• KSBB Journal
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• v.26 no.5
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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.

• Food Science and Preservation
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• v.10 no.1
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• pp.80-88
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• 2003

In order to establish the enzymatic hydrolysis system improving of taste and flavor in the preparation of soy protein hydrolysates using the enzymes with excellent hydrolytic ability and different hydrolysis pattern of soy protein, Degree of hydrolysis(DH) and surface hydrophobicity under the optimal conditions of enzyme reaction, hydrolysis patterns by the SDS electrophoresis and sensory evaluation of soy protein hydrolysates by enzyme reactions were investigated. Four enzyme reactions were highly activated at pH 7.0, 45$^{\circ}C$ under the optimal conditions. As result of changes on the pattern of soy-protein hydrolysates by SDS-electrophoresis, high molecular peptides of hydrolysates by No. 5(Mucor circinelloides M5) and No. 16(Bacillus megaterium B16) enzymes were slowly decrease and 66KD band of these were remained after 3hours reaction. Production of low molecular peptides of hydrolysates by No. 4(Aspergillus oryzae M4) and No. 95(Bacillus subtilis YG 95) enzymes were remarkably detected during the proceeding reactions. As results of HPLC analysis, low molecular peptides of 15∼70KD were mainly appeared during the proceeding enzyme reactions. And, the more DH was increased, the more SDS-surface hydrophobicity was decreased. Hydrolysates by No. 4 enzyme was not only the highest DH of all hydrolysates, but the strongest bitter taste in a sensory evaluation. Sweat taste among the hydrolysates showed little difference. But, when combinative enzymes were treated, combinative enzyme of No. 4(Aspergillus oryzae M4)and No. 16(Bacillus megaterium B16) showed the strongest sweat taste. In conclusion, we assumed that it will be possible to prepare the hydrolysates having functionality when soy-protein were hydrolyzed by these specific enzymes.

• Korean Journal of Poultry Science
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• v.35 no.2
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• pp.153-162
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• 2008

This experiment was conducted to evaluate the dietary effects of mixture of probiotics and colistin on laying performance, intestinal microflora and egg yolk cholesterol in laying hens. One hundred sixty 50-wk-old Hy-Line Brown layers were divided into four treatments and fed a commercial diet (Control) or experimental diets containing 0.2% mixture of probiotics or probiotics and colistin mixture (T1, Bacillus subtilis + Aspergillus oryzae + Lactobacillus plantarum; T2, Bacillus subtilis + Aspergillus oryzae; T3, Bacillus subtilis + Aspergillus oryzae+colistin) for 8 wk. No significant differences were found in laying performance and liver weight among the groups. The Haugh unit of treated groups were significantly improved (P<0.05) compared to that of control, but eggshell qualities were not changed by the treatments. The cecal ammonia concentration was significantly decreased in both T1 and T3 groups. The number of coli forms in cecal content and feces were significantly reduced in all treated groups compared to that of Control (P<0.05). The egg yolk cholesterol contents in the groups fed the diet containing mixture of probiotics and colistin were reduced in comparison with that of Control. In conclusion, dietary supplementation of mixture of probiotics and colistin improved quality of egg albumen, and reduced the egg cholesterol contents. They also reduced intestinal coli forms without harmful effects on overall productive and physiological responses in laying hens.

• Korean Journal of Agricultural Science
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• v.22 no.2
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• pp.188-196
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• 1995

To obtain useful mould strain in soybean fermentation industry, we collected conventional Meju all over the Korea and tested existance of aflatoxin in Meju collected. Also, we measured distribution of microorganism and enzyme activity of Meju and isolated Aspergillus oryzae O4-5 as a industrially useful mould. The results obtained were summarized as follows: 1. Aflatoxin was not detected by EZ-Screen Test Kit and HPLC analysis in various Meju sample collected all over the Korea from 1994.12 to 1995.2. 2. Colony forming units(CFU) of mould, yeasts, aerobe, and anaerobe were $1.3{\times}10^4-2.8{\times}10^6$, $1{\times}10^2-1.5{\times}0^6$, $2.0{\times}10^7-8.0{\times}10^8$ and $3.0{\times}10^6-7.3{\times}10^8$ per gram of Meju, respectively, indicating that CFU inter Meju samples were varied with big difference. 3. The activities of ${\alpha}$-amylase and gluco-amylase were 5-80 units and 2-34 units per g of Meju, respectively. It was shown that enzyme activity was varied depending on where the Meju was collected. 4. The activities of acidic, neutral and alkaline protease were 5-33 units, 5-302 units and 5-363 units per g of Meju, respectively. Acidic protease of Improved Meju made by D-Company was higher than that of conventional Meju as 66 units per g of Meju. 5. CNU O4-5 strain selected as a noble strain could produce amylase and protease in high level, and identified as a strain that belongs to Aspergillus oryzae. 6. The activities of acidic and neutral protease of Aspergillus oryzae CNU O4-5 strain isolated were about 10% higher than those of Aspergillus oryzae JM which has been used in soybean fermentation industry. Amylase activity of CNU O4-5 strain was similar to Aspergillus oryzae JM.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.3
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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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• v.29 no.4
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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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• v.14 no.4
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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 $50^\circ{C}$.

• The Korean Journal of Mycology
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• v.51 no.3
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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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• v.37 no.2
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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.

• The Korean Journal of Food And Nutrition
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• v.8 no.3
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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.