Elis Andersson

Bioconversions in aqueous two-phase systems

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.

α-amylase production in aqueous two-phase systems with Bacillus subtilis

Abstract The production of α-amylase (1,4-α- d -glucan glucanohydrolase, EC 3.2.1.1) by Bacillus subtilis has been studied in repeated batch fermentations in aqueous two-phase systems. In a phase system composed of PEG 600, 8% (w/w), PEG 3350, 5% (w/w)/Dextran T 500, 2% (w/w), 82% of the enzyme partitioned to the top phase. The enzyme concentration in the top phase reached 0.85–1.35 U ml−1 during the fermentations compared with 0.58 U ml−1 in the reference fermentation. In the phase system composed of PEG 3350, 9% (w/w)/Dextran T 500, 2% (w/w), 73% of the enzyme partitioned to the top phase. However, the enzyme concentration in this phase system reached only 0.35 U ml−1 in the top phase. The bacterial cells were microscopically observed to partition totally to the bottom phase in the aqueous two-phase system used. The results are discussed in relation to recirculation of cells by immobilizing to a solid matrix. Extraction of the product to the top phase and the effect of the phase polymers, especially PEG, on the production are also discussed.

Enzymatic conversion in aqueous two-phase systems: deacylation of benzylpenicillin to 6-aminopenicillanic acid with penicillin acylase

The conversion of benzylpenicillin (BP) to 6-aminopenicillanic acid (6-APA) using penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) has been studied in aqueous two-phase systems. In a system composed of 8.9% (w/w) PEG 20000/7.6% (w/w) potassium phosphate the enzyme was almost completely partitioned to the bottom phase (K < 0.01), which allowed repeated batch conversions, recirculating the enzyme several times. The initial specific productivities were 0.31–1.47 μmol 6-APA mg protein−1 min−1 in repeated conversions over five steps. The yield obtained from the top phase was 0.47–0.71 mol 6-APA mol BP−1. The results are discussed in relation to recirculating the enzyme by immobilizing it to a solid matrix. Despite the high phosphate concentration in the bottom phase the system needs to be titrated in order for the reaction to proceed. Titration of the top phase alone protected the enzyme from denaturation by strong alkali used for the titration.

Enzyme action in polymer and salt solutions. I. Stability of penicillin acylase in poly(ethylene glycol) and potassium phosphate solutions in relation to water activity

The stability of penicillin acylase (penicillin aminohydrolase, EC 3.5.1.11) was studied in poly(ethylene glycol) and potassium phosphate solutions. Enzyme stability measured as the half-life of the enzymatic activity and the transition temperature determined by differential scanning calorimetry, correlated well. The enzyme stability could not be related to the water activity as a measure of solute-solvent interaction. It seems to be related more to the concentration of the solutes and much less to the molecular weight of poly(ethylene glycol). The stabilizing effect of poly(ethylene glycol) is also discussed in terms of poly(ethylene glycol)-protein interactions.

Solvent Production by Clostridium Acetobutylicum in Aqueous Two-Phase Systems

One way to overcome product inhibition in biological conversions is by means of extraction. This has proven successful, with the aid of aqueous two-phase systems, in the bioconversion of cellulose (1). Aqueous two-phase systems are especially suitable for biological conversion processes because the high water content of both phases makes them compatible with biological material. This paper concerns the production of acetone and butanol in aqueous two-phase systems using Clostridium acetobutylicum. This system was chosen because at a total solvent concentration of less than 2% the products have a strong inhibitory effect on the microorganism.

High concentrations of PEG as a possible uncoupler of the proton motive force: α-Amylase production with Bacillus amyloliquefaciens in aqueous two-phase systems and PEG solutions

Summaryα-Amylase production with Bacillus amyloliquefaciens was investigated in two different aqueous two-phase systems and in polyethylene glycol (PEG) 600 solutions of different concentrations. The cells did not partition totally to the bottom phases of the aqueous two-phase systems, and the enzyme production was repressed in both systems as well as in PEG 600 solutions. Concomitantly, the cultivation time was prolonged, indicating an increased maintenance metabolism. The surface properties of cells grown in 200 g/kg PEG 600 were investigated by phase partitioning and compared to the surface properties of Bacillus subtilis, which under these conditions showed increased α-amylase production. The cells of B. amyloliquefaciens partitioned to the top phase in a PEG-dextran system, whereas the cells of B. subtilis partitioned to the bottom phase. The results are discussed in relation to water activity, oxygen transfer rate and PEG-induced changes of the surface properties of the cells. The possible role of PEG as an uncoupler of the proton motive force at high concentrations is also discussed.

α-Amylase production with Bacillus subtilis in the presence of PEG and surfactants

Summaryα-Amylase production with Bacillus subtilis was studied in the presence of PEG 600 and PEG 3350 as well as different surfactants: Trixon X-100, Tween 80, CTAB (cetylammonium-bromide) and SDS (sodiumdodecylsulphate) at concentrations resulting in comparable decreases in surface tension. Only PEG 600, at a concentration of 200 g/kg, was found to increase the enzyme production. Cell growth estimated as optical density at 620 nm and viable counts were not influenced by either PEG or surfactants. The results are discussed in relation to α-amylase production in aqueous two-phase systems.

The Influence of PEG on α-Amylase Production with Bacillus Speciesa

The recirculation of cells in biotechnical processes can generally be considered to have the following advantages: a clean cell-free product stream is obtained, substrate is saved, high cell densities are obtained, lower amounts of waste materials are produced, and a continuous process can be designed. However, these advantages must be weighed against the effects of recirculation on process stability, activity, product concentration, productivity, and the cost for the recirculation. Here, the recirculation in aqueous two-phase systems with cells of Bacillus sp. for a-amylase production will be discussed, with special focus on clean product streams, product concentrations, and productivities, and on how the polymers of the phase systems influence the production.

Synthesis of Oligosaccharides and Peptides using Hydrolytic Enzymes at Decreased Water Activity

It has frequently been observed in studies of enzymatic hydrolysis of carbohydrates that the enzymes show transglycosylative activity.I4 Instead of completely hydrolyzing the carbohydrates to monosaccharides the glycolytic enzymes also catalyze the formation of oligosaccharides. Similarly, proteolytic enzymes have been used to catalyze the so-called plastein synthesis. The reaction has been much studied in order to improve the functional properties of protein hydrolysates, especially for the removal of bitter The striking feature in these reports is that the synthesis reactions are always observed a t unusually high concentrations of sugars or small peptides. Considering the enzymatic reactions