Affinity separation by protein conjugated IgG in aqueous two-phase systems using horseradish peroxidase as a ligand carrier

A novel affinity separation method in an aqueous two-phase system (ATPS) is suggested, using protein conjugated IgG as a ligand. For verification of the proposed approach, horseradish peroxidase (HRP) and human IgG was used as a ligand carrier and affinity ligand, respectively. The partition of the affinity ligand, human IgG, was controlled by the conjugation of HRP. Two ATPSs, one consisting of potassium phosphate (15%, w/w) and polyethylene glycol (PEG, M.W. 1450, 10%, w/w) and the other of dextran T500 (5%, w/w) and PEG (M.W. 8000, 5%, w/w), were used. The conjugated human IgG-HRP favored a PEG-rich top phase, whereas human IgG, rabbit anti-human IgG and goat anti-mouse IgG preferred a salt or dextran-rich bottom phase. Using the conjugated human IgG-HRP, rabbit anti-human IgG was successfully separated into a PEG-rich top phase from the mixture with goat anti-mouse IgG. The appropriate molar ratio between human IgG-HRP and rabbit anti-human IgG was around 3:1 and 1:1 for the salt and dextran-based ATPS, respectively. The dextran-based ATPS showed a better recovery yield and purity than the salt-based ATPS for the range of test conditions employed in this experiment. The yield and purity of the recovered rabbit anti-human IgG were 90.8 and 87.7%, respectively, in the dextran-based ATPS, while those in the salt-based ATPS were 78.2 and 73.2%.     

Design of acoustic cell settler for filtering and recycling microbial cells

An acoustic cell settler (ACS) using ultrasound at cells of 3 MHz was used to recycle Saccharomyces cerevisiae in a fermenter. The locations of both the inlet and outlet in the acoustic cell settler, which have a relatively long distance between the transducer and reflector, were optimized. A tilted settler was designed to make up for the defect in the horizontal ACS, which has a low recovery ratio. The tilted ACS gave a recovery ratio of yeast cells of about 5 during the most period of operation, which was twice that of the horizontal ACS.     

Interconversion and disproportionation reaction among cyclodextrin homologues in an aqueous two-phase-forming solution

Interconversion reactions of cyclodextrin glycosyltransferase (CGTase) among cyclodextrin (CD) homologues were experimentally investigated using each CD as a substrate in an aqueous, two-phase-forming polymer solution of dextran and polyethylene glycol. Degradation rate of -CD was highest and that of -CD was lowest among -, - and -CD with Bacillus macerans CGTase. Degradation of each CD was accelerated with dextran, while decelerated with polyethylene glycol.     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

Influence of ionic liquids as additives on sol–gel immobilized lipase

The immobilization of lipase from Candida rugosa, using ionic liquids as additives to protect the inactivation of lipase by released alcohol and shrinking of gel during sol–gel process, was investigated. The influence of various factors, such as structure of ionic liquids, content of ionic liquids and types of precursor in the sol–gel process on the activity and stability of immobilized lipase was also studied. The highest hydrolytic activity of immobilized lipase was obtained when the hydrophilic ionic liquid, [C₂mim][BF₄], was used as an additive, while the highest stability of immobilized lipase was obtained by using hydrophobic ionic liquid, [C₁₆mim][Tf₂N]. Therefore, the binary mixtures of these ionic liquids as additives were used to obtain the optimal immobilized lipase, which shows both high activity and stability. The hydrolysis and esterification activities of lipase co-immobilized with the mixture of 1:1 at molar ratio of [C₂mim][BF₄] and [C₁₆mim][Tf₂N] were 10-fold and 14-fold greater than in silica gel without ionic liquids (ILs), respectively. After 5 days incubation of this immobilized lipase in n-hexane at 50 °C, 84% of initial activity was remained, while the residual activity of the lipase immobilized without ILs was 28%.     

Adverse effect of chloride impurities on lipase-catalyzed transesterifications in ionic liquids

The adverse influence of chloride impurities on the lipase-catalyzed transesterification in ionic liquid is described. The activity of lipase from Rhizomucor miehei exponentially decreased with increasing Cl⁻ content in 1-octyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl] amide, [Omim][Tf₂N], and the activity of lipase in [Omim][Tf₂N] mixture containing 2% [Omim] [Cl] was only about 2% of the activity in pure [Omim][Tf₂N]. The activity of lipase from Candidantarctica linearly decreased at about 5% with every 1% increase in [Omim][Cl] with there being no activity in [Omim][Tf₂N] containing about 20% [Omim][Cl].     

Effect of ionic liquids on enzymatic synthesis of caffeic acid phenethyl ester

Although caffeic acid phenethyl ester (CAPE), an active flavonoid, plays an important role in the antioxidant activity of honeybee propolis, the isolation of CAPE from honeybee propolis is time-consuming due to wide variety of impurities present. Therefore, biochemical method to synthesize CAPE was investigated in this study. Since ionic liquids (ILs) possess some unique characteristics as appreciated alternatives to conventional solvents for certain biotransformation, the effect of ILs as reaction media for enzymatic synthesis of CAPE was assessed. Several factors including substrate molar ratio, and reaction temperature affecting the conversion yield of lipase-catalyzed CAPE synthesis were also investigated. Reaction yields were significantly higher in hydrophobic ILs than in hydrophilic ILs (almost zero). Among nine hydrophobic ILs tested, the highest conversion of synthetic reaction was obtained in 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]). A reaction temperature of 70 °C was found to give high conversion. In addition, optimal substrate molar ratio between phenethyl alcohol and caffeic acid (CA) was decreased significantly from 92:1 to 30:1 when ILs were used instead of isooctane.     

On-line recovery of large molecules from mixtures using reciprocating size exclusion chromatography

Blue Dextran, a standard large molecule, was successfully recovered on-line from the aqueous mixture solution with nickel nitrate using a novel reciprocating size exclusion chromatography. After 7 cycles of repeating operations of frontal mode, 70% of Blue Dextran in the feed was isolated as a pure solution. On-line recovery of large molecules from the mixture is an unusual trial, comparing to the routine practice of filtration where small molecule is isolated from the mixture.     

PCR analysis of four length-polymorphic loci in Korean population for genotyping

On human chromosomes, a short sequence of DNA is known to repeat a number of times. These repeats are called variable number of tandem repeat (VNTR) or short tandem repeat (STR) which has a short repeat core. VNTR and STR are used in the field of forensic science, evolution, and anthropology. In this work, we examined allele frequencies of one VNTR (YNZ22) and three STRs (NeuR, D21S11, Humth01) in a Korean population sample by polymerase chain reaction (PCR) followed by high-resolution polyacrylamide gel electrophoresis (PAGE) with silver stain. Subsequently, the polymorphism information content (PIC) was calculated: the highest PIC was observed in the NeuR locus (0.95680) and lowest in the Humth01 locus (0.75809).     

“Bottom-up” approach for implementing nano/microstructure using biological and chemical interactions

The “Bottom-up” approach for implementing nano/microstructure using biological self-assembled systems has been investigated with tremendous interest by many researchers in the field of medical diagnostics, material synthesis, and nano/microelectronics. As a result, the techniques for achieving these systems have been extensively explored in recent years. The developed or developing techniques are based on many interdisciplinary areas such as biology, chemistry, physics, electrical engineering, mechanical engineering, and so on. In this paper, we review the fundamentals behind the self-assembly concepts and describe the state of art in the biological and chemical self-assembled systems for the implementation of nano/microstructures. These structures described in the paper can be applied to the implementation of hybrid biosensors, biochip, novel bio-mimetic materials, and nano/microelectronic devices.