• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.13-16
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• 2001

Maltose and soluble starch were used as secondary sources of carbon for glucoamylase production by Aspergillus awamori in solid state fermentation. During batch cultivation, maltose above 2.5%(w/w) repressed glucoamylase production, but, by adding either 2.5% (w/w) maltose or 1.25% (w/w) soluble starch to fed-batch cultivations, glucoamylase activity was increased by 15% and 170% over standard medium, respectively. The data showed that maltose is a weak inducer of glucoamylase production in solid stat fermentation.

• 한국미생물·생명공학회지
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• 제24권6호
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• pp.712-716
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• 1996

The effects of non-ionic surfactants (Triton X-100 and Tween 80) on cloned glucoamylase production and secretion in recombinant Saccharomyces cerevisiae culture were studied. Even though the extracellular glucoamylase activity was increased by addition of Tween 80 due to the increase of the cell mass, Tween 80 did not play a role in the increase of glucoamylase secretion. On the addition of Triton X-100 addition, the secretion efficiency was increased while the cell growth was inhibited. Triton X-100 was added to the culture broth after 24 hr of culture to minimize the inhibition of the cell growth, and consequently the glucoamylase activity in the culture broth was increased by 12%.

• 한국미생물·생명공학회지
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• 제19권5호
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• pp.497-503
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• 1991

Packed bed column과 continuous stirred tank reactor에서 고정화된 glucoamylase의 catalytic activity를 비교하였다. Settling chamber를 이용한 연속애화, 당화 공정을 사용하여 액화된 전분으로부터 포도당을 연속적으로 생산하였다. . Chitin에 고정화된 glucoamylase에 의한 당화실험에 있어서는 dextrin의 농도가 100g/l일 때, 체류시간 20분 동안 20의 당화수율을 나타냈다.

• 한국미생물·생명공학회지
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• 제28권4호
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• pp.195-201
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• 2000

Glucoamylase of Saccharomyces diastaticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (ml : Pro-18 longrightarrowLeu-18, m2 : Tyr-13 longrightarrowLeu, m3 : Ser-9longrightarrowLeu-9, m4 : Asn-5 longrightarrowPro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP in N-terminal domain and hydrophobic domain. n mutant 4, a polar amino acid was replaced by a helix - breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Ser-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.489-493
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• 1988

Starch로부터 ethanol을 직접적으로 발효 생산할 수 있는 새로운 효모 균주를 개발하고자 glucoamylase 생성균으로 알려진 Saceharomyces diastaticus의 glucoamylase gene을 cloning vector를 사용하지 않고 S, cerevisiae에 transformation시켰다. Li$_2$SO$_4$, 처리로써 competent화 한 S, cerevisiae의 Intact cells을 recipient로 하여 BamHI으로 partial digestion한 S, diastaticus의 chromosomal DNA를 transformation시키고 starch를 유일한 탄소원으로 함유한 최소 배지상에서 starch 자화능을 marker로 하여 transformant를 선별한 결과, 8.5$\times$$10^{-7}$ 빈도로 transformant를 얻었다. Transformant의 특성을 recipient 및 donor와 비교하기 위해 copper resistance와 당 발효능을 조사한 결과, donor인 S, diastaticus와 동일한 성질로서 표현된 maltose와 starch 발효능을 제외하고는 800ppm 농도까지 생육 가능한 copper resistance와 galactose 발효능 등에 있어서는 recipent와 동일하게 나타났다. 또한 transformant가 생성하는 glucoamylase의 그 작용에 있어서의 최적온도와 최적pH를 조사하여 본 바 각각 pH5.0, 50C로서 donor의 glucoamylase와 동일함을 알 수 있었다.

• 미생물학회지
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• 제18권4호
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• pp.161-171
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• 1980

A mutant strain having increased productivity of both enzymes, protease and amylase, was obtained from A. flavus KU 153, isolatd from South Korea for its high protease production by successive ultra-violet light irradiation, Two glucoamylases from the mutant strain selected were purified from wheat branculture by successive salting out, followed by dialysis and column chromatography, and their characteristics were compared with those of the wild strain. Glucoamylase production of the mutant selected was increased about 3.3 times compared with the wild strain, and 2.1 times compared with the parental strain, ${\alpha}-amylase$ activity of the mutant selected was about 2 times hugher than that of the wild strain or the parental strain. Protease and cellulase productivities of the muant selected were all alike compared with those of the highly proteolytic mutant, the parental strain. Therefore, it was considered that the back mutation on the protease production did not occurred in the formation process of the glucoamylase producing mutant. Total activities of glucoamylase I and II from the mutant selected were 2.86 and 3.65 times higher compared with those from the wild strain, respectively. Considering the optimal pH-thermal stability and Km-Vmax value of glucoamylase I and II from both strains, wild and mutant, it was deduced that the characteristics of glucoamylase I and II from the wild strain did not altered during the mutation process. Therefore, it was concluded that the selected mutant did not induce the formation of another glucoamylase isozyme, or the changes in the characteristics of the glucoamylase, but induce the productivity of the same glucoamylase I and II by the action of regulatory gene.

• 환경위생공학
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• 제5권1호
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• pp.49-64
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• 1990

Glucoamylase from the culture filtrate of Aspergillus nicer was purified by ammonium sulfate precipitation, aceton precipitation, DEAE-cellulose ion exchange chromatography and Sephadex G-50 gel fillration. Glucoamylase was secreted into the medium upon growth on glucose, sucrose or a variety of other hexose sugars or hexose sugar polymers and little or no glucoamylase activity was found when glycerol or xylose was used as the carbon source. The optimum pH and temperature (or the maximum enzyme activity were found to be 5.0 and $50^{\circ}C$, respectively. The enzyme was considerably thermostable, for no loss of activity was observed when the enzyme was preincubated at $60^{\circ}C$ for 30 min. The enzyme activity was inhibited by 20 mM of $Hg^{2+}$, $Fe^{2+}$. The km value for starch was 0.045%.

• 한국미생물·생명공학회지
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• 제16권4호
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• pp.320-326
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• 1988

Aspergillus sp.로부터 추출한 glucoamylase를 Sephacryl S-200과 DEAE-Sephacel 이온교환 칼럼을 통과시킨 후, 2개의 Isozyme으로 분리하였다. 분리된 glucoamylase isozyme(GI, GII)의 효소학적특성을 각각 조사하였다. GI 및 GII isozyme의 활성최적 pH 및 온도는 pH4.5 및 $65^{\circ}C$였다. 분리된 효소는 pH 3-7 사이에서 안정했으며, 또한 55$^{\circ}C$ 이하에서 안정하였다. Hg$^{++}$는 효소 황성을 완전히 저해하였으나, 글리세롤은 효소의 안정성을 크게 증가시켰다. 효소반응의 활성화에너지는 GI의 경우 10,62 kcal/mole이었으며, GII의 경우에는 10.23kcal/mole이었다. GI isozyme의 기질에 대한 Km값은, 0.62% (soluble starch), 0.32% (dextrin), 1.03%(glycogen)이었으며, GII isozyme의 경우에는, 0.66%(soluble starch) 0.23%(dextrin), 그리고 0.14% (glycogen)이었다.

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.519-523
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• 1989

Aspergillus sp.로부터 glucoamylase를 생성키 위한 조건을 검토하였다. 곰팡이는 탄소원으로 soluble starch, 질소원으로 yeast extract를 사용했을 때 최대 효소생성을 얻을 수 있었다. 그리고, 탄소원 및 질소원의 농도에 따른 효소생성은 soluble starch를 5%, yeast extract를 1% 수준으로 사용했을 때 효소생성은 극대화하였다. 또한 배지의 초기 pH를 6.0, 그리고 배양온도를 28$^{\circ}C$로 유지했을 때 효소생성이 높았다. 고체배지를 사용했을 때에는, 밀기울 배지가 glucoamylase 생성을 위해서 가장 효과가 좋았다.

• 미생물학회지
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• 제41권2호
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• pp.146-151
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• 2005

전분 이용이 가능한 산업용 Saccharomyces cerevisiae균주를 개발하기 위해 alcohol dehydrogenase 유전자 프로모터(ADClp)의 조절하에 발현되는 Aspergillus awamori glucoamylase cDNA 유전자(GA1)를 산업용 S. cerevisiae의 염색체에 도입하였다. 산업적 이용에 적합한 효모균주를 얻기 위해 세균 ampicillin 저항성 유전자가 제거되고 GA1 유전자와 선별 표지유전자로 S. cerevisiae aureobasidin A 저항성 유전자(AUR1-C)와 재조합 부위로 Tyretrotransposon $\delta$-서열이 포함된 integrative cassette를제조하였다. 이 $\delta-integrative$ cassette로 형질전환된 산업용 S. cerevisiae는 배지상에 glucoamylase를 생산 분비하였고 전환을 유일한 탄소원으로 하여 생장하였다. 형질전환체를 비선택배지에서 배양했을 매 삽입된 GA1유전자가 100세대까지 안정되게 유지되었다.