• KSBB Journal
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• 제26권5호
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• pp.427-432
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• 2011

In this study, a EXGA gene code for exo-β-1,3-glucanase from Aspergillus oryzae was overexpressed and secretory produced in Saccharomyces cerevisiae. To overexpress the β-1,3-glucanase, pGInu-exgA and pAInu-exgA plasmids having GAL10 and ADH1 promoter, respectively, and exoinulinase signal sequence (Inu s.s) were constructed and introduced in S. cerevisiae SEY2102 and 2805. The recombinant β-1,3-glucanase was successfully expressed and secreted into the medium and the β--1,3-glucanase activity in 2102/pGInu-exgA and 2102/pAInu-exgA strain were 5.01 unit/mL and 4.09 unit/mL, respectively. In the 2805/pGInu-exgA and 2805/pAInu-exgA strain, the β-1,3-glucanase activity showed 3.23 unit/mL and 3.22 unit/mL, respectively. Secretory efficiency in each strain reached 95% to 98%. Subsequently, the recombinant β1,3-glucanase was used for ethanol production. Ethanol productivity in 2102/pAInu-exgA strain was 0.83 g/L when pre-treated Laminaria japonica which has initial reducing sugar of 1.4 g/L was used as substrate. It is assumed that the polysaccharides of Laminaria japonica was effectively saccharified by recombinant β-1,3-glucanase, resulting in increase of ethanol productivity. These results suggested that recombinant β-1,3-glucanase was efficiently overexpressed and secreted in S. cerevisiae SEY2102 as host strain by using ADH1 promoter-Inu s.s system.

• 한국식품저장유통학회지
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• 제10권1호
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• pp.80-88
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• 2003

대두단백 가수분해 산물의 맛과 향을 개선하기 위해 효소에 의한 가수분해 system을 확립하기 위하여 단백질 가수분해 패턴이 서로 다른 효소로 생산된 단백 분해산물의 가수분해도와 표면 소수도 등을 측정하였다. 이들 분해물의 pattern을 SDS 전기영동으로 조사하였고, 효소반응에 의한 단백질분해물의 관능검사를 실시하였다. 각 균주가 생산한 단백질 분해효소의 pH 변화에 따른 효소의 활성은 No. 16 효소(Bacillu megarerium Bl6)와 No. 4효소(Aspergillus oryzae M4)는 pH 7.0에서 No. 95효소(Bacillu subtilis YG 95)와 No. 5효소(Mucor circinelloides M5)는 8.0에서 가장 높은 효소반응 활성을 보였다. 또한 반응온도에 따른 효소활성의 크기는 4가지 효소 모두 45$^{\circ}C$에서 가장 높은 활성을 나타내었다. 대두단백질 분해물의 SDS 전기영동 pattern변화에서, Bacillus megaterium Bl6과 Mucor circinelloides M5의 효소 (No. 16, No. 5)는 반응 후 분자량이 비교적 큰 peptide가 많이 생성되었으며, 효소반응 3시간 경과 후에도 분자량 66KD의 peptide를 확인할 수 있었다. 반면에 Bacillus subtilis YG 95와 Aspergillus oryzae M4의 효소(No. 95, No. 4)는 분자량 15KD∼45KD 미만의 작은 분자량의 peptide 물질이 주로 생성되었으며, 반응 2시간 경과후에는 30KD 미만의 저분자 Peptide가 주로 생성되었다. 이와 같은 결과는 HPLC 분석 결과와 일치하였다. 가수분해가 진행됨에 따라서 SDS 표면소수도가 크게 저하되었으며, Aspergillus oryzae M4 효소의 분해물의 가수분해도가 가장 높았다. 관능검사 결과, No. 4(Aspergillus oryzae M4)와 No. 95(Bacillus subtilis YG 95) 가 강한 쓴맛을 나타내었다. 각각의 효소들을 조합해서 분해한 단백분해물을 관능검사한 결과, Aspergillus oryzae M4 효소와 조합된 것의 대두단백질 분해물이 비교적 강한 쓴맛을 나타내었다. 분해물의 단맛은 시료별로 큰 차이가 나타나지 않았으나 Bacillu megarerium Bl6과 Aspergillus oryzae M4를 조합하였을 때 상대적으로 단맛의 정도가 높게 나타났다. 따라서 이와 같은 효소특성을 이용하여 대두단백질을 가수분해를 하였을 때 다양한 단백질 분해물의 제조에 이용이 가능할 것으로 사료된다.

• 한국가금학회지
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• 제35권2호
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• pp.153-162
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• 2008

본 연구는 산란계에 Lactobacillus plantarum, Bacillus subtillis와 Aspergillus oryzae 및 colistin의 복합 첨가제를 급여하였을 때 난 생산성과 난질 및 난각질에 미치는 영향과 장내 미생물 조성, 계분 암모니아 발생량 및 난황 내 콜레스테롤 함량에 미치는 영향을 함께 조사하였다. 50주령의 Hy-Line Brown 산란계 160수를 공시하여 일반 사료(Control) 또는 0.2% 복합균종 생균제를 함유하는 실험 사료(T1, Bacillus subtilis + Aspergillus oryzae + Lactobacillus plantarum; T2, Bacillus subtilis + Aspergillus oryzae; T3, Bacillus subtilis + Aspergillus oryzae + colistin)를 8주간 급여하였다. 사료 섭취량, 난 생산성 및 간의 중량 그리고 난각 강도, 난각 두께, 난각색은 처리구 간 유의성 있는 차이가 관찰되지 아니하였다. 그러나 Haugh unit는 처리구가 Control에 비해 유의하게 높았다(P<0.05). 혈중 총 콜레스테롤 농도와 HDL 콜레스테롤은 Control에 비해 모든 처리구에서 감소하는 경향이 관찰되었으나 통계적 유의차는 없었다. 맹장 내 암모니아 농도에 미치는 영향은 Control에 비해 T1 처리구와 T3 처리구에서 유의하게 감소하는 것으로 나타났다(P<0.05). 총 균수와 lactic acid bacteria는 맹장 및 분내 모두 Control과 처리구간 큰 차이가 없었고, 맹장 및 분내의 coli forms은 Control에 비해 복합 생균제 처리구가 유의하게 낮거나(P<0.05) 낮은 경향이 관찰되었다. 4 주차와 8주차의 난황 콜레스테롤 농도는 복합 생균제 처리구가 Control에 비해 유의하게 감소하거나 감소하는 경향이 나타났다(P<0.05). 난황 중성지질과 인지질의 농도는 Control과 처리구간의 차이는 없는 것으로 나타났다. 결론적으로 본 실험을 통해서 생균제인 Lactobacillus plantarum, Bacillus subtillis, Aspergillus oryzae 그리고 항생제인 colistin으로 이루어진 복합제의 급여는 생산성과 혈액 성상에 부정적인 영향이 없이 난황 콜레스테롤과 분내 암모니아 농도를 감소시키고, 장내 coli forms를 저하시키는 결과가 관찰되었다. Colistin이라는 항생제는 생균제의 활성에 부정적인 영향 없이 생균제로만 이루어진 첨가제와 같은 효과를 내었으며, 장내 미생물 균총 조절에는 보다 효과적으로 작용하는 것으로 나타났다. 따라서 복합 생균제를 급여함으로 장내 균총과 산란계 사양에 유익한 효과를 기대할 수 있으며, colistin의 병용도 생균제의 이점을 저해하지 않는 것으로 사료된다.

• 농업과학연구
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• 제22권2호
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• pp.188-196
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• 1995

장류제조에 유용성이 높은 곰팡이를 분리할 목적으로 재래식 메주를 수집하여 aflatoxin 생성 유무를 확인하고 재래식 메주의 미생물 분포와 효소 역가를 측정한 후 효소역가가 높은 균주로 Aspergillus oryzae O4-5를 분리 선정한 결과를 요약하면 다음과 같다. 1. 1994년 12월에서 1995년 2월 사이에 전국 각 지역에서 수집한 메주 중의 mycotoxin 생성을 검출하기 위하여 EZ-Screen Test Kit와 HPLC에 의해 분석한 결과 aflatoxin은 검출되지 않았다. 2. 메주 중의 곰팡이 수는 $1.3{\times}10^4-2.8{\times}10^6CFU/g$ 이었으며 효모수는 $1{\times}10^2-1.5{\times}10^6CFU/g$으로 시료별 차이가 대단히 컸다. 또한 호기성 세균수는 $2.0{\times}10^7-8.0{\times}10^7CFU/g$, 혐기성 세균은 $3.0{\times}10^6-7.3{\times}10^8CFU/g$ 이었다. 3. 메주 19 중의 ${\alpha}$-amylase는 5 - 80 units, gluco-amylase는 2 - 34 units로 지역별 메주 간의 차이가 심하였다. 4. 메주 1g 중의 산성 protease, 중성 protease 및 알칼리성 protease 역가는 5 - 33 units, 5 - 302 units 및 5- 363 units였으며 시중제품인 D회사의 개량메주는 산성 protease가 66 unitts로 재래식 메주 보다 높았다. 5. 재래식 메주로 부터 amylase와 protease가 높아 유용성이 높은 CNU O4-5는 Aspergillus oryzae로 동정되었다. 6. 우수 균주인 CNU O4-5균주의 산성 및 중성 protease는 장류공업에서 실제 이용하고 있는 Aspergillus oryzae JM 보다 10%정도 높게 나타났으며 amlylase 역가는 비슷 하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.184-190
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• 2004

The influences of impeller types on morphology and protein expression were investigated in a submerged culture of Aspergillus oryzae. The impeller types strongly affected mycelial morphology and protein production in batch and fed-batch fermentations. Cells that were cultured by propeller agitation grew in the form of a pellet, whereas cells that were cultured by turbine agitation grew in a freely dispersed-hyphal manner and in a clumped form. Pellet-grown cells showed high levels of protein production for both the intracellularly heterologous protein (${\beta}$-glucuronidase) and the extracellularly homologous protein (${\alpha}$-amylase). The feeding mode of the carbon source also influenced the morphological distribution and protein expression in fed-batch fermentation of A. oryzae. Pulsed-feeding mainly showed high protein expression and homogeneous distribution of pellet whereas continuous feeding resulted in less protein expression and heterogeneous distribution with pellet and dispersed-hyphae. The pellet growth with propeller agitation paralleling with the pulsed-feeding of carbon source showed a high level of protein production in the submerged fed-batch fermentation of recombinant A. oryzae.

• Journal of Microbiology and Biotechnology
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• 제29권4호
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• pp.577-586
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• 2019

The engineered Aspergillus oryzae has a high NADPH demand for xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, and is also the main enzyme involved in NADPH regeneration. The open reading frame and cDNA of the putative A. oryzae G6PDH (AoG6PDH) were obtained, followed by heterogeneous expression in Escherichia coli and purification as a his6-tagged protein. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed maximal activity at pH 7.5 and the optimal temperature was $50^{\circ}C$. This enzyme also had a half-life of 33.3 min at $40^{\circ}C$. Kinetics assay showed that AoG6PDH was strictly dependent on $NADP^+$ ($K_m=6.3{\mu}M$, $k_{cat}=1000.0s^{-1}$, $k_{cat}/K_m=158.7s^{-1}{\cdot}{\mu}M^{-1}$) as cofactor. The $K_m$ and $k_{cat}/K_m$ values of glucose-6-phosphate were $109.7s^{-1}{\cdot}{\mu}M^{-1}$ and $9.1s^{-1}{\cdot}{\mu}M^{-1}$ respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate, steady-state, ordered BiBi mechanism, where $NADP^+$ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae to improve its xylose utilization and yields of valued metabolites.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.863-867
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• 2004

$\alpha$-Galactosidase from A. oryzae DR-5 was induced in the presence of melibiose, raffinose, galactose, and locust bean galactomannan. The enzyme was purified to homogeneity by precipitation with acetone followed by ion-exchange chromatography using DEAE-Sephacel. The purified enzyme showed a single band in both nondenaturing-PAGE and SDS-PAGE. The enzyme was a glycoprotein in nature by activity staining. The molecular weight of the purified enzyme was 93-95 kDa by SDS-PAGE. The enzyme exhibited the optimum pH and temperature at 4.7 and $60^\circ{C}$, respectively. $\alpha$-Galactosidase activity was strongly inhibited by $Ag^{2+}, Hg^{2+}, Cu^{2+}$, and galactose. EDTA, 1,10-phenanthraline, and PMSF did not inhibit the enzyme activity, whereas N-bromosuccinimide completely inhibited enzyme activity. Investigation by TLC showed complete hydrolysis of stachyose and raffinose in soymilk in 3 h at pH 5.0 and $50^\circ{C}$.

• 한국균학회지
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• 제51권3호
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• pp.219-227
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• 2023

This study was conducted to analyze the distribution and characteristics of fungal species in meju using the traditional method. Fungal distribution in meju was investigated using metagenomic and morphological analyses, based on which Aspergillus flavus/oryzae strains were identified as the dominant fungi in all meju samples, followed by Pichia, Rhizopus and Lichtheimia spp. As A. flavus/oryzae was dominant, we further evaluated the aflatoxin production ability and enzymatic activity of the isolates. Thin-layer chromatography and polymerase chain reaction revealed that the A. flavus/oryzae strains isolated from meju are non-aflatoxigenic fungi. Based on the analyses of amylase and protease activities, strains with high activities of amylase or protease were identified, which are proposed to be used as starters for meju fermentation.

• Journal of Microbiology
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• 제37권2호
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• pp.80-85
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• 1999

The purification system of glucoamylase (glucan 1,4-${\alpha}$-glucosidase, EC 3. 2. 1. 3), some characteristics of the purified enzyme and hydrolysis rate of various raw starch were investigated through several experiments. The enzyme was produced on a solid, uncooked wheat bran medium of Aspergillus oryzae NR 3-6 isolated from traditional Korean Nuruk. The enzyme was homogeneously purified 6.8-fold with an overall yield of 28.3% by the criteria of disc- and SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 48 kDa by SDS-PAGE. The optimum temperature and pH were 55$^{\circ}C$ and 4.0, respectively. The enzyme was stable at a pH range of 3.0∼10.0 and below 45$^{\circ}C$. Enzyme activity was inhibited about 27% by 1mM Hg2+. The hydrolysis rate of raw wheat starch was shown to be 17.5-fold faster than the hydrolysis rate of soluble starch. The purified enzyme was identified as glucoamylase because the product of soluble starch by the purified enzyme was mainly glucose by thin layer chromatography.

• 한국식품영양학회지
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• 제8권3호
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• pp.206-211
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• 1995

Three kinds of soy sauce were prepared using the brick type of conventional menu(A), the brick type of meju of Aspergillus oryzae (B) and the grain type of menu Aspergillus oryzae (C). Organic acid and fatty acid were analyzed In accordance to aging time of those products Citric acid, lactic acid, acetic acid, malonic acid, butyric acid, oxalic acid, and propionic acid were dejected in all kinds of soy sauce. The content of lactic acid was shown higher than those of any other organic acids. The content of lactic acid was much higher at beginning of preparation and at 180 days in soy sauce B than any other conditions. The content of acetic acid was much higher at beginning of preparation, at 120 days in soy sauce C and at 180 days in soy sauce B than any other conditions. The content of citric acid was highest at beginning preparation in soy sauce C, and that was highest in soy sauce B except beginning preparation to 120 days. Myristic, palmitic, stearic, oleic, linoliic, linolenic, arachidonic acid were detected in all kinds of soy sauce after 180 days. The content of oleic acid were shown 32.59∼53.79% in soy sauce B and in soy sauce C. The content of stearic acid was shown 49.7oA In soy sauce A. Linolinec acid and arachidonic acid were detected in only soy sauce C.