Characterization of aqueous two-phase systems based on polydisperse phase forming polymers: Enzymatic hydrolysis of starch in a PEG-starch aqueous two-phase system

A new method for determination of the binodial of an aqueous two-phase system, using spectrophotometric measurements of the turbidity, is described in this article. The method is especially designed to characterize phase systems composed of polydisperse phase components. It gives information about the area representing the transition from homogeneous solution to a two-phase system. The two-phase systems studied were first a conventional Dextran T40-polyethylene glycol 20M (PEG 20M) system, then a less-well-defined phase system based on PEG 20M and partially hydrolyzed starch. The PEG 20M-starch system could be changed with respect to the volume ratio between the phases with time using hydrolytic enzymes, and the possibility of using the glucose released from the starch polymer is pointed out. Then the system is transformed to an extractive biconversion where the bottom phase polymer also served as the substrate.     

Enzymatic cellulose hydrolysis in an attrition bioreactor combined with an aqueous two-phase system

Cellulose was hydrolyzed in the attrition bioreactor (ABR) with enzyme recycling by employing an aqueous two-phase system (composed of dextran and polyethylene glycol) and an ultrafiltration unit. The ABR combines wet ball milling and enzymatic hydrolysis in one process step. The cellulase enzymes were more stable in the two-phase system than in the normal buffer solution. With the initial substrate concentration (Solka Floe BW200) of 40 g/L and intermittent addition of cellulose, sugar was semicontinuously produced at dilution rates of 0.06 h(-1) and productivities of 2.1 g/L h, which is approximately a 10-fold increase of the previously reported values performed in a regular stirred reactor with an aqueous two-phase system. The conversion of the substrate was 86%.     

Integration of bioconversion and downstream processing: starch hydrolysis in an aqueous two-phase system

Integration of bioconversion and the first step(s) of down stream processing can be used as a means to increase the productivity of bioprocesses. This integration also gives the possibility to run the bioconversion in a continuous mode. We demonstrate the use of an aqueous two-phase system in combination with ultrafiltration to accomplish this. Conversion of native starch to glucose by alpha-amylase and glucoamylase was carried out in an aqueous two-phase system in connection with a membrane filtration unit. In this way, a continuous stream of glucose in buffer solution was obtained; the phase-forming polymers as well as the starch-degrading enzymes were recycled, and clogging of the ultrafiltration membrane was avoided. The process was carried out continuously in a mixer-settler reactor for a period of 8 days. The enzyme activities in the top and bottom phases and in the mixing chamber were monitored intermittently throughout the experiment. The optimum pH, temperature, and ionic strength for the activity of the enzyme mixture were determined. The settling time of phase systems containing varying amounts of PEG, crude dextran, and solid starch was studied. The activity and stability of enzyme mixtures was studied both in buffer medium and in the medium containing the polymers. The enzymes were found to be more active and stable in medium containing polymers than in the buffer solutions.     

The use of fractionation in a polyethylene glycol-dextran two-phase system for the testing and purification of restriction endonucleases

A simple procedure was developed for testing and purification of restriction endonucleases Msp I, Pst I, Bam HI, Pvu I, Pvu II that includes biomass destruction, fractionation of cell-free extracts in the aqueous two-phase (polyethylene glycol-dextran) system and chromatography oh phosphocellulose. Optimal conditions for the fractionation of Msp I, Pst I, Bam HI, Pvu II, EcoR I, EcoR II, BspR I, Alu I were chosen. For separation of Pvu I and Pvu II gel filtration through biogel A-0.5 m was additionally introduced.     

Enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis

The enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis (commercial enzyme Termamyl 300 L Type DX) have been experimentally studied at pH 7.5, within the temperature range of 37-75 degrees C, at initial substrate concentrations of between 0.25 and 2.00 g/L, and enzyme concentrations of between 0.575 x 10(-4) and 13.8 x 10(-4) g/L. To follow the reaction a procedure based on the iodometric method for measuring alpha-amylase activity was used. The kinetics of the enzymatic hydrolysis was fitted to the Michaelis-Menten equation using the integral method, taking into account that the thermal deactivation of the enzyme follows a second-order kinetic. These parameters were fitted to the Arrhenius equation obtaining activation energies of 24.4 and 41.7 kJ/mol and preexponential factors of 734.9 g/L and 1.74 x 10(8) min(-1) for K(M) and k, respectively.     

Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system

In this paper, an integrated process involving the mixed ionic liquids/water two-phase system (MILWS) is proposed to improve the efficiency for enzymatic hydrolysis of penicillin G. First, hydrophilic [C4mim]BF4 (1-butyl-3-methylimidazolium tetrafluoraborate) and NaH2PO4 salt form an ionic liquids aqueous two-phase system (ILATPS), which could extract penicillin from its fermentation broth efficiently. Second, a hydrophobic [C4mim]PF6 (1-butyl-3-methylimidazolium hexafluoraphosphate) is introduced into the ionic liquids-rich phase of ILATPS containing penicillin and converses it into MILWS. Penicillin is hydrolyzed by penicillin acylase in the water phase of MILWS at pH 5. The byproduct phenylacetic acid (PAA) is partitioned into the ionic liquids mixture phase, while the intended product 6-aminopenicillanic acid (6-APA) is precipitated at this pH. In comparison with a similar butyl acetate/water system (BAWS) at pH 4, MILWS exhibits two advantages. (1) The selectivity between PAA and penicillin is greatly optimized at pH 5 by varying the mole ratio of [C4mim]PF6/[C4mim]BF4 in MILWS, whereas in BAWS the unalterable nature of the organic solvent restricts the optimized pH for maximum selectivity between PAA and penicillin at pH 4. (2) The pH for 6-APA precipitation in BAWS is 4, whereas it shifts to pH 5 in MILWS due to the complexation between negatively charged 6-APA and the cationic surface of the ionic liquids micelle. As a result, the removal of the two products from the enzyme sphere at relatively high pH is permitted in MILWS, which is beneficial for enzymatic activity and stability in comparison with the acidic pH 4 environment in BAWS.     

Novel polyethylene glycol/levan aqueous two-phase system for protein partitioning

Biotechnology Techniques -     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. Partition of cellulases from Trichoderma reesei

The partitioning of endo-beta-glucanase, exo-beta-glucanase, and beta-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one-sidedly partitioning the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enzyme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-beta-glucanase to 0.02 and for beta-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose, At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-beta-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.     

Metal affinity extraction of human hemoglobin in an aqueous polyethylene glycol-sodium sulfate two-phase system

Summary We have developed a protein extraction technique which uses metal affinity ligands in PEG/salt aqueous two-phase systems. Cu(II)IDA-PEG will partition proteins according to their surface histidine contents in two-phase systems formed from sodium sulfate and polyethylene glycol. The nearly complete separation of human hemoglobin and human serum albumin in a single stage is presented as a demonstration of the effectiveness of metal affinity extraction in PEG/salt systems.     

Steroid bioconversion in a novel aqueous two-phase system

A novel aqueous two-phase system, based on polyethyleneglycol (PEG) and monosodium glutamate, was tested for the 1-dehydrogenation of hydrocortisone-based substrates. This system led to higher substrate solubilities and biocatalyst/steroid separation levels when compared with alternative systems. The addition of short-chain monohydric alcohols resulted in higher solubilities and more favourable partition coefficients for the tested substrates. Bioconversion activities in PEG/glutamate systems with 2,5% (v/v) methanol were comparable to those measured in monophasic buffer-methanol medium.     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. II. Semicontinuous conversion of a model substrate, solka floc BW 200

A model substrate, Solka Floc BW 200, was semicontinuously hydrolyzed in an aqueous two-phase system based on crude dextran and polyethylene glycol over a period of more than 450 h. With an initial concentration of 75 g/L and intermittent addition of cellulose an average concentration of 50 g/L sugar was semicontinuously produced at dilution rates of 0.006-0.012 h(-1). The conversion of substrate varied between 49 and 66%. The enzyme consumption measured as FPU/g reducing sugar (RS) produced could be reduced by a factor two when compared to a batch process since, in the aqueous two phase system investigated, the enzyme could be recycled two times.     

Enzymatic preparation of 20 beta-hydroxysteroids in a two-phase system.

The behavior of 20 beta-hydroxysteroid dehydrogenase in a two-phase system consisting of an emulsion of water with an immiscible organic solvent was investigated. The effect of several organic solvents on the stability, activity, and kinetic profile of the enzyme was considered. The most suitable system for carrying out the enzymatic reaction proved to be water-butyl acetate. The production of high quantitied of 20 beta-hydroxysteroids in 100% yield using catalytic amount of cofactor was achieved by coupling the 20 beta-hydroxysteroid dehydrogenase-and the alcohol dehydrogenase-catalyzed reactions.     

Enzymatic production of enantiopure ketoprofen in a solvent-free two-phase system

Enzymatic hydrolysis conducted in a medium composed of solely substrate is considered to resolve racemic ketoprofen esters. In a system composed of two components, the pure liquid substrate (organic phase) and water (aqueous phase), hydrolysis products can be efficiently removed from the reaction mixtures. Accordingly, in this study we designed a solvent-free two-phase system for the enantioselective enzymatic hydrolysis of ketoprofen esters. In order to further optimize this system, the influences of various factors, such as the pH of the aqueous phase, temperature, enzyme content, and the alcohol chain length of esters, were examined on conversion and enantiomeric excess. 1N NaHCO₃ was identified as the most efficient aqueous phase for the extraction of ketoprofen. Changes in the amount of enzyme did not significantly affect the maximum conversion or the enantiomeric excess. On the other hand, ketoprofen esters with shorter alcohol chains displayed higher initial reaction rates and conversions in solventless media. In the case of ketoprofen propyl ester, for example, the productivity of the solvent-free two-phase system was about 10–100 times higher than that obtained to date for ketoprofen esterification with alcohols in organic solvents. The enantioselectivities obtained in solvent-free media were similar to those obtained for the enantioselective esterification of ketoprofen in organic solvents.     

双水相生物催化技术的研究进展

双水相生物催化是一种高效且易于放大的生物催化技术,可以有效解决传统生物催化过程中产物浓度低、产物和副产物的抑制、以及生物催化剂难以回收等缺点。介绍了该技术的操作工艺及设备研究及其在抗生素、激素、肽类和有机化舍物酶法合成中的应用研究现状,展望了该技术的可能发展方向。     

Enzymatic dehydrogenation of steroids by beta-hydroxysteroid dehydrogenase in a two-phase system

The catalytic activity of β-hydroxysteroid dehydrogenase on 3β- or 17β-hydroxysteroids was studied when the reaction was carried out in a two-phase system where the enzyme and the cofactor were in the water phase and the substrate was predominantly in the organic phase.With some organic solvents the enzyme displayed its activity over a long period. Large amounts of steroids could be transformed in small volumes, using low enzyme concentrations.The kinetics of the reaction in the two-phase system as a function of substrate, enzyme concentrations, and pH as well as the equilibrium position were examined.     

A kinetic expression for hydrolysis of soluble starch by glucoamylase

As the hydrolysis of starch by glucoamylase proceeds with stepwise removal of glucose units from the nonreducing ends of the starch chain, the number of available substrate molecules is essentially unchanged in the course of the degradation. In view of this aspect, a simple practical kinetic expression, which consists of a modified Michaelis-Menten form with product inhibition, is presented for the hydrolysis of soluble starch. It is assumed that the values of kinetic parameters V(m) and K(m) vary linearly from the values for starch toward those for maltose. The applicability of this kinetic expression is verified through the simulation with the experimental results for the hydrolysis of two soluble starches with different average molecular weights of 3 x 10(4) and 3 x 10(6).     

Extractive bioconversions in aqueous two-phase systems: enzymatic hydrolysis of casein proteins

Partition coefficients in poly(ethylene glycol)/dextran aqueous two-phase systems are reported for mixed-casein and its components, alpha, beta and kappa casein. Rates of casein proteolysis by alpha-chymotrypsin and by trypsin are reported in single-phase and aqueous two-phase reactor systems. The advantages resulting from selective partitioning of substrates, enzymes, and products are examined in terms of relative volumetric reaction rates.     

Enzymatic hydrolysis of soluble cellulose derivatives as measured by changes in viscosity

Observation of changes in fluidity is presented as a method for following the enzymatic hydrolysis of soluble cellulose derivatives. The activity of different cell-free enzyme preparations may be compared by this method, providing certain precautions are observed. In general, results obtained by use of the fluidity method are similar to those obtained using the reducing sugar technique, indicating that the same enzyme system is measured by the two methods. Changes in the DP of the substratum may be followed within certain limits of molecular size. Results indicate that a random splitting of CMC occurs during enzymatic hydrolysis, with a concomitant decrease in intrinsic viscosity and an increase in reducing sugars. Certain inadequacies of the cellulose-cellobiose-glucose theory, together with more recent findings, have led to the postulation of an alternate explanation of the mechanism of cellulose hydrolysis.