• Journal of Applied Biological Chemistry
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• 제57권4호
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• pp.313-320
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• 2014

Black liquor (BL) is a by-product of rice straw pulping process. It is a low costs raw material for production value-adding proteins and enzymes, which has been paid more and more attention to reduce its environmental pollution. Mixed cultures of micelial fungi, Trichoderma reesei Northern Regional Research Laboratory (NRRL)11236, Trichoderma reesei NRRL 6165 and Aspergillus niger strains NRC 5A, NRC 7A, and NRC 9A were evaluated for their ability to produce xylanase using crude hemicellulose (CHC) prepared from BL and peat moss as an inert support under solid state fermentation (SSF). The most potent strains, A. niger NRC 9A (818.26 U/g CHC) and T. reesei NRRL 6165 ($100.9{\pm}57.14$ U/g CHC), were used in a mixed culture to enhance xylanase production by co-culturing under SSF. In the mixed culture, xylanase production ($1070.52{\pm}12.57$ U/g CHC) was nearly1.3 and 10.6-fold increases over the activities attained in their monocultures, A. niger NRC 9A and T. reesei NRRL 6165, respectively. Optimization of the culture parameters of the mixed culture SSF process, concentration of ammonium sulfate and corn steep liquor, CHC/peat moss ratio, inoculum size and ratios of the two strains, initial pH value, initial moisture content and incubation time, exhibited a significant increase ($2414.98{\pm}84.02$ U/g CHC) in xylanase production than before optimization.

• The Plant Pathology Journal
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• 제27권4호
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• pp.342-348
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• 2011

Bacillus subtilis EBS05, an endophytic bacteria strain isolated from a medicinal plant Cinnamomum camphor, can produce antagonistic compounds that effectively inhibit plant pathogenic fungi. The greenhouse experiments showed that wheat sharp eyespot disease (WSED) was reduced by 91.2%, 88.2% and 43.0% after the treatment with fermentation broth, bacteria-free filter and a fungicide fludioxonil, respectively. The culture broth of strain EBS05 can more effectively control WSED than can fludioxonil. The fermentation broth and bacteria-free filter ability to suppress WSED was not significantly different, suggesting that an active secreted substance played a major role in controlling WSED. Separation and purification of the active compounds was carried out by serial processes, including hydrochloric acid (pH 2.0) treatment, methanol extraction and Sephadex LH-20 column chromatography, silica gel column chromatography and reverse-phase high-pressure liquid chromatography (HPLC), respectively. The purified compounds, one of active peaks in the HPLC spectrum, were obtained from the collection. Analysis of the chemical structures by time-of-flight mass spectrometry (TOF-MS) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS) showed that the active substances produced by the endophytic bacteria EBS05 are mixture of the ${\beta}$-hydroxy-C12~C15-$Leu^7$ surfactin A isomers with 1035.65 Da, 1021.64 Da, 1007.63 Da and 993.65 Da molecular weights, respectively.

• Journal of Microbiology and Biotechnology
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• 제24권5호
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• pp.585-591
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• 2014

To evaluate the potential utility of pretreatment of raw biomass with a complex microbial system, we investigated the degradation of rice straw by BMC-9, a lignocellulose decomposition strain obtained from a biogas slurry compost environment. The degradation characteristics and corresponding changes in the bacterial community were assessed. The results showed that rapid degradation occurred from day 0 to day 9, with a peak total biomass bacterium concentration of $3.3{\times}10^8$ copies/ml on day 1. The pH of the fermentation broth declined initially and then increased, and the mass of rice straw decreased steadily. The highest concentrations of volatile fatty acid contents (0.291 mg/l lactic acid, 0.31 mg/l formic acid, 1.93 mg/l acetic acid, and 0.73 mg/l propionic acid) as well as the highest xylanse activity (1.79 U/ml) and carboxymethyl cellulase activity (0.37 U/ml) occurred on day 9. The greatest diversity among the microbial community also occurred on day 9, with the presence of bacteria belonging to Clostridium sp., Bacillus sp., and Geobacillus sp. Together, our results indicate that BMC-9 has a strong ability to rapidly degrade the lignocelluloses of rice straw under relatively inexpensive conditions, and the optimum fermentation time is 9 days.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.803-813
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• 2015

The growth of Irpex lacteus F17 and manganese peroxidase (MnP) production in a selfdesigned tray bioreactor, operating in solid-state conditions at a laboratory scale, were studied. The bioreactor was divided into three layers by three perforated trays. Agroindustrial residues were used both as the carrier of bound mycelia and as a nutrient medium for the growth of I. lacteus F17. The maximum biomass production in the bioreactor was detected at 60 h of fermentation, which was consistent with the CO₂ releasing rate by the fungus. During the stationary phase of fungal growth, the maximum MnP activity was observed, reaching 950 U/l at 84 h. Scanning electron microscopy images clearly showed the growth situation of mycelia on the support matrix. Furthermore, the MnP produced by I. lacteus F17 in the bioreactor was isolated and purified, and the internal peptide sequences were also identified with mass spectrometry. The optimal activity of the enzyme was detected at pH 7 and 25℃, with a long half-life time of 9 days. In addition, the MnP exhibited significant stability within a broad pH range of 4-7 and at temperature up to 55℃. Besides this, the MnP showed the ability to decolorize the polymeric model dye Poly R-478 in vitro.

• Journal of Microbiology and Biotechnology
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• 제26권3호
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• pp.627-636
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• 2016

The causative agent of pandemic cholera, Vibrio cholerae, infects the anaerobic environment of the human intestine. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly induced during anaerobic respiration with trimethylamine N-oxide (TMAO) as an alternative electron acceptor. However, the molecular mechanism of TMAO-stimulated CT production is not fully understood. Herein, we reveal that CT production during anaerobic TMAO respiration is affected by glucose fermentation. When the seventh pandemic V. cholerae O1 strain N16961 was grown with TMAO and additional glucose, CT production was markedly reduced. Furthermore, an N16961 Δcrp mutant, devoid of cyclic AMP receptor protein (CRP), was defective in CT production during growth by anaerobic TMAO respiration, further suggesting a role of glucose metabolism in regulating TMAO-mediated CT production. TMAO reductase activity was noticeably decreased when grown together with glucose or by mutation of the crp gene. A CRP binding region was identified in the promoter region of the torD gene, which encodes a structural subunit of the TMAO reductase. Gel shift assays further confirmed the binding of purified CRP to the torD promoter sequence. Together, our results suggest that the bacterial ability to respire using TMAO is controlled by CRP, whose activity is dependent on glucose availability. Our results reveal a novel mechanism for the regulation of major virulence factor production by V. cholerae under anaerobic growth conditions.

• 한국미생물·생명공학회지
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• 제17권2호
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• pp.88-93
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• 1989

Xylan성 biomass로부터 직접 alcohol을 얻고자 xylose 발효효모 X-6-41 균주의 NTG mutant인 X-6-41-1(his-) 균주와 xylanase 분비효모인 XB-33 의 NTG mutant XB-33-37(Arg-) 균주를 세포융합 시켰다. 원형질체 생성조건은 KYPX(XB-33), KYPD(X-6-41)에서 대수증식기 말기까지 증식한 세포를 집균하여 zymolyase(0.25mg/$m\ell$), cellulase (4mg/$m\ell$)와 100mM 2-mercaptoethanol 처리시 protoplast화 율은 X-6-41 경우 80%, XB-33인 45%로 나타났다. 선별된 융합체의 탄소자화능은 parent의 자화능을 서로 보완하였음을 확인하였고 xylan에서의 alcohol 발효는 2% xylan에서 발효 15 일만에 총당에 대해 0.28% alcohol 농도를 나타내었다. Parent와 fusant의 xylanase 활성은 완전배지 보다 최소배지상에서 높았는데 이것은 xylose 발효효모(X-6-41)의 경우 isoleucine에 의한 xylanase production inhibition 효과와 일치하였다.

• Food Science and Biotechnology
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• 제15권2호
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• pp.214-219
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• 2006

A bacterial strain, initially identified as B1-3, was isolated from cheonggukjang, a traditional Korean dish made from fermented soybeans. Using the Biolog system and 16S rRNA sequence analysis, we identified B1-3 as Bacillus mojavensis. We manufactured a quickly fermented soybean (QFS) food product using the B. mojavensis, and guided by their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging ability. We isolated substances with antioxidative activity from it. Using mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques, we isolated 4 compounds from the ethyl acetate (EtOAc)-soluble neutral fraction of methyl alcohol (MeOH) extracts of the QFS food product (genistein, daidzein, 3R,4R-3-methyl-3,4-dihydroxy-2-pentanone, and 3S,4R-3-methyl-3,4-dihydroxy-2-pentanone) and 3 compounds from its acidic fraction (4-hydroxyphenylacetic acid, genistin, and daidzein). Two compounds from the neutral fraction (3R,4R-3-methyl-3,4-dihydroxy-2-pentanone and 3S,4R-3-methyl-3,4-dihydroxy-2-pentanone) were not detected in nonfermented soybeans (NFS) or in the filtrate of the LB broth used to culture B. mojavensis. However, they were detected in the filtrate of the same broth when it contained 2% glucose. These results suggest that these 2 compounds were derived from glucose (or other saccharides) in the soybean during fermentation. One compound that was found in the acidic fraction (4-hydroxyphenylacetic acid) was readily detected in NFS, but not in the culture broth. This suggests that 4-hydroxyphenylacetic acid was derived from NFS. We concluded that the antioxidative activity of cheonggukjang is a result of the interactions between soybean components and the microorganisms used in the fermentation of cheonggukjang.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Journal of Applied Biological Chemistry
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• 제55권1호
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• pp.19-25
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• 2012

Green tea leaves were fermented for 15 and 30 days with Monascus pilosus which is known to produce functional statins (TMs), and the content of various biochemical constituents such as total polyphenol (TP), total flavonoid (TF), theaflavin, and thearubigin were analyzed and compared with that of non-fermented green tea (GT) and Pu-erh Chinese post-fermented tea (PU). In addition to the electron donating ability (EDA), ferric iron reducing power (FIRP), xanthine oxidase (XO) inhibitory activity, superoxide dismutase (SOD)-like activity, iron chelating activity (ICA) and hydrogen peroxide contents were also measured and compared with that of GT and PU. Content of TP and TF in the water and ethanol extracts in TMs were lower than those in GT and PU. Theaflavin and thearubigin contents of water and ethanol extracts in TMs were higher than those of GT. And, these components were increased depending on the period of fermentation. While, EDA and FIRP of TMs were lower than those of GT, XO inhibitory activity of TMs was higher than non-fermented tea. While, ICA of TMs was slightly higher than GT and PU, the content of hydrogen peroxide in TMs was markedly lower than GT. This results suggested that the green tea fermented by M. pilosus was valuable for oxidative stress-induced diseases by decreasing hydrogen peroxide, and forming theaflavins and thearubigins with functionality of genus Monascus.

• 한국환경농학회지
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• 제17권1호
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• pp.39-42
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• 1998

사탕수수 찌거기를 여름느타리에 의해 발효한 후 부산물의 추출물로부터 생물학적 활성을 조사하기 위하여, 항종양성, 콩에 있어서의 glyceollin 유도성 및 완두에 있어서의 줄기 성장성을 관찰하였다. 알콜추출분획 및 DMSO분획물은 자실체 추출분획보다는 약했지만 매우 좋은 항종양성을 나타내었다. 콩의 자엽실험에서는 모든 추출물이 식물체로 하여금 병원성 감염을 방지할 수 있는 glyceollin 유도성을 갖고 있었다. 또한 IAA 유도 줄기성장 실험에서는 양성반응을 보인 모든 실험군에서 IAA와 추출물간의 유도 상승효과가 있는 것으로 나타났다.