• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1559-1565
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• 2014

Cellulase and xylanase are main hydrolysis enzymes for the degradation of cellulosic and hemicellulosic biomass, respectively. In this study, our aim was to develop and test the efficacy of a rapid, high-throughput method to screen hydrolytic-enzyme-producing microbes. To accomplish this, we modified the 3,5-dinitrosalicylic acid (DNS) method for microwell plate-based screening. Targeted microbial samples were initially cultured on agar plates with both cellulose and xylan as substrates. Then, isolated colonies were subcultured in broth media containing yeast extract and either cellulose or xylan. The supernatants of the culture broth were tested with our modified DNS screening method in a 96-microwell plate, with a $200{\mu}l$ total reaction volume. In addition, the stability and reliability of glucose and xylose standards, which were used to determine the enzymatic activity, were studied at $100^{\circ}C$ for different time intervals in a dry oven. It was concluded that the minimum incubation time required for stable color development of the standard solution is 20 min. With this technique, we successfully screened 21 and 31 cellulase- and xylanase-producing strains, respectively, in a single experimental trial. Among the identified strains, 19 showed both cellulose and xylan hydrolyzing activities. These microbes can be applied to bioethanol production from cellulosic and hemicellulosic biomass.

• Korean Journal of Microbiology
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• v.50 no.1
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• pp.33-39
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• 2014

One hundred forty four bacterial colonies which were able to degrade crude oil were isolated from soil samples that were contaminated with oil in Daejeon area. Among them, one bacterial strain was selected for this study based on its emulsification activity, growth rate and surface tension activity, and this selected bacterial strain was identified as Pseudomonas sp. HN37 through physiological- biochemical tests and analysis of its 16S rRNA sequence. Pseudomonas sp. HN37 utilize the several aliphatic hydrocarbons, 3,5-dinitrosalicylic acid and 2,4-dichlorophooxyacetic acid as a sole carbon source. And this bacterial strain showed a high resistance to antibiotics such as ampicillin and chloramphenicol, as well as heavy metals such as Ba, Cr, Li and Mn. Also, it was found that the optimal pH and temperature for the cell growth, surface tension, and emulsification activity of Pseudomonas sp. HN37 were pH 6.0-9.0 and $30^{\circ}C$, respectively. The emulsification and surface tension activity was reached the maximum to 1% (V/V) crude oil and 1% (W/V) NaCl concentration. The surface tension of the culture broth was decreased from 62 to 27 dyne/cm after fifteen hours of inoculation in LB media.

• Journal of Korean Society of Forest Science
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• v.39 no.1
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• pp.57-63
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• 1978

Enzymatic hydrolysis of the substrate from Alnus hirsuta (Spach) Rupr (8-14years) was investigated using cellulase preparations of Trichoderma viride Pers. ex. Fr. SANK 16374 and conduced on the optimum reaction conditions of the cellulase on saccharification. The crude cellulase was produced by the submerged culture process and produced in the culture fluid was salted out quantitatively by the use of ammonium sulfate. The method of delignification from wood(Saw dust) was treated by the peracetic acid (PA) method. Reducing sugar was determined by the dinitrosalicylic acid (DNS) method. The results were summerized as follows; 1. The optimum pH of cellulase was 5.0 and the range of stability with respect to pH was generally from 4.0 to 6.0 2. The optimum temperature of cellulase was generally $40^{\circ}C$, but reducing sugar formation did not show significent differences at 5% levels in the reaction temperature from $40^{\circ}C$ to $50^{\circ}C$. 3. The redusing sugar were increased with increase of cellulase concentration. 4. The reducing sugar were decreased with increase of substrate concentration. 5. Fructose was a very good inhibitor of the enzyme from Trichoderma viride, but glucose inhibition was generally weak.

• Journal of Korean Society of Forest Science
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• v.41 no.1
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• pp.1-6
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• 1979

1. The study was conducted on the optimum condition of the treated substrate with dilute sulphuric acid solution and cellulase for saccharification. The wood (saw dust) of Alnus hirsuta Rupr. (10~15 years) was treated with 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, $H_2SO_4$ solution at $1.5kg/cm^2$ for 15min., 30min., 45min., and 60min., followed by thermal treatment at $190^{\circ}C$ for 30min., and screening with 60 mesh sieve, after which to 0.5 grams of each sample was added 0.5ml cellulase solution, and 50ml 0.1M acetic acid buffer solution (pH 5.0), after incubating at $40^{\circ}C$ for 96hr. 2. The crude cellulase of Trichoderma viride Perx. ex. Fr. SANK 16374 was produced by the submerged culture process and produced in the culture fluid was salted out quantitatively by the use of ammonium sulfite. 3. Reducing sugar was determined by the 3.5-dinitrosalicylic acid (DNS) method. 4. The reducing sugar was increased with increase of the sulphuric acid concentration and saw dust was treated with 1.5% $H_2SO_4$ solution at $1.5kg/cm^2$ for 45min. showed the best saccharification (16.0%). The reducing sugar formation did not show statistically significant in 5% levels by thermal treatment time 45min. and 60min. 5. The substrate for cellulase which was treated with 0.9% $H_2SO_4$ solution at $1.5kg/cm^2$ for 60min. showed the best reducing sugar formation (23.6%). And did not show significant difference in 5% levels at 0.9%, 1.2%, and 1.5% $H_2SO_4$ solution.

• Journal of Korean Society of Forest Science
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• v.38 no.1
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• pp.13-18
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• 1978

In this study, enzymatic hydrolysis of the holocellulose from Alnus hirsuta (Spach) Rupr. (8-14 yr's) was investigated using crude cellulase preparations of Trichoderma viride Pers. ex. Fr. SANK 16374. And conducted on the optimum condition of the treated substrate for saccharification. A strain of Trichoderma viride Pers. ex. Fr. SANK 16374 was found to be highly efficient for the cellulase productivity, especially in the submerged culture process. The culture medium used in this experiment was prepared from an extract of wheat bran consisting also of $KH_2PO_410$, $(NH_4)_2$ $SO_4$ 3, $NaNO_3$ 3, and $MgSO_4$ $7H_2O$ 0.5g/l. Cellulose powder (Toyo filter paper, 60 mesh) was found to be an importent factar for inducing the cellulase formation. And the cellulase produced in the culture fluid was salted out quantitatively by the use of ammonium sulfate (Fig. 1) Reducing sugar was determined by the Dinitrosalicylic acid (DNS) method, using reagents prepared according to the method of Sumner (1925). The results obtained were summerized as follows; 1. The method of delignification were treated by the Peracetic acid (PA) method, according to the method of Toyama (1970). The yield of holocellulose were decreased in accordance with increasing concentration of Peracetic acid solution; delignification of Alnus hirsuta Rupr. with 20% Peracetic acid was satisfied for 48 hours and 40%~60% peracetic acid was satisfied for 24 hrs: 2. The substrate (holocellulose) was changed easely into fine powder with enzymatic hydrolysis and cellulase exhibits optimum activity on the reducing sugar formation from substrate at the range of 60-100 mesh. 3. The reducing sugar formation increased in accordance with increasing dry temperature on holocellulose substrate was found to be $190{\pm}5^{\circ}C$. 4. The optimal heat treated time of holocellulose substrate was found to be 45 min. for the reducing sugar formation showed the best products. The reducing sugar formation did not show statisticaly significent diflerences at 5% levels by heat treated time for 45 min. and 60 min.

• KSBB Journal
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• v.22 no.1
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• pp.16-21
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• 2007

Although Energy demands of modern society increase rapidly, current energy would be exhausted shortly. Therefore development of bio-ethanol production process from cellulose containing materials was extremly demanded. Therefore development of highly functional cellulase is requisite for this purpose. In this study cellobio-hydrolase (CBH1) gene from Trichorderma reesei was used to increase cellulase activity by directed evolution and highly functional cellobio-hydrolase was obtained and characterized.

• Journal of Korean Society of Forest Science
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• v.49 no.1
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• pp.1-5
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• 1980

In this study, enzymatic saccharification of substrates from Alnus hirsuta Ruper (8-14 years). Quercus acutissima Carruthers, Betula platyphylla var. japonica Nera, Populus euramericana Guiner and Platanus orientalis L. were investigated using crude cellulase preparations of Trichoderma viride Pers. ex. Fr. SANK 16374, and conduced on the optimum treated conditions of the cellulase sacchrification and reactivation of residue of digested substrates. The Trichoderma viride cellulase was produced by the submerged culture process and produced in the culture fluid was salted out quantitatively by the use of ammonium sulfate. The method of dilignification from wood (5 species) was treated by the peracetic acid(PA) method. The reducing sugar was determined by the dinitrosalicylic acid (DNS) method. 1. The results of tests carried out for 96 hr. (Figure 1), show conclusively the initial substrates from 5 species ($S_3$) which has been rendered highly reactive form and the mean rate of reducing sugar was 28.3 %. 2. The results of tests carried out for 96 hr., the reactivation of residue of digested substrates (improvement in the quality of the substrate through preheating in air at $190^{\circ}C$. for 45 min. followed by milling was (60 mesh size) at the same substrate level, increased concentrations of cellulase at the same substrate level, and increased concentrations of cellulase increases the rate of hydrolysis considerably. 3. Figure 1. shows conclusively that the residue of digested substrates ($S_1$ dried at $60^{\circ}C$) which has been rendered extremly resistant to cellulase action can be reactivated into a highly reactive form ($S_2$), almost comparable to that of the initial substrates ($S_3$). And the reducing sugar formation did not show statistically significent differences at 5% levels by initial substrates and the residue of digested substrates (preheating in air at $190^{\circ}C$. for 45 min. fallowed by milling was (60 mesh size).

• Korean Journal of Food Science and Technology
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• v.21 no.4
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• pp.480-485
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• 1989

Changes of color intensity and components during browning reaction of water extracts from white tail ginseng were investigated. Temperature dependence was described by the Arrhenius relationship with an activation energy of 16kcal/mole. Temperature sensitivities$(Q_{10}\;value)$ for water extracts of ginseng was 1.90 between $70^{\circ}C\;and\;80^{\circ}C$, 1.57 between $80^{\circ}C\;and\;90^{\circ}C$ and 1.46 between $90^{\circ}C\;and\;100^{\circ}C$. pH value of the solution treated at $90^{\circ}C\;and\;100^{\circ}C$ slightly increased with an increase in reaction time. Among ginseng saponins ginsenoside-Re was most unstable against heat-treatment, white diol group saponins were more stable against heat-treatment. Hydrogen donating activity (reducing activity for ${\alpha},\;{\alpha}'-diphenyl-{\beta}-picrylhydrazyl$) and 3,5-dinitrosalicylic acid(DNS) positive substances of browning reaction products increased in proportion to the length of browning reaction time and temperature, whereas folin positive substances decreased by heat-denaturation of ginseng protein at initial reaction time and then increased thereafter.

• Journal of Life Science
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• v.25 no.6
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• pp.680-685
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• 2015

Previously, cellulase and xylanase producing microorganism, Bacillus subtilis NC1, was isolated from soil. Based on the 16S rRNA gene sequence and API 50 CHL test the strain was identified as Bacillus subtilis, and named as B. subtilis NC1. We cloned and sequenced the genes for cellulase and xylanase. Plus, the deduced amino acid sequences from the genes of cellulase and xylanase were determined and were also identified as glycosyl hydrolases family (GH) 5 and 30, respectively. In this study to optimize the medium parameters for cellulase production by B. subtilis NC1 the RSM (response surface methodology) based on CCD (central composite design) model was performed. Three factors, tryptone, yeast extract, and NaCl, for N or C source were investigated. The cellulase activity was measured with a carboxylmethyl cellulose (CMC) plate and the 3,5-dinitrosalicylic acid (DNS) methods. The coefficient of determination (R²) for the model was 0.960, and the probability value (p=0.0001) of the regression model was highly significant. Based on the RSM, the optimum conditions for cellulase production by B. subtilis NC1 were predicted to be tryptone of 2.5%, yeast extract of 0.5%, and NaCl of 1.0%. Through the model verification, cellulase activity of Bacillus subtilis NC1 increased from 0.5 to 0.62 U/ml (24%) compared to the original medium.