• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.206-215
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• 2020

Trichoderma reesei is the major filamentous fungus used to produce cellulase and there is huge interest in promoting its ability to produce higher titers of cellulase. Among the many factors affecting cellulase production in T. reesei, the mycelial phenotype is important but seldom studied. Herein, a close homolog of the Neurospora crassa COT1 kinase was discovered in T. reesei and designated TrCOT1, which is of 83.3% amino acid sequence identity. Functional disruption of Trcot1 in T. reesei by RNAi-mediated gene silencing resulted in retarded sporulation on potato dextrose agar and dwarfed colonies on minimal medium agar plates containing glucose, xylan, lactose, xylose, or glycerol as the sole carbon source. The representative mutant strain, SUS2/Trcot1i, also displayed reduced mycelia accumulation but hyperbranching in the MM glucose liquid medium, with hyphal growth unit length values decreased to 73.0 ㎛/tip compared to 239.8 ㎛/tip for the parent strain SUS2. The hyperbranching phenotype led to slightly but significantly increased cellulase secretion from 24 to 72 h in a batch culture. However, the cellulase production per unit of mycelial biomass was much more profoundly improved from 24 to 96 h.

• Microbiology and Biotechnology Letters
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• v.29 no.2
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• pp.72-77
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• 2001

Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.406-412
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• 1998

The endoglucanase gene, egl6, of Trichoderma sp. was connected with the yeast ADH1 promoter, and the resultant plasmid, pVT-C4, was introduced into three S. cerevisiae host strains (YNN27, 2805, and SEY2102). Among each 80 transformants, the cell growth and expression level of endoglucanase were compared in test-tube cultivation, and three respective transformants for each host cells showing the highest expression level and cell growth were selected. When three recombinant yeast cells were batchwise cultivated for 48 hr in flask, the total activities of endoglucanase expressed were about 1140 unit/l with 2805/pVT-C4, 1020 unit/l with SEY2102/pVT-C4, and 590 unit/l with YNN27/pVT-C4. Irrespective of host strain, about 80% of the expressed endoglucanase was detected in the extracellular medium. In addition, it was also found that the recombinant enzyme was secreted into the culture medium as two major forms of lightly and heavily glycosylated proteins.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.68-72
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• 2002

To obtain a yeast mutant with high RNA content and high growth rate, Saccharomyces cerevisiae MTY62 was mutated with ethylmethane sulfonate. Among the selected mutants that were sensitive to the high concentration of KCl, M40-10 strain was finally selected due to its rapid cell growth and high RNA content in the tube and baffled-flask cultures. In the batch culture of M40-10 mutant, the maximum specific growth rate ($\mu_{max}$) of $0.38 h^{-1}$ , RNA concentration of 3210 mg-RNA/1, and RNA content of 183 mg-RNA/g-DCW were obtained, which were 23%, 15%, and 12% increased levels, respectively, compared to those of MTY62 parent strain. The intermittent fed-batch culture of M40-10 strain resulted in the maximum cell concentration of 35.6 g-DCW/1, RNA concentration of 5677 mg/1, and RNA content of 160 mg-RNA/g-DCW. Through the constant fed-batch culture, the maximum cell concentration of 46.4 g-DCW/1, RNA concentration of 6270 mg-RNA/1, and RNA content of 135 mg-RNA/g-DCW were obtained. At the 20 h culture time in the fed-batch cultures of M40-10 strain, the cell and RNA concentrations were increased by 30% and 10%, respectively, over the parent strain MTY62. In addition, it was also found that the accumulated RNA within the mutant cell was not degraded until the end of fed-batch cultivation, indicating that the M40-10 cell is a mutant with weak acidic RNase activity.y.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.40-46
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• 2006

A bacterial strain producing high level of a phytase was isolated from cattle feces and identified as Bacillus subtilis, and designated as Bacillus sp. CF 5-26. The production of the phytase from Bacillus sp. CF 5-26 reached the highest level after 72 hours at $37^{\circ}C$. The optimum condition of the media for the production of phytase was 10% rice bran extract, 0.1% whey protein powder, $0.01%\;CaCl_{2},\;0.01%\;KH_{2}PO_4$. The phytase was purified 20.3 folds with ethanol precipitation, Sephadex G-100, CM Sepharose CL-6B and Sephacryl S-100-HR column chromatography. The molecular weight of the purified enzyme was estimated to be 66 kDa on SDS-polyacrylamide gel electrophoresis. The purified phytase activity was stable up pH 5.0, 7.0, 11.0 and the remaining activity was 50% when it was treated at $100^{\circ}C$ for 1 hour. The substrate specificity of phytase was most active against sodium phytate and inositol polyphosphate compound. And the phytase hydrolysed tripolyphosphate and pyrophosphate a little. The Km value for the sodium phytate was 0.64 mM and the Vmax value was $4.41\;{\mu}mol/min$.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1670-1674
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• 2007

Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as monomers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity ($K_D=1.02{\times}10^{-7}\;M$). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and playa critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.

• Microbiology and Biotechnology Letters
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• v.35 no.1
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• pp.66-72
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• 2007

Yam has been recognized as healthy food due to its various biological activities, such as anti-obesity, antimicrobial, anticancer and immune-stimulation activities. In this study, antibacterial activities of 800 more natural plant extracts against yam-putrefactive psychrotrophic bacterium, Pseudomonas rhodesiae YAM-12, were evaluated to select a natural preservative, which is useful to long-term storage of yam and fresh-cut production. Finally, the clove oil was selected, and applied for the production of yam fresh-cut. The 1% of clove oil treated fresh-cut showed minor color changes during 31 days storage at $4^{\circ}C$ and the microbial growth was not detected. When the artificially contaminated fresh cut by dipping with P. rhodesiae YAM-12 suspension for 3 min was treated 1% clove oil, the microbial growth was identified to $10^4$ CFU/g from $10^3$ CFU/g with minor color changes. Whereas, treatment of sterilized water, or 100 ppm NaOCl into artificially contaminated fresh cut showed severe putrefaction $(10^8\;CFU/g)$ and color changes. Considering the previous reports that clove oil has antimicrobial, antioxidation, and antithrombosis activity, the use of clove oil into the yam fresh cut will provide market safety and consumer acceptability by prevention of microbial putrefaction and its biological activity.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.340-349
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• 2009

A CJ9 bacterial strain, which showed antifungal and antibacterial activities, was isolated from meju and identified as Bacillus polyfermenticus based on Gram staining, biochemical properties, as well as its 16S rRNA sequence. B. polyfermenticus CJ9 showed the antimicrobial activity against the various pathogenic molds, yeasts, and bacteria. The antibacterial activity was stable in the pH 5.0~9.0, but the activity was lost at $37^{\circ}C$ for 24 hr. The antifungal activity was stable in the pH range of 3.0~9.0 and reduced at $121^{\circ}C$ for 15 min, but antifungal activity was not completely destroyed. The antibacterial activity was completely inactivated by proteinase K, protease, trypsin, and $\alpha$-chymotrypsin. The antifungal activity was also completely inactivated by protease and $\alpha$-chymotrypsin, and reduced its activity by proteinase which indicated that the antifungal and antibacterial compounds have proteineous nature. The apparent molecular mass of the partially purified antifungal compound, as indicated by using the direct detection method in Tricine-SDS-PAGE, was approximately 1.4 kDa. The molecular mass of the antibacterial compound could not be determined because of its heat-liable characteristic.

• Microbiology and Biotechnology Letters
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• v.36 no.1
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• pp.72-81
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• 2008

The effect of genistein on melanin synthesis was studied using in vitro and in vivo model. Genistein inhibited melanin synthesis in cultured melan-a cells dose dependently. Tyrosinase activity was decreased by genistein treatment in melan-a cells, but genistein did not inhibit tyrosinase directly. Genistein did not affect the expression of tyrosinase in melan-a cells. Genistein inhibited the activity of ${\alpha}$-glucosidase in virtro and the glycosylation of tyrosinase in melan-a cells. The resulting unsaturated glycosylation of tyrosinase makes it unstable and disturb correct transportation. To further clarify the effect of genistein on the melanogenesis, we established UVB-induced hyperpigmentation on the shaved backs of brown guinea pigs. The animals were exposed to UVB radiation once a week for three consecutive weeks. Genistein (1 and 2%) or vehicle alone as a control were then topically applied to the hyperpigmented areas daily. Genistein showed significant lightening effect on the UVB-induced hyperpigmentation in five weeks. Depigmenting effect was prominent in 2% genistein treatment with Fontana-Masson staining. In conclusion, genistein may be a useful agent for skin whitening.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.74-74
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• 2001

난자 동결방법의 선별은 보다 효과적인 난자은행의 개발에 필수 불가결한 중요한 요소이다. 이전의 연구에서 마우스의 난자를 ethylene glycol과 electron microscope grid를 이용한 유리화 동결법으로 동결 융해한 결과 기존의 slow freezing 방법에서보다 높은 생존율과 배발달율이 나타남을 관찰하였다. 그러나 동결융해후의 난자는 방추사와 염색체의 이상성이 대조군에 비해 높은 빈도로 나타나 융해후의 배발달율을 감소시키는 것으로 보고되었다. 이에 본 연구에서는 유리화동결법동안 항동해제에 Cytoskeleton system을 안정화시키는 cytoskeleton stabilizer인 taxol을 첨가시킨후 동결시켰을때 생존율과 발달율을 개선시킬 수 있는지 알아보고자 본 실험을 시행하였다. ICR mouse의 성숙란을 채취하여 연구목적에 따라 taxol을 첨가시키지 않은 대조군과 첨가시킨 실험군으로 분류하였다. 동결방법은 난자를 1.5 M ethylene glycol (EG)에 2분 30초간 노출시킨후 5.5 M EG와 1 M sucrose가 첨가된 동결액에 20초간 노출시킨 후 Grid에 난자를 부착시킨후 직접 액체질소에 침지하여 동결하였다. 동결후 난자는 5단계로 융해를 실시한 후 정자와 체외수정을 시킨 후 수정된 난자는 modified P1 배약액에 124 h까지 발달율을 관찰하였고, 배양 후 발달된 배반포는 대조군과 실험군, 각각 4마리의 발정동기화된 recipient에 이식을 시행하였다. 배발달율은 대조군에 비해 실험군에서 4세포기 (48 vs. 84.4%), 8세포기 (34% vs. 70.6%), 상실배 (26% vs. 58.6%) 그리고 배반포 발달율은 (24% vs. 58.6%)로 높게 관찰되었다. 배아이식후 대조군과 실험군에서 각각 2 마리가 임신이되어 정상적인 산자를 분만하였다. 따라서 항동해제에 taxol의 첨가는 동결 융해후의 난자의 배발달율을 증진시킬 수 있었다..8%로 나타나 난할율 및 배반포 발생율에 있어서 융합조건에 따라 큰 차이는 없었으나 1.9㎸/cm, 30$\mu\textrm{s}$ 2회의 조건이 다른 조건들에 비하여 유의적으로 낮았다. 따라서, 체세포와 수핵란 세포질간의 융합율과 배반포 발생에 미치는 영향은 전압보다는 시간에 더 크게 받음을 알 수 있었으며, 이와 같은 결과에서 융합시 시간을 오래 주는 것보다 전압을 높이는 것이 수핵난자의 세포질에 상해를 줄이고 이후 배반포 발생에 유리할 것으로 사료되었다.면에서도 더욱 더 활발할 것으로 기대된다. 배란후 72시간째에 초음파진단기를 이용하여 난소의 난포발달을 조사한 결과 , 대조구와 bFF처리구에 비해 AI처리구에서 발달난포가 유의적으로 많은 것을 확인하였다. 이상과 같은 결과로, Anti-inhibin serum은 한우 자체에서 분비하는 Inhibin을 특이하게 억제하여 Inhibin에 의해 억제되는 FSH분비가 촉진됨으로써 난포발달과 estrogen의 농도가 촉진되는 것으로 사료되어 anti-inhibin serum이 한우의 과배란유기 효과가 있는 것으로 사료된다.정량 분석한 결과이다. 시편의 조성은 33.6 at% U, 66.4 at% O의 결과를 얻었다. 산화물 핵연료의 표면 관찰 및 정량 분석 시험시 시편 표면을 전도성 물질로 증착시키지 않고, Silver Paint 에 시편을 접착하는 방법으로도 만족한 시험 결과를 얻을 수 있었다.째, 회복기 중에 일어나는 입자들의 유입은 자기폭풍의 지속시간을 연장시키는 경향을 보이며 큰 자기폭풍일수록 현저했다. 주상에서 관측된 이러한 특성은 서브스톰 확장기 활동이 자기폭풍의 발달과 밀접한 관계가 있음을 시사한다.se that were all low in two aspects, named "the Nonsignificant group". And the issues were high risk perception in general setting and