Heterogeneous Biocatalysis Research Articles

Turbidimetric analysis of the enzymatic hydrolysis of polyethylene terephthalate nanoparticles

Abstract The heterogeneous enzymatic hydrolysis of polyethylene terephthalate (PET) by a polyester hydrolase (TfCut2) from Thermobifida fusca KW3 was determined by measuring the change of intensity of transmitted light due to the scattering effect of PET nanoparticles immobilized in an agarose gel. Nanoparticles with a mean diameter between 100 and 160 nm were prepared from PET samples of different crystallinity to provide a large surface area for the adsorption of the enzyme. The turbidity decrease of the PET nanoparticle suspensions was correlated to the surface erosion process resulting from the enzymatic degradation, and enabled a direct estimation of the kinetic parameters of the enzymatic hydrolysis of PET based on a model for heterogeneous biocatalysis. A comparison of the hydrolysis rate constants and the adsorption equilibrium constants of the enzymatic hydrolysis of PET nanoparticles prepared from recycled PET granulate, film and fibres showed that the biodegradability of PET was mainly influenced by the mobility of the polyester chains, which determined the affinity and accessibility of the ester bonds to the enzyme. Differential scanning calorimetric analysis of the partially hydrolyzed PET nanoparticles provided indirect evidence for an endo-type hydrolytic mechanism of TfCut2 in the heterogeneous degradation of aromatic polyesters.

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml−1, 290 UI g−1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.

Separation, Immobilization, and Biocatalytic Utilization of Proteins by a Supramolecular Membrane

Membrane separation of biomolecules and their application in biocatalysis is becoming increasingly important for biotechnology, demanding the development of new biocompatible materials with novel properties. In the present study, an entirely noncovalent water-based material is used as a membrane for size-selective separation, immobilization, and biocatalytic utilization of proteins. The membrane shows stable performance under physiological conditions, allowing filtration of protein mixtures with a 150 kDa molecular weight cutoff (∼8 nm hydrodynamic diameter cutoff). Due to the biocompatibility of the membrane, filtered proteins stay functionally active and retained proteins can be partially recovered. Upon filtration, large enzymes become immobilized within the membrane. They exhibit stable activity when subjected to a constant flux of substrates for prolonged periods of time, which can be used to carry out heterogeneous biocatalysis. The noncovalent membrane material can be easily disassembled, purified, reassembled, and reused, showing reproducible performance after recycling. The robustness, recyclability, versatility, and biocompatibility of the supramolecular membrane may open new avenues for manipulating biological systems.

Enzyme stability studies in microplates and stirred mini-bioreactors as representative scaling systems in heterogeneous biocatalysis

Enzyme stability studies in microplates and stirred mini-bioreactors as representative scaling systems in heterogeneous biocatalysis

Heterogeneous biocatalysis by enzyme/mesoporous silicas

Heterogeneous biocatalysis by enzyme/mesoporous silicas

Application of NMR Spectroscopy and Imaging in Heterogeneous Biocatalysis

Heterogeneously catalyzed enzymatic glucose isomerization was considered as a model process to extend the application of nuclear magnetic resonance (NMR) and magnetic resonance imaging techniques to the studies of biocatalytic processes and heterogeneous biocatalysts. It has been demonstrated that the T 2 times of glucose are different for its aqueous solution in the pores of an unmodified porous support and in a heterogeneous biocatalyst, comprising bacterial cells immobilized on the same support. This observation has been used to map the spatial distribution of the active component within a packed bed of biocatalyst in a model reactor. 13C NMR spectroscopy was applied to follow the progress of glucose isomerization catalyzed by the heterogeneous biocatalyst in a batch reactor. The utilization of proton spin decoupling and nuclear Overhauser effect was shown to be necessary to obtain high signal-to-noise ratio in the natural abundance 13C NMR spectra of a glucose–fructose syrup present in the packed bed of biocatalyst. The spectra thus obtained were suitable for the quantification of the glucose-to-fructose ratio achieved in the biocatalytic reaction.

Catalysis: concepts and green applications

INTRODUCTION Green and sustainable development What is catalysis and why is it important? Tools in catalysis research Exercises THE BASICS OF CATALYSIS Catalysis is a kinetic phenomenon Practical approaches in kinetic studies An overview of the basic concepts in catalysis Exercises HOMOGENEOUS CATALYSIS Metal complex catalysis in the liquid phase Homogeneous catalysis without metals Scaling up and down: Pros and cons Click chemistry and homogeneous catalysis Exercises HETEROGENEOUS CATALYSIS Classic gas/solid systems Liquid/solid and liquid/liquid catalytic systems Advanced process solutions using heterogeneous catalysis Exercises BIOCATALYSIS The basics of enzymatic catalysis Applications of enzyme catalysis Developing new biocatalysts: Better than nature's best Non-enzymatic biocatalysts Industrial examples Exercises COMPUTER APPLICATIONS IN CATALYSIS RESEARCH Computers as research tools in catalysis Modelling catalysts and catalytic cycles Predictive modelling and rational catalyst design An overview of data mining methods in catalysis Exercises

The effects of engineering design on heterogeneous biocatalysis in microchannels

The results of a numerical study of the fundamental interactions of engineering design and micromixing on conversion in packed microchannels are presented. Previously, channel-based microreactors made of molded silicon plastic were designed, fabricated, and experimentally tested. These reactors have enzymes immobilized on the channel walls by various methods including layer-by-layer nano self-assembly techniques. They also contain molded packing features to add reactive surface area and to redistribute the fluid. An arbitrary but intuitively sensible packing arrangement was initially chosen and used in experimental studies. The current computer simulation study was undertaken to understand how static laminar mixing affects the conversion efficiency. The reactors previously used experimentally have been simulated using CFD-ACE+multiphysics software (ESI CFD Inc., Huntsville, AL). It is found that packing significantly increases conversion when compared with empty channels over the entire flow rate range of the study (0.25<Re<62.5). The boost in conversion has an optimal point near Re=20 for the particular geometry examined.

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.

Prediction of water adsorption curves for heterogeneous biocatalysis in organic and supercritical solvents

In the framework of heterogeneous biocatalysis in non-aqueous media, knowledge of water adsorption curves is necessary for the choice of operating conditions. Indeed, water content plays a crucial role in the performance of such enzymatic reactors. A method to estimate water adsorption curves in biphasic solid-fluid systems is proposed here. It is grounded on the hypothesis that the water adsorption of the solid is not dependent on the solvent and on the theoretical evaluation of water activity in the solvent. It is tested in the case of 3-methyl-1-butanol, 5-methyl-2-hexanone and supercritical carbon dioxide as solvents and an immobilized enzyme, Lipozyme ®, as the solid phase. The method proved to be in good agreement with experiments. The method uses only a single experimental curve but necessitates the development of numerical modeling of thermodynamics. However, a simplified method, restricted to the range—hydrophobic to weakly hydrophilic solvent—is also proposed and tested.

A countercurrent adsorptive reactor for acidifying bioconversions

A countercurrent adsorptive reactor, based on the segregation tendency of dense sorbent particles through a liquid fluidized bed of light biocatalyst particles, is described for the acidifying production of the key pharmaceutical intermediate 6-Aminopenicillanic acid from Penicillin G. Mass transfer limited release of hydroxide from an ion exchange resin with simultaneous separation of the product by ion exchange, enabled axial control of the pH and product inhibition. The complexity of the countercurrent Trickle Flow Reactor requires a mathematical model, incorporating the effects of hydrodynamics, heterogeneous biocatalysis and multicomponent ion exchange. Such a model is presented and the parameters in the Three Phase-liquid, biocatalyst, sorbent-Model were determined from independent experiments. Model predictions gave an adequate description of experimental reactor performance. Using this model, the sensitivity of key parameters in the model, such as sorbent to liquid feed ratio and ion exchange selectivity and reactor optimization are readily studied.

Polymers for the immobilization of whole cells and their application in biotechnology

AbstractImmobilization of whole cells has become an efficient tool for biosynthesis, biotransformation and analysis. High cell density, high operational stability, easy handling, propably in continuous systems, and multiple reuse are important advantages of immobilized cells. In comparison to other methods like adsorption, crosslinking and encapsulation, the entrapment within a polymeric network is the most widely applied technique in heterogeneous biocatalysis. For immobilizing mammalian cells nearly exclusively the adsorption onto micorcarriers is used. Requirements for the polymers to be used in such immobilized cell systems are discussed in detail.

Size-exclusion effect of a substrate upon kinetics of trypsin immobilized on porous bead cellulose. 1. Influence of distribution coefficient of a substrate

A model of heterogeneous biocatalysis, in which kinetics and partition effects are connected via the size-exclusion principle, was worked up experimentally and theoretically. The present paper shows that the maximum relative activity of trypsin (EC 3.4.21.4) immobilized on porous bead (spherical) cellulose is directly proportional to the available distribution coefficient of the substrate. Providing that the excess of substrate is not sufficient (e.g. S/ K m ≈ 1) to safeguard saturated enzyme kinetics, the originally linear relationship of R a versus K av turns to an exponential one, without any dependence upon the manner of enzyme immobilization. It is suggested that the above may be a result of partition resistance and that the main factors determining the shape of the R a versus K av relation in conditions of substrate shortage are the size and geometry of the matrix. The physical characteristics of the porous carrier as well as the manner of covalent immobilization of the enzyme are all reflected in the constants applied in the derived equations.

Polymeric catalysts: A contribution to improve the ecology of chemical processes

Polymeric catalysts can typically be considered as being a polymeric carrier, to which a catalytically active species - well known in homogeneous catalysis - has been attached in an appropriate fashion. This heterogenization of homogeneous catalysis is of increasing importance due to certain advantages with respect to the ecology of industrial chemical processes. It is the intention of this contribution to discuss two approaches thus illustrating the general statement more specifically. Approach one is the application of ion exchange resins, generally in their protonated form, to perform acid catalyzed reactions in a solid matrix. Approach two is the immobilization of whole, living microbial cells to be applied in heterogeneous biocatalysis.

Soluble and immobilized enzyme technology in bioconversion of barley starch

Homogeneous and heterogeneous biocatalysis were both investigated as tools for barley starch syrup production. Barley starch was first liquefied by soluble heat-stable Bacillus sp. α-amylase EC 3.2.1.1 (1,4-α- d-glucan glucanohydrolase) Termamyl 60 L at 95°C, pH 6.5, to obtain slurries of varying DE- values up to ≈37. Alternatively, it was extruded with a Creusot-Loire BC 45 twin-screw extruder at 25% moisture, 150°C, for denaturation. After cooling and adjusting the pH to 4.5 or grinding, respectively, the pretreated starch was saccharified either by soluble or by immobilized Aspergillus niger glucoamylase EC 3.2.1.3 (1,4-α- d-glucan glucohydrolase) at 60°C, pH 4.5, to obtain glucose syrup of up to DE 96. The course of hydrolysis was followed by automated Biogel P-2 chromatographic analysis. Glucoamylase was immobilized either on a phenol-formaldehyde resin Duolite S 761 or on silanized Spherosil porous silica beads. Barley glucose syrup obtained was further continuously converted to high fructose syrup by a packed bed reactor of Actinoplanes missouriensis whole cell glucose isomerase (EC 5.3.1.5) Maxazyme entrapped within α-cellulose beads. We could conclude that barley starch may be used as an alternative raw material for biocatalytic starch syrup production.