Jolanta Bryjak

Glucoamylase, α-amylase and β-amylase immobilisation on acrylic carriers

Abstract In this paper immobilisation effectivity of α-amylase, β-amylase and glucoamylase on copolymers of butyl acrylate and pentaerhitrite triacrylate or ethylene glycol dimethacrylate was evaluated. Special attention was focused on copolymers having three kinds of anchor groups: OH, COOH, and NH 2 . The anchor groups were generated on polymer matrices by aminolysis with ethylenediamine. Particular carrier with the same structure was activated by glutaraldehyde, divinyl sulfone or carbodiimide procedure. The first method was found to give enzyme-carrier preparations of the highest activities. From seven investigated carriers the copolymer of butyl acrylate and ethylene glycol dimethacrylate was found as the best for three amylolytic enzymes immobilisation. The activities of selected immobilised enzyme preparations and thermal and pH stability were determined. These efforts resulted in selection of preparation based on one polymer matrix to form dual enzyme systems: α-amylase and β-amylase, or α-amylase and glucoamylase.

Plasma modified polymers as a support for enzyme immobilization II. Amines plasma

Polysulfone films were modified by ammonia, n-butylamine and allylamine remote plasma using various sample-toplasma distances. Contact angle measurements, FTIR-ATR and XPS spectroscopy proved the presence of polar, including amine, groups on the modified surface. Presence of argon in the plasma environment made the plasma more stable and in most cases left the surface more hydrophilic but with a lower amount of nitrogen moieties on it. Glucose isomerase was successfully immobilized on the plasma-treated samples. Its activity correlates well with the concentration of C–N bonds on the surface. The highest enzyme activity was achieved for samples treated with allylamine/Ar plasma close to the plasma edge. � 2003 Elsevier Ltd. All rights reserved.

Plasma modified polymers as a support for enzyme immobilization 1. Allyl alcohol plasma

The paper describes deposition of plasma polymerized allyl alcohol on polysulfone film. It is shown that film surface becomes more hydrophilic after plasma treatment independently on presence of argon in a reaction mixture. The chemistry of the new surface layer was established by FTIR-ATR and ESCA spectroscopy. The substrate placed close to the plasma edge was the most hydrophilic but the amount of hydoxyl groups was not the highest there. Presence of argon stabilized the plasma but the deposited layer contained relatively less oxygen-bearing functionalities. The plasma treated polymer was subjected to xylose isomerase immobilization. For this purpose the divinylsulfone method was adapted. The studies revealed no correlation between the surface hydrophilicity and efficiency of immobilization.

Modelling of potato starch saccharification by an Aspergillus niger glucoamylase

The kinetics of hydrolysis of a soluble potato starch catalysed by free glucoamylase was investigated in a batch reactor. Seventeen progress curves of product concentration, obtained at varying initial substrate and enzyme concentrations, were fitted simultaneously using six different models of saccharification process. A good description of the whole course of the progress curves was obtained using a six-parameter model based on a kinetic equation, which contained product and substrate inhibition terms. An essential feature of the model was that starch was structured into susceptible and resistant fractions that differed in the rate constants of hydrolysis. The model parameters were estimated with a good accuracy and were in a good agreement with the available literature information.

Very stable silica-gel-bound laccase biocatalysts for the selective oxidation in continuous systems.

Cerrena unicolor laccase was immobilized by adsorption and covalent bonds formation on silica-gel carriers, functionalized with different organosilanes and surface densities. The effects of protein concentration, pH value of the coupling mixture and the enzyme purity on immobilization efficiency of the best carrier, moderately modified (0.75 mmol/g carrier) with 3-aminopropyltriethoxysilane were investigated. Activity of the best biocatalysts, expressed in ABTS oxidation, was 4028 U/mL of the carrier or 3530 U/mg of bound protein. Properties of immobilized laccase were determined to find excellent thermal stability improvement; t(1/2) for freely suspended enzyme was 2-3 min at 80 degrees C, whereas after immobilization over 100 min. Kinetic experiments in both batch and packed-bed reactors gave only four times lower k(cat)/K(m) value than for the native enzyme. A packed-bed reactor with silica-gel-bound laccase beads appeared to be very efficient in ABTS oxidation and its exceptional potentials were shown in the continuous decolorization of indigo carmine for 18 days without loss in activity. This system offers perfect ability to degrade recalcitrant dyes, but we can also envisage its use, with ABTS acting as a mediator, in regeneration of nicotinamide cofactors.

Immobilization of lipase on various acrylic copolymers

Abstract Polymer carriers with various degrees of hydrophobicity, porosity, and polarity were studied for immobilization of lipase from Candida rugosa. Copolymers and terpolymers were prepared by suspension polymerization of six monomers, ethyl and butyl acrylate, acrylonitrile, methyl methacrylate, 2-hydroxypropyl methacrylate and vinyl acetate, and three crosslinking agents, divinylbenzene, ethylene glycol dimethacrylate and trimethylolpropane triacrylate, in the presence of various diluents. Immobilization of lipase was carried out via glutaraldehyde chemical binding or by adsorption. It was found that the best results were obtained for carriers of butyl acrylate, ethyl acrylate or 2-hydroxypropyl methacrylate crosslinked with ethylene glycol dimethacrylate.

IMMOBILISATION OF TRYPSIN ON ACRYLIC COPOLYMERS

Abstract Twenty-six carriers from three series of acrylic copolymers were evaluated for trypsin immobilisation. The following monomers were used to prepare polymer matrices: ethylene glycol dimethacrylate, trimethylolpropane triacrylate, ethyl acrylate, butyl acrylate, hydroxypropyl methacrylate, acrylonitrile, and divinylbenzene. The carriers with trimethylolpropane triacrylate as the cross-linking agent were selected as the most profitable matrices for expressing enzyme activity. Considering the influence of inert diluent on the carriers superstructure and trypsin immobilisation it was shown that cyclohexanol as co-diluent produced a good microenvironment for enzyme activity while toluene created an unsuitable carrier structure. The most effective carrier was a copolymer of acrylonitrile/trimethylolpropane triacrylate synthesised in the presence of cyclohexanol and 2-ethylhexanol. A comparison of the properties of the enzyme-carrier preparation and native trypsin demonstrated that the binding of the protein to the carrier caused an increase of the storage and pH-stability of the bound enzyme.

Storage stabilization of enzyme activity by poly(ethyleneimine)

The effect of mixing penicillin acylase with poly(ethyleneimine) is discussed. The properties of the polymer-enzyme system were evaluated for a wide range of enzyme concentrations (0.3–45.5 mg/cm3) and poly(ethyleneimine) concentrations (0.0001–10% wt). It was shown that addition of poly(ethyleneimine) to crude enzyme preparation caused precipitation of ballast protein and stabilization of the enzyme fraction remaining in the supernatant. The soluble fraction had stable activity for 21 days storage at 37 °C while the native enzyme lost about 80% of its initial activity. Additionally, it was ascertained that the polymer very slightly affected the properties of penicillin acylase in the PEI-enzyme preparations. Finally, possible ways of using the polymer-enzyme preparations in a membrane reactor are suggested.

Synthesis and properties of acrylic carriers from acrylonitrile, vinyl acetate and divinylbenzene terpolymers

Abstract Two series of carriers based on acrylonitrile (AN)-vinyl acetate (VA)-divinylbenzene (DVB) terpolymers were obtained by aminolysis of nitrile groups to N-alkylamino ones. In the first series, the terpolymers differed in the content of VA units (7.5, 10, 20, and 30 mol.%). The modifying diluents used in the polymerization stage were: hexadecane (HD) and toluene (T). In the second series, the modifying diluents were: cyclohexanol (C), octanol (O), decanol (D), 2-ethylhexanol (E), and benzyl alcohol (B). The content of VA units was constant (20 mol.%). The IR spectra revealed that the aminolysis of nitrile groups of the terpolymers to N-ethylamino ones was accompanied by hydrolysis of the ester groups. The carriers with hydroxy groups, however, did not swell more than those obtained from AN-DVB copolymers. The concentration of amino groups was ∼ 1.8 mmol/g and the porosity was in the range 0.1–0.5, depending on the kind of diluent used in polymerization. The structural parameters of the carriers were characterized by the inverse GPC method with dextran solutions. The pore size (mean pore diameter) and its distribution width decreased with increasing VA unit content for the series HDT-7.5-HDT-30. In the second series, the porosity characteristics depended on the molecular shape of the thermodynamically bad diluent used. Acylase immobilized on those carriers had smaller activity than that immobilized on previously described carriers, whereas for glucoamylase and peroxydase the results of immobilization were very promising.

Immobilization of penicillin acylase on acrylic carriers

Penicillin acylase obtained from E. Coli (E. C. 3.5.1.11) was covalently bound via glutaric aldehyde to acrylic carriers crosslinked with divinylbenzene or ethylene glycol dimethacrylate. The best enzymatic preparation was obtained by using ethyl acrylate/ ethylene glycol dimethacrylate copolymer. 1 cm3 of the carrier bound 6.4 mg of protein, having 72% activity in relation to the native enzyme. The preparation lost only 10% of its initial activity after 100 d of storage at 4°C. A negligible effect of immobilization on the enzyme activity at different temperatures or pH as well as significant increase of the stability of the immobilized enzyme at elevated temperatures were observed.