The reciprocating size exclusion chromatography (RSEC) was operated with swing between two temperatures in a synchronous way with flow direction to recover a large solute on-line from the mixture, in addition to the small solute concentration. The concentration of small solutes in RSEC with a temperature swing was made possible by taking advantage of the temperature-dependent swelling properties of the porous gel. After 7 cycles of frontal mode operation, 76% of Blue Dextran in the feed was recovered and nickel nitrate in the feed reservoir was concentrated by 13%.
Plasmalogen that contains choline was concentrated from bovine heart to 95% purity from an initial value of 45%, and ethanolamine-containing plasmalogen from bovine brain to 99% purity from initial 64% using phospholipase A1 from Serratia sp. MK1 in a two-phase system. In the two-phase system, Ca2+ was not required and Tris/HCl buffer and butyl acetate were used as an aqueous phase and a solvent phase of the two-phase system, respectively.
Solutes of different size in a mixture solution were separated on-line, using a semi-continuous reciprocating size exclusion chromatography. The band of fast-moving large molecules was isolated during the first half cycle, while the band of slow-moving small molecules was isolated during the second half cycle. After 7 cycles of frontal mode operation, 89% of the Blue Dextran in the feed was isolated as a pure solution. Vitamin B12 of constant concentration was also isolated as a pure solution.
A ColE1-compatible vector was constructed to expressed His-tagged fusion protein in Escherichia coli cells. The vector designated pYH31 is derived from pREP4 and pQE31. It was designed to express simultaneously in the same cell, a His-tagged fused protein and a non His-tagged protein from separate plasmids. The vector was used to express both subunits of the biphenyl dioxygenase oxygenase component together in the same E. coli clone.
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.
A mathematical model was found to represent the enzyme yield (Y) as a function of pH (X1) and temperature (X2): Y=36.89+10.83X1−12.17X2−6X1X2−17.24 X12−13.24X22. The optimum values for pH and temperature to attain the maximum b-xylosidase recovery (42%) were 3.3 and 24 °C, respectively.
Expression of Brevibacillus brevis CD162 cyclodextrin glycosyltransferase (CGTase) gene using pET22b(+) vector in Escherichia coli BL21(DE3) resulted in the formation of inactive inclusion bodies under the usual induction conditions. However, by lowering the induction temperature to 30 °C and/or adding 0.5 M mannitol as an osmolyte, the formation of insoluble aggregates was prevented and about a 34-fold increase (8.51 U ml−1) in biologically active soluble form was achieved after 6 h induction. The active CGTase enzyme was estimated to comprise as much as 24% of the total soluble proteins. In addition, other polyols such as glycerol, erythritol, xylitol, sorbitol, and arabitol showed similar effects with mannitol on the production of active CGTase enzyme.
A cyanobacterium, Synechocystis sp. PCC 6803, was immobilized by entrapment in poly(vinylalcohol) bearing styrylpyridinium groups. Its properties in a single-compartment micro-photoelectrochemical cell using platinum electrodes in potentiosatic mode were compared with the native material. The operational activity was measured in the presence of an electrolytic solution containing 20 mM sodium phosphate, 0.15 mM NaCl and 1 mM MgCl2. The best conditions of use are pH 7.0, 38 °C and a 2,5-dichlorobenzoquinone concentration equal to 350 μM with native cyanobacteria or pH 6.5, 25 °C and 500 μM 2,5-dichlorobenzoquinone after entrapment. Using this procedure, the photocurrent could be inhibited by pollutants such as Diuron or HgCl2. After entrapment, the detection limits (corresponding to a 10% inhibition) were respectively 0.5 μM and 50 μM for Diuron and HgCl2 after five minutes of incubation. A permeabilization technique was used to increase sensitivity of the procedure to the detection of HgCl2 (25% inhibition with 50 μM after five minutes of incubation).
The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.
New cover filter sampling unit was tested in fungal fermentations in the presence of antifoam agents producing sticky fungal polysaccharides. Cover filter unit long-term filter capacity depends on the intensity of the pumping capacity of impeller as well as on the position of the filter unit.
To clarify the dynamic behavior of the anaerobic acid reactor in response to pH changes, a continuous cultivation was performed. By stepwise shifting the culture pH in the acid reactor from 6.0 to 8.0, the main products were changed from butyric acid to acetic and propionic acids. This phenomenon was reproducible, reversible and was not affected by the dilution rate. It was considered that the dominant microbial populations changed in the acid reactor due to the pH shift.
A sugar mixture containing fructooligosaccharides and isomaltooligo-saccharides was produced. Sucrose was converted to fructooligosaccharides by a commercial enzyme preparation. The sugar mixture contained kestose (33.5%), nystose (13.3%), fructofuranosyl nystose (5.7%), glucose (20.9%), and unreacted sucrose (26.6%). The unreacted sucrose was converted to isomaltooligosaccharides by reacting the sugar mixture with Leuconostoc mesenteroides B-512FM dextransucrase. The final product comprised fructooligosaccharides (kestose, nystose, fructofuranosyl nystose), isomaltooligosaccharides (isomaltose through isomaltodecaose), glucose, and fructose.
Coagulation of Skeletonema costatum biomass was induced at alkaline pH in enriched sea-water to concentrate the microalgal suspensions by decantation. Incubation of the concentrated cultures at 15 °C after return at pH 7.5 preserved cell viability and increased the yield of lipid extraction and the 20:5(n-3) fatty acid content of the diatom biomass.
We propose a novel effective method for a continuous peptide synthesis in an aqueous/organic biphasic medium using a pulsed column reactor. N-Formyl-l-aspartyl-l-phenylalanine methyl ester was enzymatically synthesized continuously. With this extractive method using a pulsed column reactor, we can synthesize peptides with a stable performance even if a peptide (or a peptide-amino acid complex) is precipitated due to its high hydrophobicity.
A rapid, continuous, colorimetric enzyme assay for penicillin G acylase has been developed. The assay measures the formation of the acidic products of penicillin G hydrolysis by following the decrease in pH using Phenol Red as an indicator. The activity measured is directly proportional to the amount of enzyme added to the assay, having a linear relationship with an R2 value of 0.9994.
A new airlift reactor was used to culture Catharanthus roseus cells, in which the draft tube was made up of polyurethane foam and acted as the immobilizing matrix. The reactor was connected in series to an adsorbent column with a neutral polymeric resin which absorbs these alkaloids. The synthesis of alkaloid was stimulated by adding the resin column and the total content of alkaloid secreted by cells reached 380 mg/L, which was 4.5 times of that in the control experiment. Meanwhile, most of the intracellular alkaloid produced by Catharanthus roseus was secreted into the medium.
To assess the biopotential for remediation, microbiological ex situ activities were examined in a subsurface site contaminated in the past with fluctuating hydrocarbon amounts. A novel method for estimating the proportion of hydrocarbon degraders was used. There was not in all cases a correlation between the results of chemical analysis and microbiological activities. However, the level of soil respiration (between 440 and 515 μg CO2/g dry weight h−1) correlated well with hydrocarbon contamination of the subsurface.
A precultivation technique for microorganisms exhibiting overflow metabolism is presented. It is based on a low initial medium volume in the fermenter. The medium feed contains all the nutrients, but is diluted with respect to sugar, the concentration of which determines the biomass concentration at the end of the preculture. By this method, effects of varying activity of the inocula from shake flask cultures are minimised and the metabolic state, i.e. oxido-reductive, oxidative growth on sugar plus overflow metabolite or oxidative growth on sugar alone, can be controlled at the start of the main fermentation.
Galactose oxidase was co-immobilised with peroxidase by drop-coating on the surface of a graphite electrode with adsorbed ferrocene. This system offers low detection limit – 0.51 mg galactose l−1 and fast response: 44 s in phosphate buffer or 25 s in borate buffer. Optimal working potential for galactose detection was 150 mV vs. SCE (saturated calomel electrode) with optimal pH of 7.85. The storage stability was highly improved, more than 12 times in comparison to control without stabilisers, by addition of DEAE-dextran and inositol. During repeated assays for 5.25 h, signal dropped only to 95% of original one. The response was linear in phosphate buffer in the range 1–110 mg l−1, while in borate buffer linear range was extended to 3–210 mg l−1 because of chelating effect of borate.
Each of the four nucleotides (A, C, G and T) was introduced as the base following the stop codon to investigate the effect of this fourth base on translational termination efficiency during the heterologous expression of human erythropoietin (hEPO) in E. coli. The efficiency of peptide chain termination in E. coli was markedly dependent on the fourth base. The choice of the fourth base was crucial to prevent the expression of undesirable proteins due to the translational infidelity such as frameshifting and stop codon read-through, and translational termination efficiency could be improved with adenosine as the fourth base.
