Chul Ho Kim

Sequential acid-/alkali-pretreatment of empty palm fruit bunch fiber.

Pretreatment processes are key technologies for generating fermentable sugars based on lignocellulosic biomass. In this study, we developed a novel method for empty palm fruit bunch fiber (EPFBF) using sequential pretreatment with dilute acid and then alkali. Dilute sulfuric acid was used in the first step, which removed 90% of the hemicellulose and 32% of the lignin, but left most of the cellulose under the optimum pretreatment condition. Sodium hydroxide was then applied in the second step, which extracted lignin effectively with a 70% delignification yield, partially disrupting the ordered fibrils of the EPFBF and thus enhancing the enzyme digestibility of the cellulose. The sequentially pretreated biomass consisted of 82% cellulose, less than 1% hemicellulose, and 30% lignin content afterward. The pretreated biomasses morphologically revealed rough, porous, and irregularly ordered surfaces for enhancing enzyme digestibility. These results indicate that the sequentially acid/alkali-pretreated EPFBF could be broadly useful as a novel biomass.

Production of 3-hydroxypropionic acid through propionaldehyde dehydrogenase PduP mediated biosynthetic pathway in Klebsiella pneumoniae

The pduP gene encodes a propionaldehyde dehydrogenase (PduP) was investigated for the role in 3-hydroxypropionic acid (3-HP) glycerol metabolism in Klebsiella pneumoniae. The enzyme assay showed that cell extracts from a pduP mutant strain lacked measurable dehydrogenase activity. Additionally, the mutant strain accumulated the cytotoxic intermediate metabolite 3-hydroxypropionaldehyde (3-HPA), causing both cell death and a lower final 3-HP titer. Ectopic expression of pduP restored normal cell growth to mutant. The enzymatic property of recombinant protein from Escherichia coli was examined, exhibiting a broad substrate specificity, being active on 3-HPA. The present work is thus the first to demonstrate the role of PduP in glycerol metabolism and biosynthesis of 3-HP.

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l−1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 106), as determined by HPLC while high molecular weight levan (>6 × 106) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.

Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography

The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to > 98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60 nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine.

Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli.

L‐amino acid deaminases catalyze the deamination of natural L‐amino acids. Two types of L‐amino acid deaminase have been identified in Proteus species. One exhibits high levels of activity toward a wide range of aliphatic and aromatic L‐amino acids, typically L‐phenylalanine, whereas the other acts on a relatively narrow range of basic L‐amino acids, typically L‐histidine. In this study, we cloned, expressed, and characterized a second amino acid deaminase, termed Pm1, from P. mirabilis KCTC 2566. Homology alignment of the deduced amino acid sequence of Pm1 demonstrated that the greatest similarity (96%) was with the L‐amino acid deaminase (LAD) of P. vulgaris, and that homology with Pma was relatively low (72%). Also, similar to LAD, Pm1 was most active on L‐histidine, indicating that Pm1 belongs to the second type of amino acid deaminase. In agreement with this conclusion, the Vmax and Km values of Pm1 were 119.7 (μg phenylpyruvic acid/mg/min) and 31.55 mM phenylalanine, respectively, values lower than those of Pma. The Pml deaminase will be very useful industrially in the preparation of commercially valuable materials including urocanic acid and α ‐oxoglutarate. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

β-Glucan enhanced apoptosis in human colon cancer cells SNU-C4

The apoptotic effect of bacteria-derived β-glucan was investigated in human colon cancer cells SNU-C4 using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR) expressions of Bcl-2, Bax, and Caspase-3 genes, and assay of caspase-3 enzyme activity. β-Glucan of 10, 50, and 100 µg/mL decreased cell viability in a dose-dependent manner with typical apoptotic characteristics, such as morphological changes of chromatin condensation and apoptotic body formation from TUNEL assay. In addition, β-glucan (100 µg/mL) decreased the expression of Bcl-2 by 0.6 times, whereas the expression of Bax and Caspase-3 were increased by 3.1 and 2.3 times, respectively, compared to untreated control group. Furthermore, the caspase-3 activity in the β-glucan-treated group was significantly increased compared to those in control group (P < 0.05). Bacterial derived β-glucan could be used as an effective compound inducing apoptosis in human colon cancer.

Growth of the oleaginous microalga Aurantiochytrium sp. KRS101 on cellulosic biomass and the production of lipids containing high levels of docosahexaenoic acid

We examined the growth of a novel oleaginous microalga, Aurantiochytrium sp. KRS101, using cellulosic materials as nutrients, and the resultant production of lipids containing high levels of docosahexaenoic acid (DHA). The microalgal strain could grow using either carboxymethylcellulose or cellobiose as a carbon source, and produced lipids containing high levels of DHA (49–58% of total fatty acids). In line with this growth behavior, carboxymethylcellulase and cellobiohydrolase activities were evident in both cell-free lysates and culture broths. Additionally, an industrial cellulosic biomass, palm oil empty fruit bunches (POEFB), a by-product of the palm oil industry, were utilized by the microalgal strain for cell growth and lipid production.

Levan production by use of the recombinant levansucrase immobilized on titanium-activated magnetite

Abstract Levansucrase from Zymomonas mobilis expressed in Escherichia coli was immobilized onto the surface of magnetite. Optimum conditions for the immobilization were, pH 4.0; immobilization reaction time of 30 min and 8 U of enzyme per gram of matrix. Immobilization yield was 75% under optimized conditions. The maximum production yield of levan by the immobilized levansucrase was about 42% on a fructose released basis with sucrose as substrate at 100 g/l. Thermal stability of the levansucrase increased by immobilization procedure. Furthermore, immobilized enzyme showed the less polymerizing activity in levan formation, producing the low-molecular weight (MW) levan. The molecular weight of levan can be controlled by using the immobilized levansucrase in high yield. Immobilized levansucrase retained 61% of the original activity after five repeated uses.

Synthesis of methyl β-D-fructoside catalyzed by levansucrase from Rahnella aquatilis

Abstract Methyl β-D-fructoside(MF) was formed from sucrose and methanol by a transfructosylation reaction using recombinant levansucrase from Rahnella aquatilis . The increase in the yield of MF formation was achieved by increasing methanol concentration. The enzyme stability at higher concentrations of methanol was maintained by lowering the reaction temperature. The optimum temperature and sucrose concentration for MF formation was 10°C and 50 gL −1 respectively and the yield of MF was 70%.

Production of D-β-hydroxyisobutyric acid from isobutyric acid by Candida rugosa

Abstract Production of d -β-hydroxyisobutyric acid ( d -HIBA) from isobutyric acid (IBA) was investigated using Candida rugosa IFO 0750. Cell growth and d -HIBA production decreased as the substrate concentration increased. A considerable degradation of d -HIBA was observed when the substrate, IBA, was depleted in the medium. Specific production rate of d -HIBA increased as glucose concentration decreased, while the conversion yield of IBA to d -HIBA showed an opposite trend. With a controlled feeding of IBA and glucose, a high titer of d -HIBA (100 g/l) could be obtained by a fed-batch cultivation of Candida rugosa.