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Enoch Y. Park,Vipin Kumar Deo,Tatsuya Kato,Naoko Asari 한국생물공학회 2005 Biotechnology and Bioprocess Engineering Vol.10 No.3
Insect cell transformants, stably expressing human 1,3-N-acetylglucosaminyltransferase 2 (3GnT2) as the green fluorescent protein (GFPuv)-fused protein, were efficiently isolated on Western blot by the quantification of the densitometric intensity of the fusion protein. From almost 150 transformants containing the fusion gene linked to three different types of signal sequence, two transformants, Tn-pXme4a and -pX28a, were successfully selected, showing 8.3 and 8.6 mU/mL 3GnT activity, respectively. This method requires a screening time almost one-half that required in the isolation of stably transformed cells with high expression levels, and at the same time allows the handling a large number of transformants.
Enoch Y. Park,Mi Sun Kwon,Takashi Dojima 한국생물공학회 2003 Biotechnology and Bioprocess Engineering Vol.8 No.2
Three insect cell lines, Sf9, Sf21 and Tn5B1-4, and four different kinds of serum free media (SFM), Sf 900 II, EX-CELL 420, EX-CELL 405 and Express Five, were used to compare the nutrient consumption, byproduct formation, production of recombinant protein and protease activity in suspension cultures. The Sf 900 II SFM was appropriate for the cell growth and protein production of the Sf9 and Sf21 cell lines. When the Tn5B1-4 cell line was grown in the Express Five SFM, the specific growth rate was 1.6 fold higher than those of either the Sf9 or Sf21 cell lines. The glucose and glutamine consumption rates per cells, were 4 and 2.3 times higher than those of the Sf9 cell line, respectively. The overall yield coefficients of the lactate and ammonium ion were 2.8 and 1.5 times higher compared to those of the Sf9 cell line, respectively. The maximum specific β-galactosidase production rate was 4.5 fold that of the Sf9 cell line, a 3 times higher protease activity per cell.
Waste paper sludge as a potential biomass for bio-ethanol production
Enoch Y. Park,Joni Prasetyo 한국화학공학회 2013 Korean Journal of Chemical Engineering Vol.30 No.2
This review describes the utilization of paper sludge (PS), which is waste from the pulp and paper industry. Its advantages make PS the cellulosic biomass with the most potential for bio-refinery research and applicable for industrial scale. Some of the grain based biofuels and chemicals have already been in commercial operation, including fuel ethanol or biochemical products. Unfortunately, research and application of PS are yet in their infancy and suffer from large scale because of low productivity. Reviewing the many researches that are working at the utilization of PS for bio-refineries could encourage the utilization of PS from laboratory research to be applied in industry. For this reason,PS usage as industrial raw material will be effective in solving the environmental problems caused by PS with clean technology. In addition, its conversion to bio-ethanol could offer an alternative solution to the energy crisis from fossil fuel. Two methods of PS utilization as raw material for bio-ethanol production are introduced. The simultaneous saccharification and fermentation (SSF) using cellulase produced by A. cellulolyticus and thermotolerant S. cerevisiae TJ14 gave ethanol yield 0.208 (g ethanol/g PS organic material) or 0.051 (g ethanol/g PS). One pot bioethanol production as a modified consolidated biomass processing (CBP) technology gave ethanol yield 0.19 (g ethanol/g Solka floc) and is considered to be the practical CBP technology for its minimizing process.
Empirical Evaluation of Cellulase on Enzymatic Hydrolysis of Waste Office Paper
Park, Enoch Y.,Ikeda, Yuko,Okuda, Naoyuki The Korean Society for Biotechnology and Bioengine 2002 Biotechnology and Bioprocess Engineering Vol.7 No.5
Enzymatic hydrolysis of waste office paper was evaluated using three commercial cellulases, Acremonium cellulase, Meicelase, and Cellulosin T2. Varying the enzyme loading from 1 to 10% (w/w) conversion of waste office paper to reducing sugar was investigated. The conversion increased with the increase in the enzyme loading: in the case of enzyme loading of 10% (w/w), Acremonium cellulase yielded 79%conversion of waste office paper, which was 17% higher compared to Meicelase, 13% higher than that of Cellulosin T2. Empirical model for the conversion (%) of waste office paper to re-ducing sugar (x) was derived from experimental results as follow, x = $kE^{m}t^{(aE+b)}$ where k, m, a, and b de-note empirical constants. E indicates initial enzyme concentration.
Enoch Y. Park,Shin Kanamasa,Satoshi Tajima 한국생물공학회 2007 Biotechnology and Bioprocess Engineering Vol.12 No.2
For this study, we hypothesized that mitochondrial NAD+-dependent isocitrate dehydrogenase 1 (ICDH1) and isocitrate lyase (ICL1) were important enzymes for riboflavin synthesis in the fungus Ashbya gossypii. Here, the genes encoding ICDH1 and ICL1 were disrupted in order to analyze the enzymes’ functions on riboflavin production by the fungus. The riboflavin production resulting from these disruptants was markedly decreased compared to the concentration produced by its parental strain when cultured in a rich nutrient medium used to optimize riboflavin production. Furthermore, when comparing the transcription levels of the genes encoding ICDH1 and ICL1, between wild-type A. gossypii and an itaconate resistant mutant of A. gossypii obtained by UV irradiation, the mRNA levels in the mutant were 1.8- and 2.0-fold higher than those in the wild-type strain, respectively. These results indicate that ICDH1 and ICL1 are key enzymes for riboflavin synthesis in A. gossypii.
Kanamasa, Shin,Tajima, Satoshi,Park, Enoch Y. Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.2
For this study, we hypothesized that mitochondrial $NAD^+-dependent$ isocitrate dehydrogenase 1 (ICDH1) and isocitrate lyase (ICL 1) were important enzymes for riboflavin synthesis in the fungus Ashbya gossypii. Here, the genes encoding ICDH1 and ICL1 were disrupted in order to analyze the enzymes' functions on riboflavin production by the fungus. The riboflavin production resulting from these disruptants was markedly decreased compared to the concentration produced by its parental strain when cultured in a rich nutrient medium used to optimize riboflavin production. Furthermore, when comparing the transcription levels of the genes encoding ICDH1 and ICL1, between wild-type A. gossypii and an itaconate resistant mutant of A. gossypii obtained by UV irradiation, the mRNA levels in the mutant were 1.8- and 2.0-fold higher than those in the wild-type strain, respectively. These results indicate that ICDH1 and ICL1 are key enzymes for riboflavin synthesis in A. gossypii.
Palaniyandi Muthukutty,Tatsuya Kato,Enoch Y. Park 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.3
Using human papillomavirus (HPV) as a subunit vaccine and its manipulation of surface loops is current trending research. Since the atomic model of L1 protein conformations were deciphered, their manipulations of epitopes bring multivalent vaccines. Here, in the present study, we have manipulated antigenic loops of HPV 6b L1capsid proteins in the amino acid regions 174 ~ 175(L1:174EGFP) and 348 ~ 349 (L1:348EGFP) with whole enhanced green fluorescent protein(EGFP), expressed in the silkworm larva using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid technology. The expressed proteins were partially purified using sucrose density-gradient centrifugation and size-exclusion chromatography (SEC). The display of EGFP in virus-like particles (VLPs) was confirmed by immuno-fluorescence microscopy, Western blots and immune-transmission electron microscopy (immuno-TEM). There was higher expression of EGFP incorporated L1:174EGFP than L1:348EGFP. Hydrodynamic diameter of VLPs was corroborated by dynamic light scattering,confirming the size of expected range of around 160 nm and substantiating the incorporation of EGFP. From immuno-TEM, each L1:EGFP VLP formed small particles,suggesting that small particles of L1:EGFP fusion protein were aggregated. Our study illustrates that incorporation of whole protein can efficiently form chimeric VLPs, without hindering the conformation. HPV L1 protein accommodated a whole protein on its antigenic loop as a small particle, but an inserted whole protein was unstable.