Earticle

Hyaluronidase is an enzyme that catalyzes the hydrolysis of hyaluronic acid (1). Hyaluronidase isolated in mammalian testis has been used in medicine in conjunction with local anesthetic to speed its dispersion (2-3). There are many animal-derived clinical grade hyaluronidases available but all have potential BSE problem. Only one recombinant hyaluronidase was approved in 2005 by US FDA.In this study, we have established the high yield purification process for natural hyaluronidase from Korean bovine testis. Solubilizing bovine testis with detergent containing buffer, supernatant was treated with ammonium sulfate and acid. Precipitates were then further purified by ion-exchange chromatography. About 120,000 units of hyaluronidase were collected from 200 g of bovine testis. The activity of isolated hyaluronidase was determined by the test method in USP-NF and 56 kDa protein was identified as bovine hyaluronidase by Western blot analysis. Isolated natural hyaluronidase from bovine testis has been compared with recombinant bovine hyaluronidase in E. coli. The recombinant bovine hyaluronidase will be used for a safe biopharmaceutical once its safety and efficacy are confirmed.

Traditional cellulose production from cellulosic biomass for industrial purpose was complicated. The pulping process from cellulosic biomass caused various problems such as the exhaustion of forest resources and environmental pollution; exclusion of polychlorinated dioxins, furans, persistent organics, sulfur oxides, nitrogen oxides, carbon monoxide etc. These toxic chemical materials cause air, water, soil pollution. However, bacterial cellulose produced by acetobacters strains is free from lignin, hemicelluloses and thus, it does not need chemical chlorine bleaching process. Bacterial cellulose has unique properties including an ultra-fine fiber network, high purity, and high crystallinity. It shows more than five times higher tensile strength and six times higher elasticity coefficient than plant cellulose. With these unique physico-chemical properties bacterial cellulose is used for various industrial purposes. In this study, the effect of water soluble oligosaccharide on production of bacterial cellulose was investigated in the view point of metabolic pathway. We could get high improvement in bacterial cellulose productivity by adding 0.5-4% (w/v) water soluble oligosaccharide to the medium. BC water holding capacity was improved.

Accurate prediction of high-performance liquid chromatography (HPLC) retention time has been a challenge for many analytical researchers including biochemists and bioengineers. There were many trials to rationally analyze and predict HPLC retention time, and some of them showed reasonable results. However, existing methods need too many parameters (five or more) to build a reliable model, which made it difficult to use these methods efficiently. In this study, we tried to find a novel model that can analyze HPLC retention time using less than five parameters. Some chemical properties related to electrical characteristics can be major factors that determine HPLC retention time, especially when ion-exchange column is used. Also, they can be easily computed with various programs and functions. In this study we focus on how Surface-Electrostatic Potential (SEP), Polarity, and charge dispersion of chemicals affect the HPLC retention times. We also considered molar volume of chemicals to reflect the effect of molecular size.

Aptamers are artificial nucleic acid ligands, specifically generated to recognize target molecules, such as amino acids, drugs, proteins or other molecules. Some aptamers can even discriminate optica isomers of a target molecule such as L-arginine, L-tyrosinamide, and D-vasopressin. These artificial ligands have mostly been screened from combinatorial libraries of synthetic nucleic acids by a method, known as systematic evolution of ligands by exponential enrichment (SELEX), comprising an in vitro iterative process of adsorption, recovery and amplification. Verapamil, a calcium entry blocker, has been used in the treatment of hypertension, angina pectoris and cardiac arrhythmia. Verapamil formulations for drug administration usually consist of a blend of R-(+) and S-(−) enantiomers pharmacokinetic and/or pharmacodynamic properties of which are radically different. In addition, an overdose injection of verapamil may cause serious side effects. Therefore, identification of aptamers specifically cognitive of S-(−)-verapamil would facilitate an efficient separation of verapamil enantionmers, furthermore rendering controlled administration of S-(−)-verapamil or close monitoring of drug concentration in order to avoid toxic effects. In the present study, owing to the recent advances in SELEX methodology, we took advantage of chromatography-based in-vitro selection using FPLC to select aptamers capable of specifically binding to S-(−) verapamil from an 86-bp random single-stranded DNA pool. Pre-negative and negative screening were implemented with bare column and R-(−)-verapamil immobilized column in the recycle mode to eliminate the non-specific binders before the main screening. The percentage of ssDNA eluted measured for the fractionated DNA pool following each round of main selection increased with selection rounds, indicating the successful enrichment of aptamers with enhanced affinity to the target. Finally, an aptamer sequence, exhibiting the highest affinity and specificity to S-(−) verapamil following the assessment of Kd for the enriched aptamer sequences, was demonstrated to effect a high-efficiency chiral separation of verapamil enantiomers.

Bacilli are widely used as functional probiotics, feed and food additives. In this work, Bacillus sp. LS 1-2, was isolated from Korean transitional soybean paste foods and had similar with 16S rRNA of Bacillus sp. about 98%. LS 1-2 was cultured in citrus-processingwaste containing 5% solid as a culture medium and produced an antimicrobial agent against both of the gram-positives and negatives at adequate culture conditions (pH, Temperature, Culture period and etc). An antimicrobial agent produced by LS 1-2 was purified by alcohol precipitation, silica gel open column, prep TLC and prep HPLC. The antimicrobial activity was detected in the fermentation supernatant of LS 1-2. The active substance had more activity through the each purification step. The active substance was not sensitive to lipase, proteinase K, amylase, catalase. So this antimicrobial agent was expected as a new antimicrobial agent. In order to evaluate commercial value, the minimum inhibitory concentration of purified antimicrobial agent from Bacillus sp. LS 1-2 was compared with commercialized antimicrobial agents (Colistin, Chlortetracycline, Ampicillin) against Staphylococcus aureus and Escherichia coli O-157.

For the recovery of ammonium lactate by nanofiltration, TFC-SR2 membrane was selected through a permeation test with single-salt and glucose solutions. For the optimization of operating conditions under various operating pressures (50~250 psig.), the effects of lactate concentration (0.5~2.0 M), flow rate (0.5~2.5 L/min), temperature (15~35℃), and pH (5.0~8.0) on the solution flux and lactate rejection were investigated with model solutions. The optimal lactate concentration was found to be 2.0 M, flow rate 1.5 L/min, temperature 30℃, and initial pH 7~8. For a model solution containing 1.0 M ammonium lactate at an operating pressure of 200 psig, the rejections of lactate, acetate, phosphate, sulfate and total protein were 22.7%, 6.9%, 47.7%, 91.5% and 94.4%, respectively under the optimal conditions. Finally, ammonium lactate was recovered from an actual fermentation broth containing 1.5 M lactate under the optimal conditions. At an operating pressure of 200 psig, the rejections of lactate, acetate, phosphate, sulfate and total protein were 19.4%, 9.8%, 43.4%, 69.8% and 97.1%, respectively.

Many bacterial species are known to produce an extracellular heteroor homopolysaccharides, which are attached to the bacterial cell or secreted into the environment. Microbial saccharides produced by bacteria have potential applications in stabilizing, in increasing the viscosity, in promoting adhesion and water retention properties. Gluconacetobacter hansenii PJK are cellulose producing strains which also produce water soluble oligosaccharides (WSOS) as a by- product. Agar plates were used to study the effect of agar surface in the production of WSOS along with BC. It was found that by using the flasks coated with agar plates, not only the BC production was increased but the production of WSOS was also sufficiently increased compared to the control. The experiments were carried out in both day wise and batch wise manners. Initially, the production of WSOS was almost double in the reactors containing the agar plates compared to the control in day wise as well as in the batch wise experiments. However, the production was decreased with successive batches. The results revealed that besides providing surface effect the agar itself played an active role in the production of WSOS.

Streptococcus pneumoniae is a major cause of invasive infection in young infants and older adults. Capsular polysaccharide (CPS) produced by S. pneumoniae is a virulence factor. CPS has been used as a vaccine. Purification of CPS is complicate process and requires many kinds of reagents and column chromatography method. In this study, purification process of CPS from S. pneumoniae serotype 19A was simplified by ethanol fractionation and pH adjustment as following: First the cultured broth was removed a precipitant by ethanol fractionation (50~80%, v/v). The precipitant with less than 50% and more than 80% of ethanol contained most of the contaminated proteins. Precipitations in both ethanols 50% and 80% were performed at neutral pH (7.0) by NaOH adjustment. Small carbohydrates and various contaminated cell hydrolyzates were easily removed by dialysis with dH2O.

Biomineralization of calcium carbonate was studied in the solution containing aspartic acid (Asp). Peristaltic pump controled feeding velocity of Asp or Na2CO3 solutions. Morphology and growth of crystals were varied by changing feeding velocity of Asp or Na2CO3 solution into CaCl2 solution. The ratio of calcite and vaterite crystals was observed in accordance with the reaction time. CaCO3 crystals were analyzed by X-ray Diffraction (XRD), Field Emission Scanning Electron Microscope (FE-SEM) and Fourier Transform Infrared Spectrometry (FT-IR). XRD was used to select the intensities and crystal structure of specific calcium carbonate. SEM was employed for the analysis of the morphology of the precipitation and particle size. Two kinds of crystals were identified by FT-IR spectrum. Vaterite morphologies were affected by the Asp in the crystallization solution. Excess amount of Asp induced vaterite morphologies. Various morphologies of CaCO3 were made by changing feeding velocity of Asp and Na2CO3 solutions.

Fractional precipitation is a simple, efficient method for pre-purifying paclitaxel extracted from plant cell cultures. However, the fractional precipitation process has been inherently problematic due to the lengthy precipitation time (~3 days) that is required. An improved fractional precipitation process could significantly reduce the precipitation time by increasing the purity of crude extract and the surface area available for precipitation. Glass beads (7 mm) were used to increase the surface area, and the optimal surface area per working volume (ie. volume of reaction solution) (S/V) for achieving the highest purity and yield of paclitaxel possible was found to be 0.428 mm-1. The content of paclitaxel dissolved in methanol that can be processed during fractional precipitation was evaluated, and it was established that up to 0.9% (w/v) pure paclitaxel content could be processed. This improved pre-purification process serves to inimize solvent usage and the size and complexity of the high performance liquid chromatography operation required for paclitaxel purification.

DNA is increasingly received a lot of interests from a variety of industry because it is easy to understand the origin of human genome through analytical studies. Adenine and Cytosine are important substances which consist of nucleotides in DNA sequence. To attain sensitive separation, reverse phase-high performance liquid chromatography (RP-HPLC) is analytical tool separation of polar compounds. Eater, methanol and acetonitrile are used as mobile phase of RP-HPLC and C18 or C8 non-polar group on silica surface adopted as stationary phase. Purpose of this study is to separate adenine and cytosine as well as to get adsorption isotherm by RP-HPLC experiments and Aspen simulation.

Humans have different characteristics such as skin colors, physical constitutions, and disease incidences. Most of them are caused by single nucleotide polymorphism (SNP), a DNA sequence variation occurring when a single nucleotide in the genome differs between members of species. For multiple SNP analysis, we studied a technique named single base extension-end labeled free solution electrophoresis (SBE-ELFSE). ELFSE needs drag-tags that give uniform drag force to target DNAs in free solution. General requirements for the optimum drag-tags are high water-solubility, monodispersity, and charge neutrality. To fulfill these requirements, we designed and produced repetitive polypeptide drag-tags. We designed repetitive polypeptides sequenced as [GAGASGAGATGAGASGAGAT]n and expressed them with protein expression vector, pET32a. We are attempting SNP analysis using these repetitive polypeptides conjugated with DNAs which have different sizes. We expect that fast and accurate SNP analysis is accomplished based on our study.

Proteins have wide applications in the medical, industrial and agricultural fields. They are usually produced in huge quantities in host cells such as Escherichia coli. However, high-level expression of recombinant proteins in E. coli often results in accumulation of insoluble aggregates known as inclusion bodies (IBs), thus requiring further solubilization, refolding and purification procedures to achieve functionally active products. Molecular chaperones have been applied successfully to refold various proteins both in vivo and in vitro, opening a new era in protein refolding. However, the exact function of individual molecular chaperones and the interaction between target protein and molecular chaperones are still unclear. We demonstrated that refolding cocktail comprising ClpB/DnaKJE with or without the presence of ATP regeneration system and a refolding additive, PEG, could significantly enhance the refolding efficiency of heat-denatured malate dehyrogenase (MDH). To further clarify the individual or synergistic roles of each chaperone, various molecular chaperones including His-ClpB, His-DnaK, His-DnaJ, His-GrpE, His-Gro EL, His- Gro ES, and His-Trigger Factor were recently cloned, expressed and purified through IMAC, respectively. Currently, chaperoning efficiencies of these His-tagged molecular chaperones are being studied systematically using heat- or chemicaldenatured model proteins of different aggregate size. It is expected that our study will provide a better understanding of chaperone-assisted refolding process, thereby facilitating engineering implementation of a novel refolding strategy, folding-likerefolding approach based on refolding cocktail.

In this study, we aimed to isolate agarase using chromatographic method from the fermentation broth of bacteria, GNUM-0812-2, which was isolated and selected from several sites along the coastal regions of Tongyeong and Jindong located at South Korea. After 4-day culture of this strain, crude enzyme extracts were obtained from supernatant by ammonium sulfate precipitation followed by membrane dialysis. A anion exchange resins, DEAE sepharoseTM(Amersham Biosciences, Sweden) were packed into column and then adsorption and elution conditions of enzyme were optimized in batch-type experiments. Adsorptions of enzyme were investigated at different equilibrium pH and pH 7.2 was found to be suitable. The ratios between resin and amount of loaded enzyme were also optimized. The elution performances of bind enzyme were compared at different concentrations of NaCl in elution buffer. Enzyme activity was monitored quickly by high throughput screening system developed by our group. Protein concentration was measured using commercial Assay kit (PIERCE). SDS-PAGE and Zymographies [1] were used for identifying the purified agarase