Showing papers by "Byung-Gee Kim published in 2005"

Revisit of aminotransferase in the genomic era and its application to biocatalysis

Abstract: Aminotransferases (ATs) have useful applications in the chemical industry because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity and regioselectivity and broad substrate specificity Abundant protein sequence databases and new powerful tools such as advanced computational structure modeling, multiple sequence analysis, and in vitro evolution have made it possible to understand the detailed reaction mechanisms of various ATs and to isolate and design novel enzymes for unnatural substrates This, in turn, suggests that developing new integrated approaches to screen ATs are possible, but at the same time poses formidable technical challenges Here, this paper reviews the use of family profile analysis to find the correlation between the type of ATs and their substrate specificities, the relation between the 3-D structures of ATs and their substrate specificities, and enzyme engineering for the synthesis of unnatural substrates

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

Abstract: A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant omega-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, omega-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using omega-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.

Microaffinity purification of proteins based on photolytic elution: toward an efficient microbead affinity chromatography on a chip.

Abstract: A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated.

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal

Abstract: The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal.

Novel Method for Detection of Butanolides in Streptomyces coelicolor Culture Broth, Using a His-Tagged Receptor (ScbR) and Mass Spectrometry

Abstract: γ-Butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.

Microfluidic chip for biochemical reaction and electrophoretic separation by quantitative volume control

Abstract: We have developed a microfluidic integrated chip with all the fluidic manipulations required for biochemical reaction and electrophoretic separation. The microchip was fabricated using standard photolithography, isotropic wet etching, liftoff patterning of fluorocarbon film, and polydimethylsiloxane replica molding. The fabricated microfluidic chip (46 mm × 25 mm) was composed of two nanoliter-metering microchannel networks, a serpentine mixing channel, a sample injector, and an electrophoretic separation channel. In particular, a novel Y-shaped sample injector was developed as an important interface for the successful combination of the biochemical reaction with electrophoretic separation. A nanoliter-sized sample was pneumatically injected into the separation channel using the novel Y-shaped sample injector. This consisted of a planar hydrophobic valve and two vertical hydrophobic valves. The performance of the hydrophobic valves for fluidic manipulations was predicted using capillary pressure derived from the relationship of the work performed by the capillary pressure (d U p ), the surface free energy of the system (d U s ), and Young's equation. In addition, we were able to explain in detail the fluidic manipulation schemes for the biochemical reaction and subsequent separation. We also successfully demonstrated the enzyme reaction of β-galactosidase using fluorescein di-β- d -galactopyranoside as a substrate and the electrophoretic separation of its reaction products.

Characterization of GDP-mannose Pyrophosphorylase from Escherichia Coli O157:H7 EDL933 and Its Broad Substrate Specificity

Abstract: GDP-mannose pyrophosphorylase gene (ManC) of Escherichia coli (E. coli) O157 was cloned and expressed as a highly soluble protein in E. coli BL21 (DE3). The enzyme was subsequently purified using hydrophobic and ion exchange chromatographies. ManC showed very broad substrate specificities for four nucleotides and various hexose-1-phosphates, yielding ADP-mannose, CDP-mannose, UDP-mannose, GDP-mannose, GDP-glucose and GDP-2-deoxy-glucose.

The ribostamycin biosynthetic gene cluster in Streptomyces ribosidificus: comparison with butirosin biosynthesis.

Abstract: A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.

"In-gel patch electrophoresis:" a new method for environmental DNA purification.

Abstract: Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple, and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and polymerase chain reaction (PCR) amplifications.

Screening ofExiguobacterium acetylicum from soil samples showing enantioselective and alkalotolerant esterase activity

Abstract: About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (E app>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.

The effects of ftsZ mutation on the production of recombinant protein in Bacillus subtilis.

Abstract: In this paper, the possibility of using a mutation of ftsZ as a pseudo-spore mutant is investigated. ftsZ, which is essential for cell division and sporulation of Bacillus subtilis, was placed under the spac promoter, which is inducible with isopropyl thiogalactose (IPTG). Cell growth of the ftsZ mutant and its β-galactosidase activity under the aprE promoter were compared with the wild type. In the presence of 1 mM IPTG, cell growth of the ftsZ mutant was almost the same as that of the wild type and its sporulation frequency was slightly lower than that of the wild type. However, under uninduced conditions, cell growth of ftsZ mutant was severely impaired. When induced with 0.2 mM IPTG, the ftsZ mutant showed about 13 times higher β-galactosidase activity than the wild type. When the ftsZ mutant was used for secretory production of subtilisin, only three times higher extracellular subtilisin activity was measured, compared with the wild type. By real-time PCR investigation, it was revealed that the ftsZ mutant intracellular mRNA level for subtilisin was more than 16 times higher, compared with the wild type. However, it appears that the secretion pathway is somewhat damaged in the ftsZ mutant. These results suggest that the cell division mutant can also be used like a sporulation mutant to produce recombinant proteins, with a precise control of cell growth and induction.

Fabrication, Estimation and Trypsin Digestion Experiment of the Thermally Isolated Micro Teactor for Bio-chemical Reaction

Abstract: This paper describes design, fabrication, and application of the silicon based temperature controllable micro reactor. In order to achieve fast temperature variation and low energy consumption, reaction chamber of the micro reactor was thermally isolated by etching the highly conductive silicon around the reaction chamber. Compared with the model not having thermally isolated structure, the thermally isolated micro reactor showed enhanced thermal performances such as fast temperature variation and low energy consumption. The performance enhancements of the micro reactor due to etched holes were verified by thermal experiment and numerical analysis. Regarding to 42 percents reduction of the thermal mass achieved by the etched holes, approximately 4 times faster thermal variation and 5 times smaller energy consumption were acquired. The total size of the fabricated micro reactor was 37×30×1 ㎣. Microchannel and reaction chamber were formed on the silicon substrate. The openings of channel and chamber were covered by the glass substrate. The Pt electrodes for heater and sensor are fabricated on the backside of silicon substrate below the reaction chamber. The dimension of channel cross section was 200×100 ㎛². The volume of reaction chamber was 4 ㎕. The temperature of the micro reactor was controlled and measured simultaneously with NI DAQ PCI-MIO-16E-1 board and LabVIEW program. Finally, the fabricated micro reactor and the temperature control system were applied to the thermal denaturation and the trypsin digestion of protein. BSA(bovine serum albumin) was chosen for the test sample. It was successfully shown that BSA was successfully denatured at 75℃ for 1 min and digested by trypsin at 37℃ for 10 min.

A microfabricated device with integrated nanoelectrospray source for capillary electrophoresis and mass spectrometry

Abstract: In this study, a new method is described for integrating an electrospray ionization interface to a mass spectrometer with a capillary electrophoresis channel. We have fabricated the ESI-MS device composed of the metal emitter tip, allowing the generation of an efficient nanospray for protein detection, and CE separation channel monolithically in a glass microchip. A triangular-shaped gold emitter tip was formed by electroplating at the end of the separation channel. As an ESI source, this emitter structure aided the formation of a stable Taylor cone. It is easily fabricated by MEMS technology and more robust than that of silica or polymer recently reported. Moreover, this approach is less involved than applying a conductive coating to the exit end to establish electrical contact. As such, the interface is less dependent upon the longevity or durability of such coating, factors that have been consideration in the sheathless interfaces. The spraying stability was evaluated and the ESI-MS experiment was performed by spraying standard peptides for mass spectrometric analysis. The spraying was stable, with a relative standard deviation of 2.9%. The CE/ESI-MS analysis was performed by separating and spraying standard peptide mixture of Bradykinin 1-5, Bradykinin 1-8, and Angiotensin I. Each peptide was separated successfully and singly-charged peaks and doubly-charged peaks of each peptide were detected, respectively. Direct comparisons with conventional ESI-MS system using glass or fused silica emitters showed very similar performance with respect to signal intensity and stability.

Thermal denaturation and trypsin digestion of protein on a temperature controllable microchip for MALDI-TOF MS analysis

Abstract: This paper describes thermal denaturation and trypsin digestion of protein on a microchip as an alternative application of a temperature controllable microchip. Analysis of the protein of small volume and low concentration, which is impossible in macro scale, could be possible with the fairly reduced process time using the proposed temperature controllable microchip. We optimized the parameters concerning thermal denaturation on a microchip such as thermal denaturation temperature, thermal denaturation time, digestion time and concentration of protein using BSA(bovine serum albumin) as a reference sample. Then we applied the optimized parameters to the other proteins (ovalbumin, myoglobin, hemoglobin, cytochrome C, Ubiquitin). The proposed method on a microchip in this paper needed an even shorter reaction time, smaller volume of sample and smaller concentration of sample compared to the previously presented marco scale thermal denaturation and trypsin digestion method. We could successfully acquire the thermally denatured protein in 1 minute at 85°C and the digested peptides in 10 minutes at 37°C with 3 μl/0.2 μM protein. The acquired average sequence coverages are range from 24 to 57% for the test proteins, which are sufficient for the protein identification in practical use.