• Journal of Microbiology and Biotechnology
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• v.24 no.9
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• pp.1178-1188
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• 2014

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.12-17
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• 2001

Aspergil-lus coreanus NR-15 and Aspergilus oryzae NR-2-5 from traditional Korean Nuruk were selected as parental strains producing starch hydrolysis enzyme. Xll(Arginine-) mutant from A. coreanus NR 15-1 showed high glu-doamylase activity and total acid productivity. Z6(Adenine-) mutant from A. oryzae NR2-5 showed the highest $\alpha$-amylase activity. Therefore, both XII and Z6 mutants were selected and investigated for the optimal conditions of protoplast formation for protoplast fusion. Mixture of equal amount of cellulase and driselase(10mg/ml each) was the most effective as lytic enzymes. The optimal pH and temperature for protoplast formation were 5.0 and $30^{\circ}C$, respectively. The most effective reaction for protoplast formation time was 4 hours. The maximum of protoplst for- mation of Xll mutant and Z6 mutant were $6.54$\times$10^{7}$ protoplasts/ ml and $3.04$\times$10^{ 7}$ protoplasts/ml, and the regen-eration frequencies of the protoplasts were 11.3% and 11.6%, respectively. The size of the protoplasts from X11 and Z6 mutants were 3~6 $\mu\textrm{m}$ and 4~9$\mu\textrm{m}$, respectively.

• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.539-546
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• 2020

A cellulolytic bacterial strain, S2-3, was isolated from sea water collected in Jeju island, Republic of Korea. The strain was aerobic and gram negative, and formed yellow colored colonies on marine agar medium. S2-3 cells were long rod-shaped, 0.5 × 0.25 ㎛ (width x length) in size, and did not have flagella. The optimal growth conditions for S2-3 were 30-35℃ and pH 6.5-7.0. Analysis of the 16S rRNA gene sequence of S2-3 revealed that it had the highest identity with those of Seonamhaeicola algicola Gy8 (97.08%), Hyunsoonleella udonensis JG48 (95.01%), and Aestuariibaculum scopimerae I-15 (94.86%). In phylogenetic analysis, S2-3 formed the same clade as S. algicola Gy8, implying that S2-3 belongs to the genus Seonamhaeicola. The major fatty acids (>10%) comprised C15:1 iso G (22.29%), C15:0 iso (17.71%), C17:0 iso 3OH (16.06%), and C15:0 iso 3OH (10.7%), resulting in quite different ratio of the component from those of S. algicola Gy8. Moreover, its biochemical characteristics, including acid production and enzyme activities, were different from those of S. algicola Gy8. Therefore, putting all these results together, we concluded S2-3 is distinct species from S. algicola Gy8, and thus named it Seonamhaeicola sp. S2-3. In liquid culture, S2-3 produced extracellular cellulases that can hydrolyze cellulose or cellooligosaccharides into cellobiose, which is a good enzyme resource that deserves further research.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.1
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• pp.52-59
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• 2005

The study was targeted to saccharify foodwastes with the cellulolytic and amylolytic enzymes obtained from culture supernatant of Trichoderma harzianum FJ1 and analyze the kinetics of the saccharification in order to enlarge the utilization in industrial application. T. harzianum FJ1 highly produced various cellulolytic (filter paperase 0.9, carboxymethyl cellulase 22.0, ${\beta}$-glucosidase 1.2, Avicelase 0.4, xylanase 30.8, as U/mL-supernatant) and amylolytic (${alpha}$-amylase 5.6, ${\beta}$-amylase 3.1, glucoamylase 2.6, as U/mL-supernatant) enzymes. The $23{\sim}98\;g/L$ of reducing sugars were obtained under various experimental conditions by changing FPase to between $0.2{\sim}0.6\;U/mL$ and foodwastes between $5{\sim}20\%$ (w/v), with fixed conditions at $50^{\circ}C$, pH 5.0, and 100 rpm for 24 h. As the enzymatic hydrolysis of foodwastes were performed in a heterogeneous solid-liquid reaction system, it was significantly influenced by enzyme and substrate concentrations used, where the pH and temperature were fixed at their experimental optima of 5.0 and $50^{\circ}C$, respectively. An empirical model was employed to simplify the kinetics of the saccharification reaction. The reducing sugars concentration (X, g/L) in the saccharification reaction was expressed by a power curve ($X=K{\cdot}t^n$) for the reaction time (t), where the coefficient, K and n. were related to functions of the enzymes concentrations (E) and foodwastes concentrations (S), as follow: $K=10.894{\cdot}Ln(E{\cdot}S^2)-56.768,\;n=0.0608{\cdot}(E/S)^{-0.2130}$. The kinetic developed to analyze the effective saccharification of foodwastes composed of complex organic compounds could adequately explain the cases under various saccharification conditions. The kinetics results would be available for reducing sugars production processes, with the reducing sugars obtained at a lower cost can be used as carbon and energy sources in various fermentation industries.

• Journal of Microbiology
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• v.45 no.6
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• pp.485-491
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• 2007

The effects of biological pretreatment on the Japanese red pine Pinus densiflora, was evaluated after exposure to three white rot fungi Ceriporia lacerata, Stereum hirsutum, and Polyporus brumalis. Change in chemical composition, structural modification, and their susceptibility to enzymatic saccharification in the degraded wood were analyzed. Of the three white rot fungi tested, S. hirsutum selectively degraded the lignin of this sortwood rather than the holocellulose component. After eight weeks of pretreatment with S. hirsutum, total weight loss was 10.7%, while lignin loss was the highest at 14.52% among the tested samples. However, holocellulose loss was lower at 7.81 % compared to those of C. lacerata and P. brumalis. Extracelluar enzymes from S. hirsutum showed higher activity of ligninase and lower activity of cellulase than those from other white rot fungi. Thus, total weight loss and changes in chemical composition of the Japanese red pine was well correlated with the enzyme activities related with lignin- and cellulose degradation in these fungi. Based on the data obtained from analysis of physical characterization of degraded wood by X-ray Diffractometry (XRD) and pore size distribution, S. hirsutum was considered as an effective potential fungus for biological pretreatment. In particular, the increase of available pore size of over 120 nm in pretreated wood powder with S. hirsutum made enzymes accessible for further enzymatic saccharification. When Japanese red pine chips treated with S. hirsutum were enzymatically saccharified using commercial enzymes (Cellulclast 1.5 L and Novozyme 188), sugar yield was greatly increased (21.01 %) compared to non-pre treated control samples, indicating that white rot fungus S. hirsutum provides an effective process in increasing sugar yield from woody biomass.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1681-1691
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• 2012

This work was conducted to evaluate the effect of dilute sodium hydroxide (NaOH) on barley straw at boiling temperature and fractionation of its biomass components into lignin, hemicellulose, and reducing sugars. To this end, various concentrations of NaOH (0.5% to 2%) were applied for pretreatment of barley straw at $105^{\circ}C$ for 10 min. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and Fourier transform infrared (FTIR) spectroscopy studies revealed that 2% NaOH-pretreated barley straw exposed cellulose fibers on which surface granules were abolished due to comprehensive removal of lignin and hemicellulose. The X-ray diffractometer (XRD) result showed that the crystalline index was increased with increased concentration of NaOH and found a maximum 71.5% for 2% NaOH-pretreated sample. The maximum removal of lignin and hemicellulose was 84.8% and 79.5% from 2% NaOH-pretreated liquor, respectively. Reducing sugar yield was 86.5% from 2% NaOH-pretreated sample using an enzyme dose containing 20 FPU of cellulase, 40 IU of ${\beta}$-glucosidase, and 4 FXU of xylanase/g substrate. The results of this study suggest that it is possible to produce the bioethanol precursor from barley straw using 2% NaOH at boiling temperature.