Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.     

Economic comparison of enzymatic reactors and advanced oxidation processes applied to the degradation of phenol as a model compound

Abstract

Some micropollutants present in wastewaters are barely removed in sewage treatment plants. In many cases a post-treatment process based on separation and/or oxidation has to be applied. The aim of this study was the technical and economic comparison of enzymatic technologies with other advanced oxidation processes (AOPs) for the degradation of phenol. Batch and continuous enzymatic reactors, using free and immobilized manganese peroxidase (MnP, EC 1.11.1.13), were considered. Continuous degradation of phenol in an enzymatic membrane reactor was shown to be the fastest process and degradation in a continuous reactor with immobilized enzyme involved the lowest consumption of enzyme. However, the immobilization process increased the enzyme cost 100-fold. A continuous enzymatic membrane reactor gave high degradation efficiency and may be a viable technology for phenol removal when compared with other AOPs from both technical and economic points of view.     

Model of the regulation of activity of immobilized enzymes (amylases) in soil

The preservation of activity of extracellular enzymes in soil is presently associated with their immobilization on organic or inorganic carriers. Enzyme immobilization results, however, in a significant decrease in enzymatic activity. In the present work, the mechanism responsible for promotion of the catalytic activity was revealed, as well as the favorable effect of low-molecular alkylhydrozybenzenes of the class of alkylresorcinols, which are common in soil organic matter, on stability of immobilized enzymes (exemplified by amylases) by their post-translational modification. Optimal conditions (enzyme to sorbent ratio, pH optimum, CaCl₂ concentration, and sorption time) for amylase sorption on a biological sorbent (yeast cell walls) were determined and decreased activity of the immobilized enzyme compared to its dissolved state was confirmed. Alkylresorcinols (C₇AHB) at concentrations of 1.6 to 80 mM were found to cause an increase of amylase activity both in the case of already sorbed enzymes (by 30%) and in the case of a free dissolved enzyme with its subsequent immobilization (by 50–60%). In both cases, the optimal C₇AHB concentration was 16 mM. Amylase stability was determined for C7AHB-modified and unmodified enzymes immobilized on the biological sorbent after two cycles of freezing (–20°C) and thawing (4°C). Inverse dependence was revealed between increasing stability of C₇AHB-modified enzymes and an increase in their activity, as well as higher stability of immobilized modified amylases than of the dissolved modified enzyme. Investigation of the effect of C₇HOB-modification in the preservation of activity in immobilized amylases after four freeze–thaw cycles revealed: (1) better preservation of activity by the modified immobilized enzymes compared to immobilized ones; (2) differences in the dynamics of activity loss within compared pairs, with activity of immobilized amylases decreasing after the second cycle to a lower level (42%) than activity of the modified immobilized enzymes after the fourth cycle (48%). These results demonstrate that in the preservation of activity of extracellular enzymes in soil both stabilization mechanisms are of importance: immobilization on organic carriers and modification of the enzyme conformation by low-molecular compounds with the functions of chemical chaperones.     

Cofactor regeneration for sustainable enzymatic biosynthesis

Oxidoreductases are attractive catalysts for biosynthesis of chiral compounds and polymers, construction of biosensors, and degradation of environmental pollutants. Their practical applications, however, can be quite challenging since they often require cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These cofactors are generally expensive. Efficient regeneration of cofactors is therefore critical to the economic viability of industrial-scale biotransformations using oxidoreductases. The chemistry of cofactor regeneration is well known nowadays. The challenge is mostly regarding how to achieve the regeneration with immobilized enzyme systems which are preferred for industrial processes to facilitate the recovery and continuous use of the catalysts. This has become a great hurdle for the industrialization of many promising enzymatic processes, and as a result, most of the biotransformations involving cofactors have been traditionally performed with living cells in industry. Accompanying the rapidly growing interest in industrial biotechnology, immobilized enzyme biocatalyst systems with cofactor regeneration have been the focus for many studies reported since the late 1990s. The current paper reviews the methods of cofactor retention for development of sustainable and regenerative biocatalysts as revealed in these recent studies, with the intent to complement other reviewing articles that are mostly regeneration chemistry-oriented. We classify in this paper the methods of sustainable cofactor regeneration into two categories, namely membrane entrapment and solid-attachment of cofactors.     

Laccase grafted membranes for advanced water filtration systems: a green approach to water purification technology

Purpose: Conventional wastewater treatment technologies are not good enough to completely remove all endocrine disrupting compounds (EDCs) from the water. Membrane separation systems have emerged as an attractive alternative to conventional clarification processes for waste and drinking water. Coupling of a membrane separation process with an enzymatic reaction has opened up new avenues to further enhance the quality of water. This review article deliberates the feasibility of implementing enzymatic membrane reactors has been deliberated.

Materials and methods: A comprehensive study of conventional water treatment technologies was carried out and their shortcomings were pointed out. Research findings from the leading groups working on enzyme grafted membrane based water purification were summarized. This review also comprehends the patent documents pertinent to the technology of enzyme grafted membranes for water purification.

Results: Immobilization of an enzyme on a membrane improves the performance of membrane filtration, and processes for the treatment of polluted water. Research has started exploring the potential for laccase enzymes because it can catalyze the oxidation of a wide range of substrates, structurally comparable to EDCs, by a radical-catalyzed reaction mechanism, with corresponding reduction of oxygen to water in an electron transfer process. Further, in the presence of certain mediators, the substrate range of laccases can be further enhanced to non-aromatic substrates.

Conclusions: Removal of EDCs by laccase cross-linked enzyme aggregates in fixed-bed reactors or fluidized-bed reactors and laccase immobilized ultrafiltration (LIUF) membranes are proving their worth in water purification technology. The major operational issues with the use of LIUF membranes are enzyme instability in real wastewater and membrane fouling. In view of the above-stated characteristics, laccases are considered as the most promising enzyme for a greener and less expensive water purification technology.     

Poly(vinyl alcohol) Ultrafiltration Membranes: Synthesis,Characterization, the Use for Enzyme Immobilization

An enzymatic hydrolysis in a symmetric membrane, combining reaction and separation, has been studied. PVA hydrogel was chosen because of its hydrophilicity expecting to minimize membrane fouling and concentration polarization. The membrane pores containing covalently bound enzymes serve as catalyst support. The membrane immobilization of the enzyme and the filtration mode of operating the process were chosen in order to avoid product inhibition of the enzyme. The properties of cross‐linked PVA hydrogel were investigated. The conversion of the hydrolysis of p‐nitrophenyllaurate with two different loadings of Cr lipase was evaluated. The conversion of the reaction decreased with both increasing substrate flux and initial concentration. The kinetic parameters were obtained. Compared to the free lipase, the K_m of the membrane bonded enzyme was lower and its R_max approximately the same.     

Parameters necessary to define an immobilized enzyme preparation

Biocatalytic processes continue to find increasing application in industry. Therefore enzyme immobilization has also become of increasing importance as a means of allowing enzyme containment within reactors operating in continuous mode or else separation of enzyme after use in (fed-)batch reactors, as well as potential recycle. Whilst much has been reported in the scientific literature about enzyme immobilization methods, in many cases the protocol leads to losses in enzyme activity. In this review we outline the reasons for loss of activity during immobilization and highlight suitable diagnostic tests to elucidate the precise cause and thereby methods to restore activity. The need for standardized reporting of immobilization methods is also emphasized as a means of benchmarking alternative approaches.     

Immobilized biocatalytic process development and potential application in membrane separation: a review

Biocatalytic membrane reactors have been widely used in different industries including food, fine chemicals, biological, biomedical, pharmaceuticals, environmental treatment and so on. This article gives an overview of the different immobilized enzymatic processes and their advantages over the conventional chemical catalysts. The application of a membrane bioreactor (MBR) reduces the energy consumption, and system size, in line with process intensification. The performances of MBR are considerably influenced by substrate concentration, immobilized matrix material, types of immobilization and the type of reactor. Advantages of a membrane associated bioreactor over a free-enzyme biochemical reaction, and a packed bed reactor are, large surface area of immobilization matrix, reuse of enzymes, better product recovery along with heterogeneous reactions, and continuous operation of the reactor. The present research work highlights immobilization techniques, reactor setup, enzyme stability under immobilized conditions, the hydrodynamics of MBR, and its application, particularly, in the field of sugar, starch, drinks, milk, pharmaceutical industries and energy generation.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part I: Cultivation of Pseudomonas syringae and partial purification and immobilization of 7-β-(4-carboxybutanamido) cephalosporanic acid acylase

The enzymatic transformation of 7-β-(4-carboxybutanamido)cephalosporanic acid (Gl-7-ACA) to 7-amino-cephalosporanic acid (7-ACA) is reported. The optimum conditions for cultivation of the producer strain Pseudomonas syringae, as well as the procedures for isolation, purification, and immobilization of the enzyme Gl-7-ACA acylase, are described. It is shown that when glutaraldehyde is used for immobilization of this enzyme, the yield of immobilization is low. After six hydrolyses of Gl-7-ACA to 7-ACA, the immobilized enzyme activity loss is less than 10%.     

Biospecific immobilization of mannan-penicillin G acylase neoglycoenzyme on Concanavalin A-bead cellulose

The matter of this work was to evaluate possibilities of biospecific immobilization of synthetic mannan-penicillin G acylase neoglycoconjugate on Concanavalin A support. The conjugate containing 37% (w/w) of yeast mannan was prepared. Significant biospecific interaction of this neoglycoenzyme with Con A was confirmed by precipitation method. The biospecific sorption of conjugate was investigated using Concanavalin A-triazine bead celluloses MT-100 with different content of Con A (from 1.4 to 9.8 mgCon A/gwet support). The results obtained under optimal conditions were compared with those from covalent immobilization of PGA. The sorbent capacity was observed higher for covalent binding of enzyme. On the other hand, the biospecifically immobilized neoglycoenzyme retained a greater amount of initial activity. The maximum amount of 6.6mgimmobilizedneoglycoenzyme/gwet Con A-sorbent (18.1 U/g) was achieved. The amount as well as activity of immobilized mannan-penicillin G acylase was increased by its two multiple layering on surface of sorbent (10.1mg, respectively, 23.5 U/gwet sorbent). Determined storage and operational (using flow calorimetric method) stabilities of biospecifically immobilized enzyme, were similar, possibly somewhat higher that those of covalent bound penicillin G acylase.     

Method for the stabilization and immobilization of enzymatic extracts and its application to the decolorization of textile dyes

Peroxidases from Pleurotus eryngii have been investigated for their ability to degrade recalcitrant, phenolic pollutants. The use of crude enzymatic extracts can reduce the high costs associated with enzyme purification, and enzyme immobilization can enhance enzyme stability and recovery. The present study tests the effectiveness of various conditions for crude enzyme stabilization in polyethylene glycol and glycine solutions, and immobilization on monofunctional and heterofunctional agarose solid supports. Glycine at 0.5 M at 4 °C and pH 4 was most effective stabilization agent for the crude enzymatic extracts, and enzyme immobilization efficiency was greatest for heterofunctional supports. MANA-glyoxyl heterofunctional supports were demonstrated to have the greatest enhancement of decolorization (1.3-fold) and velocity of substrate consumption (fivefold). Therefore, the application of crude enzymatic extracts to industrial processes, such as dye decolorization, represents a cost-effective alternative to purified enzymes.     

Polyvinylamine‐coated polyester fibers as a carrier matrix for the immobilization of peroxidases

Biocatalytic transformations that employ immobilized enzymes become increasingly important for industrial applications. Synthetic or natural textile fiber materials such as polyester, polyamide or viscose are support materials that are comparatively inexpensive. Contrary to traditional support materials, their flexibility enables their use in reactors of any geometry and a fast and residue‐free removal from batch reactors. In this study a permanently immobilized peroxidase (Baylase®) has been investigated on polyester felt as a solid support as a new heterogeneous catalyst system. The polyester felt was functionalized by coating with polyvinylamine and subsequent activation with glutaraldehyde as a crosslinking agent. The enzyme load on the textile surface, the activity of the immobilized protein after repeated use as well as the storage stability was evaluated. Scanning electron micrographs and UV Vis spectroscopy made it possible to verify the enzyme immobilization on the textile surface. Furthermore, the load of immobilized peroxidase was determined by ICP OE spectrometry to be 9–12 mg per gram of textile. The activity of immobilized Baylase® remained high over 35 reaction cycles and a storage period of 8 weeks.     

Enzymatic reactors for the removal of recalcitrant compounds in wastewater

Abstract

Enzymatic treatments based on oxidative enzymes, such as peroxidases, laccases and tyrosinases, have been proposed as an alternative to conventional methods to remove a broad range of contaminants present in wastewater. The aim of this study is to discuss existing technologies for the removal of pollutants based on the use of oxidative enzymes, including a discussion on the most important factors affecting the efficiency of the proposed systems. Factors involved in the catalytic cycle of the enzyme (biocatalyst, substrates and mediators), the addition of certain components to the reaction medium (additives, surfactants or solvents) as well as operational parameters (temperature, pH or agitation) will be discussed. Finally, two types of reactors: one-stage and two-stage enzymatic membrane reactors, especially designed for the treatment of micropollutants present in secondary effluents, will be described in detail.     

Intensification of biokinetics of enzymes using ultrasound-assisted methods: a critical review

The use of low intensity ultrasound has gotten surprising consideration over the last decade as a method for enhancing the catalytic activity of enzyme. Ultrasounds have the potential to significantly influence the activity of the enzymatic processes, provided that the energy input is not so high as to inactivate the enzyme. By providing the variation in parameters, various physical and chemical effects can be attained that can enhance the enzymatic reaction. Ultrasonic reactors are known for their application in bioprocesses. However, the potential of their applications is still limited broadly due to the lack of proper information about their operational and performance parameters. In this review, the detailed information about ultrasonic reactors is provided by defining the different types of reactors and number and position of ultrasonic transducers. Also, it includes mechanism of intensification and influence of ultrasonic parameters (intensity, duty cycle, and frequency) and enzymatic factors (enzyme concentration, temperature, and pH) on the catalytic activity of enzyme during ultrasound treatment.     

Immobilization materials mixed with activated sludge as column biofilters for the treatment of gaseous stream containing benzene and toluene

Activated sludge has been utilized for the treatment of volatile organic compounds (VOCs) which are emitted from industrial processes. Nevertheless, activated sludge systems often suffer from the problem caused by concentration gradients as well as pressure drops. Channeling is also a major problem in the treatment process. As the bed height of the packed activated sludge system increases, the pressure drop increases accordingly. To solve these problems, we proposed immobilized activated sludge column reactors for treating VOCs in air. The immobilization material used to mix with activated sludge was properly selected in this work. Elemental compositions of these materials were analyzed. In this study, we also proposed a VOC feed system so that more stable inlet concentrations could be achieved. Hence, the equipment and operating costs were reduced and the problem of VOCs leaking from peristaltic pumps was avoided. The moisture content of the system was well maintained and better VOC removal efficiency was achieved. With an operation condition of progressive VOC inlet concentrations, better removal efficiency of benzene and toluene was then obtained. In conclusion, by the utilization of immobilization materials selected from wastes as well as immobilized activated sludge column reactors, significant removal efficiency for both benzene and toluene was demonstrated.     

Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.     

Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose

Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD(+). In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD(+)-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.     

Kinetic studies on urea hydrolysis by immobilized urease in a batch squeezer and flow reactor

The immobilization of urease on the reticulated polyurethane foam, and the kinetic phenomenon of urea hydrolysis by the resulting immobilized urease in both batch squeezer and circulated flow reactors were studied. Urease was immobilized with bovine serum albumin and glutaraldehyde on polyurethane foam support of 7 to 15 mum thickness. The residual apparent activity of urease after immobilization was about 50%. The good hydrodynamic property and flexibility of polyurethane foam were retained in solution after immobilization. A modified biofilm reactor model was used to describe the kinetic phenomenon of urea hydrolysis in both batch squeezer and circulated flow reactors. The characteristic parameters of the reactor model for both bioreactors were obtained by combining the Rosenbrock optimization method, the Rungs-Kutta method, and the Newton-Raphson method. The best-fit results were in good agreement with the experimental data. This study suggests another application of polyurethane foam in enzyme immobilization and immobilized enzyme reactors, which offers potential for practical applications in various bioreactors. (c) 1992 John Wiley & Sons, Inc.     

Potential use of polyphenol oxidases (PPO) in the bioremediation of phenolic contaminants containing industrial wastewater

The present review emphasizes on the use of Polyphenol oxidase (PPO) enzyme in the bioremediation of phenolic contaminants from industrial wastewater. PPO is a group of enzyme that mainly exists in two forms; tyrosinase (E.C. 1.14.18.1) and laccase (E.C. 1.10.3.1) which are widely distributed among microorganisms, plants and animals. These oxidoreductive enzymes remain effective in a wide range of pH and temperature, particularly if they are immobilized on some carrier or matrices, and they can degrade a wide variety of mono and/or diphenolic compounds. However, high production costs inhibit the widespread use of these enzymes for remediation in industrial scale. Nevertheless, bench studies and field studies have shown enzymatic wastewater treatment to be feasible options for biodegradation of phenols through biological route. Nanomaterials-PPO conjugates have been also applied for removal of phenols which has successfully lower down the drawbacks of enzymatic water treatment. Therefore in this article various approaches and current state of use of PPO in the bioremediation of wastewater, as well as the benefits and disadvantages associated with the use of such enzymes have been overviewed.