Eun Gyo Lee

Improved enantioselectivity of Candida rugosa lipase towards ketoprofen ethyl ester by a simple two-step treatment

A novel two-step acetone treatment method was developed to improve the enantioselectivity of Candida rugosa lipase (CRL) towards the hydrolysis of (R,S)-ketoprofen ethyl ester. After two-step acetone treatment of crude CRL, the CRL was harvested as precipitates and used for the production of optically pure (S)-ketoprofen. The specific activity of two-step acetone-treated CRL was 2.05 kU/mg protein, which corresponds to a 2-fold increase over that of crude CRL. The two-step acetone-treated CRL was considerably more enantioselective than the crude CRL towards (R,S)-ketoprofen ethyl ester, yielding an enantiomeric excess (% eep) of about 100% and an enantiomeric ratio (E) of >100. The hydrolysis activity of acetone-treated CRL increased with an increase in reaction temperature but the enantiomeric excess was >99% regardless of reaction temperature. The production of optically pure (S)-ketoprofen was performed for 108 h in a scaled-up batch reactor containing 200 g of (R,S)-ketoprofen ethyl ester. Consequently, about 90 g of (S)-ketoprofen with an optical purity of 98% was recovered from the reaction mixture.

Enantioselective hydrolysis of racemic naproxen methyl ester by two-step acetone-treated Candida rugosa lipase

Abstract An optically pure ( S )-naproxen was produced by two-step acetone-treated Candida rugosa lipase (CRL) through enantioselective hydrolysis of racemic naproxen methyl ester. The two-step acetone-treated CRL was much more enantioselective than the crude CRL towards the hydrolysis of ( R,S )-naproxen methyl ester, yielding an enantiomeric excess (ee p ) of >98% and an enantiomeric ratio ( E ) of >100. In terms of hydrolysis reaction rate and enantioselectivity, the optimal reaction conditions were found to be 37xa0°C and pH 6.0. The scaled-up production of ( S )-naproxen was performed in a batch reactor containing 200 ml of substrate solution (50 mM MES buffer containing 200 mM ( R,S )-naproxen methyl ester). After 156 h of reaction, 38.4% of the initial ( R,S )-naproxen methyl ester was hydrolyzed, yielding an enantiomeric excess of 98% and an enantiomeric ratio of>100. Finally, ( S )-naproxen was recovered from the reaction mixture with an optical purity of 98% and a recovery yield of 95%.

Carbon nanotube-assisted enhancement of surface plasmon resonance signal.

We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody-carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM-CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For the amplification of the SPR signal, the CNT was conjugated with a polyclonal antibody, and then the conjugates were reacted with antibodies coupled with the target proteins. This amplification strategy increases the dynamic range of the immunoassays and enhances the detection sensitivity. The SPR immunoassays, combined with the CNT-assisted signal amplification method, provided a wide dynamic range over four orders of magnitude for both EPO and GM-CSF (0.1-1,000 ng/ml). The CNT amplification method is expected to realize the detection of picogram levels and a wide dynamic detection range of multiple proteins, enabling it to offer a robust analysis tool for the development of biopharmaceutical production.

Increased Genomic Integrity of an Improved Protein-Based Mouse Induced Pluripotent Stem Cell Method Compared With Current Viral-Induced Strategies

It has recently been shown that genomic integrity (with respect to copy number variants [CNVs]) is compromised in human induced pluripotent stem cells (iPSCs) generated by viral‐based ectopic expression of specific transcription factors (e.g., Oct4, Sox2, Klf4, and c‐Myc). However, it is unclear how different methods for iPSC generation compare with one another with respect to CNV formation. Because array‐based methods remain the gold standard for detecting unbalanced structural variants (i.e., CNVs), we have used this approach to comprehensively identify CNVs in iPSC as a proxy for determining whether our modified protein‐based method minimizes genomic instability compared with retro‐ and lentiviral methods. In this study, we established an improved method for protein reprogramming by using partially purified reprogramming proteins, resulting in more efficient generation of iPSCs from C57/BL6J mouse hepatocytes than using protein extracts. We also developed a robust and unbiased 1 M custom array CGH platform to identify novel CNVs and previously described hot spots for CNV formation, allowing us to detect CNVs down to the size of 1.9 kb. The genomic integrity of these protein‐based mouse iPSCs (p‐miPSCs) was compared with miPSCs developed from viral‐based strategies (i.e., retroviral: retro‐miPSCs or lentiviral: lenti‐miPSCs). We identified an increased CNV content in lenti‐miPSCs and retro‐miPSCs (29∼53 CNVs) compared with p‐miPSCs (9∼10 CNVs), indicating that our improved protein‐based reprogramming method maintains genomic integrity better than current viral reprogramming methods. Thus, our study, for the first time to our knowledge, demonstrates that reprogramming methods significantly influence the genomic integrity of resulting iPSCs.

Identification, molecular cloning and expression of a new esterase from Pseudomonas sp. KCTC 10122BP with enantioselectivity towards racemic ketoprofen ethyl ester

Abstract A newly isolated gene from Pseudomonas sp. KCTC 10122BP, encoding an esterase with enantioselectivity towards racemic ketoprofen ( rac -ketoprofen) ethyl ester, was cloned in Escherichia coli and its nucleotide sequence determined. The deduced amino acid sequence predicted an open reading frame (ORF) encoding a polypeptide of 381 amino acid residues (1143 nucleotides) with a calculated isoelectric point of pH 5.32 and molecular mass of 41,149xa0Da. The primary structure of the enzyme exhibited a significant level of homology (>31%) with those of related enzymes from various sources and an extreme homology (>81%) with five esterases from the genus Pseudomonas . The enzyme was expressed at a high level in an active form in the soluble fraction and purified to homogeneity by a successive chromatographic procedure. The purified enzyme was determined to be a monomer, plus it exhibited a strict selectivity (>99%) and high activity (2360 units/mg-protein) towards ( S )-ketoprofen ethyl ester.

Hydrodynamic extensional stress during the bubble bursting process for bioreactor system design

Cell damage, one of critical issues in the bioreactor design for animal cell culture, is caused mainly from the bubble bursting at the free surface subjected to strong extensional flows. In this work, extensive computational studies are performed to investigate bubble bursting process in great details. Extensive numerical simulations are performed for a wide range of bubble diameters (from 0.5 to 6 mm) and the surface tension values (from 0.03 to 0.072 N/m), with which effects of the bubble size and surfactant (PF68) concentration on the hydrodynamic stress are investigated. For all the cases, the maximum extensional stress appears at the instance when receding films impact each other at the bottom of the bubble. A model equation based on numerical simulations is presented to predict the maximum extensional stress as a function of the bubble diameter and the surface tension. The bubble diameter has turned out to contribute significantly the maximum hydrodynamic extensional stress. In addition, the bubble collapsed time and the affected volume around a bubble subjected to the critical extensional stress are investigated. The extensional stress estimation is reported as a function of the bubble size and the surface tension. The influence of the bubble size on the maximum stress dominates and extensional stress reaches up to the order of 104 Pa for bubble size of 0.5 mm.

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers.

Label-free measurement of cell viability via counting cells attached on affinity substrates

The commonly used trypan blue dye exclusion method and other modified cell viability methods, such as fluorescein dye and tetrazolium dye exclusion, artificially introduce toxic chemicals to cells and, thus, alter cellular organelles when measuring cell viability. Therefore, cell viability could be affected by the processes currently used to observe viability. In this study, the cell viability of Chinese hamster ovary (CHO) cells was measured by simply counting attached cells after the cultured CHO cells were attached on a Concanavalin A (Con A) substrate. The efficiency of cell attachment to Con A surfaces was different for live and dead cells allowing the cell viability of CHO cells to be measured without any chemical modifications to the cells.

Erratum to: Hydrodynamic extensional stress during the bubble bursting process for bioreactor system design

Thanh Tinh Tran 1 , Eun Gyo Lee 2 , In Su Lee 3 , Nam Sub Woo 4 , Sang Mok Han 4 , Young Ju Kim * and Wook Ryol Hwang* 1School of Mechanical and Aerospace Engineering, Research Center for Aircraft Parts Technology (ReCAPT), Gyeongsang National University, Jinju 52828, Republic of Korea 2Biotechnology Process Engineering Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Republic of Korea 3CNS Co., Ltd., Daejeon 34369, Republic of Korea 4Pohang Branch Extreme Research Plant R&D Department, Korea Institute of Geoscience and Mineral Resources, Pohang 37559, Republic of Korea