Medium engineering of phenylethanoid transfructosylation catalysed by yeast ß-fructofuranosidase.

Tyrosol and hydroxytyrosol, by-products of olive oil production, are valuable substrates for enzymatic transglycosylation that can provide products with pharmaceutical potential. Phenylethanoid fructosides are produced from sucrose and phenylethanoids by the catalytic action of ß-fructofuranosidases. This work dealt with the potential of the most abundant ß-fructofuranosidase, baker's yeast invertase, for this bioconversion. The effects of sucrose and phenylethanoid concentrations were investigated with a focus on the selectivity of phenylethanoid transfructosylation and fructoside yields. For this purpose, initial rate and progress curve experiments were carried out for the initial (hydroxy)tyrosol and sucrose concentrations of 0.072-0.3 M and 1-2 M, respectively. Reaction courses exhibited either a maximum or plateau of fructoside yield in the range of about 10-18%. The addition of deep eutectic solvents was applied in the concentration range from 5 to 70% (v/v) to investigate the possibility of shifting the reaction equilibrium towards fructoside synthesis.

Design of immobilized biocatalyst and optimal conditions for tyrosol ß-galactoside production.

Tyrosol ß-galactoside (TG) is a phenylethanoid glycoside with proven neuroprotective properties. This work deals with its biocatalytic production from tyrosol and lactose using Aspergillus oryzae ß-galactosidase in immobilized form. Six commercial carriers were examined to find the optimal biocatalyst. Besides standard biocatalyst performance characteristics, adsorption of the hydrophobic substrate on immobilization carrier matrices was also investigated. The adsorption of tyrosol was significant, but it did not have adverse effects on TG production. On the contrary, TG yield was improved for some biocatalysts. A biocatalyst prepared by covalent binding of ß-galactosidase on an epoxy-activated carrier was used for detailed investigation of the effect of reaction conditions on glycoside production. Temperature had a surprisingly weak effect on the overall process rate. A lactose concentration of 0.83 M was found to be optimal to enhance TG formation. The impact of tyrosol concentration was rather complex. This substrate caused inhibition of all reactions. Its concentration had a strong effect on the hydrolysis of lactose and all products. Higher tyrosol concentrations, 30-40 g/L, were favorable as pseudo-equilibrium concentrations of TG and galactooligosaccharide were reached. Repeated batch results revealed excellent operational stability of the biocatalyst.

Screening of Commercial Enzymes for Transfructosylation of Tyrosol: Effect of Process Conditions and Reaction Network.

Enzymatic fructosylation of organic acceptors other than saccharides brings new possibilities to synthesize molecules that do not exist in nature. The introduction of fructosyl moiety may lead to glycosides possessing enhanced physicochemical and bioactive properties which could be useful in the pharmaceutical and cosmetic industry. In this work, the regioselective synthesis of tyrosol ß-d-fructofuranoside (TF) catalyzed by ß-fructofuranosidase is investigated. In the first step, 32 commercial enzyme preparations are screened for fructoside-hydrolyzing activity. The most active preparations are subsequently examined for fructofuranosyl transfer from sucrose to tyrosol. The best candidate, Novozym 188, is chosen to study the effect of reaction conditions on the product formation in a batch reactor. The effects of substrate concentration, temperature, pH, time, and enzyme dosage on the concentration of TF produced are studied using the design of experiments methodology. The maximal product concentration of 3.8 g L-1 is achieved for the sucrose concentration of 1.5 m, tyrosol concentration of 29 g L-1 , temperature of 41 °C, and pH 5.1. Besides the main transfructosylation reaction between sucrose and tyrosol, several side reactions take place. A reaction network includes also the formation of fructooligosaccharides and the hydrolysis of sucrose and all reaction products.

Chromatographic purification of recombinant human erythropoietin.

Recombinant human erythropoietin is a valuable therapeutic protein used in the treatment of several serious diseases. It exists in different isoforms and is produced by genetically modified mammalian cells such as Chinese hamster ovary or human embryonic kidney cells. As for other biopharmaceutical drugs, a key factor for its successful industrial production is to achieve a high degree of purity and to decrease the content of critical impurities to trace amounts. This goal is achieved in the separation sequence which substantial part is formed by chromatographic steps. Therefore, downstream processing forms an essential part of production costs. This review presents the overview of published separation sequences and, analyzes the use of different types of chromatographic media such as affinity, ion-exchange, reversed-phase, hydrophobic interaction, multimodal, and size-exclusion chromatography adsorbents. Their application is discussed with regard to their place in the purification stages generally denoted as capture, intermediate purification and polishing.

A Method of Early Phase Selection of Carrier for Aspergillus Oryzae ß-Galactosidase Immobilization for Galactooligosaccharides Production.

Early phase development of industrial immobilized biocatalysts has to address the selection of the best candidates from dozens of available carriers and binding methods. This work presents a simple selection method for the immobilization of industrial-grade Aspergillus oryzae ß-galactosidase suitable for the production of galactooligosaccharides. Immobilization efficiency and yield of a variety of immobilized biocatalysts are evaluated using simple activity measurements and mathematical modeling of intraparticle kinetics and mass transfer. Activated carriers and some ion-exchange carriers provided highly active biocatalysts and are therefore selected for the investigation of GOS production in the batch reactor. Biocatalysts exhibited significant differences in the yield of galactooligosaccharides and productivity of GOS formation. Based on these process performance criteria, cost considerations are made that allow further selection of the best biocatalyst for future process optimization. The presented method of biocatalyst selection can be useful for other industrial enzymes applications.

Application of a micromembrane chromatography module to the examination of protein adsorption equilibrium.

A micromembrane chromatography module based on a 96-well plate design and enabling fast and simple separation of small amounts of proteins was used for the determination of binding capacities of lysozyme, bovine serum albumin, ovalbumin, bovine γ-globulin, and human immunoglobulin G on a hydrophobic membrane Sartobind® Phenyl. Dependence of the binding capacity of the proteins on the ammonium sulfate concentration was examined in the salt concentration range of 0.5-2.0 mol L(-1). An exponential increase of the binding capacity was observed for all proteins. Simple Langmuir one-component isotherm was found suitable for the characterization of the effect of protein concentration in all cases. A combined effect of protein and salt concentrations was expressed via the Langmuir exponential isotherm and fitted the adsorption data for three of the investigated proteins well.

Characterization of pore structure of chromatographic adsorbents employed in separation of monoclonal antibodies using size-exclusion techniques.

Structural properties of commercial chromatographic adsorbents designated for separation of monoclonal antibodies were investigated using size-exclusion techniques. A batch technique provided the specific pore volumes distributed among small, medium and large pores. Inverse size-exclusion chromatography yielded the partition coefficients of dextran solute probes in medium pores. These data were fitted with one- and two-parametric models corresponding to different pore-size distribution functions and the suitability of the individual models for the characterization of the examined materials was then assessed. The reliability of estimation of the parameters of log-normal distribution of cylindrical pores was investigated. Large uncertainties and a strong correlation of the mean pore diameter and width of distribution function were observed.

Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans.

Efficient chromatographic separation of fructosyltransferase from Aureobasidium pullulans was achieved on a preparative scale using a weak anion-exchanger Sepabeads FP-DA. The recovery yield was about 70% and the purification factor reached a value of 28. The molecular weight of the enzyme determined by size-exclusion chromatography was 570,000. The enzyme exhibited both hydrolytic and transferase activity when the latter was higher in the whole concentration range and completely dominating at higher sucrose concentrations. It was found that sucrose was the only donor of fructosyl moiety used in the transfer reaction. The initial rate method was used for the investigation of the kinetics of the action of fructosyltransferase on sucrose in the concentration range 30-2,430 mM. The initial transfructosylation rate was well fitted with a linear function of the sucrose activity where the activity coefficient was an exponentially decreasing function of the sucrose concentration.

Adsorption equilibrium of fructosyltransferase on a weak anion-exchange resin.

The adsorption equilibrium of a glycoprotein, fructosyltransferase from Aureobasidium pullulans, on an anion-exchange resin, Sepabeads FP-DA activated with 0.1M NaOH, was investigated. The adsorption isotherms were determined at 20 degrees C in a phosphate-citrate buffer with pH 6.0 using the static method. Sodium chloride was used to adjust the ionic strength in the range from 0.0215 to 0.1215 mol dm(-3) which provided conditions varying from a weak effect of salt concentration on protein binding to its strong suppression. The equilibrium data were very well fitted by means of the steric mass-action model when the ion-exchange capacity of 290 mmol dm(-3) was obtained from independent frontal column experiments. The model fit provided the protein characteristic charge equal to 1.9, equilibrium constant 0.326, and steric factor 1.095 x 10(5).