and malt-digested syrup. To reduce salt content, mustard powder (1.2%, w/w) was added to Korean traditional kochujang with 4-10% salt, and microbial characteristics, enzyme activities, and gas formation in kochujang were evaluated during fermentation for 120 days at $25^{\circ}C$. Yeast numbers of all treatments maintained 2.43-2.86 log CFU/g up to 60 days fermentation, indicating salt concentration had no effect on yeast count. Activities of ${\alpha}$- and ${\beta}$-amylases, and neutral and acidic proteases of kochujang added with mustard powder were slightly higher than those of control group. Total accumulative volume of gas produced during fermentation of kochujang without mustard powder (control group) was 5,892 mL/pack, but decreased to 34-99 mL/pack in low-salted kochujang (4 and 6% salt) added with mustard powder. Major gas produced was carbon dioxide (79-80%) with oxygen content less than 1.25%(v/v). Results indicate salt concentration of kochujang could be lowered up to 6-8% by addition of mustard powder without gas formation and quality alteration during distribution.