Chloroperoxidase Research Articles

Specific Photobiocatalytic Oxyfunctionalization Reactions

Specific oxyfunctionalization chemistry requires a delicate balance between reactivity and selectivity, and therefore still represents a major challenge in organic chemistry. Cytochrome P450 monooxygenases (P450s) are widely considered as catalysts that solve this dilemma. Here, the reactive Compound I is embedded within a defined three-dimensional protein structure, thus enabling regio-, chemo-, and enantiospecific oxyfunctionalization reactions even on nonactivated hydrocarbons. Their practical application, however, is limited to whole-cell biotransformations because of their cofactor dependency and their complex molecular architecture. However, microbial transformations are not always straightforward to perform and suffer from intrinsic disadvantages such as reactant metabolization and toxicity, and often low productivities. Great hope has been placed on the protein superfamily of heme/thiolate peroxidases to solve both challenges, the cofactor dependency as well as the complicated molecular architecture of P450s by using the hydrogen peroxide shunt. Here, the catalytically active oxoferryl species is formed directly from H2O2 instead of from reductive activation of O2. Seemingly, this allows simple circumvention of the abovementioned challenges and enables practical oxyfunctionalization procedures. Chloroperoxidase (CPO) from Caldariomyces fumago (Leptoxyphium fumago) represents the archetype of such heme/thiolate peroxidases. In fact, thus far CPO is the only peroxidase used for oxyfunctionalization reactions. Unfortunately, CPO is active in sulfoxidation reactions only, whereas its performance in hydroxylation of C H bonds and epoxidation reactions drops by several orders of magnitude. Generally, CPO only performs a few hundred turnovers prior to loss of catalytic activity (see below), and nonactivated hydrocarbons such as cyclohexane are not converted at all by CPO. Obviously, this disqualifies CPO as a broadly applicable catalyst for oxyfunctionalization chemistry. However, with more and more genome sequences becoming available, today, more than 100 putative heme/ thiolate CPO analogues can be found in nucleotide databases (Scheme 1). Hence, there is a wealth of potential alternatives to CPO that are waiting to be discovered, and they potentially have more suitable catalytic properties for oxyfunctionalization. Recently, a novel peroxidase from the basidomycetous fungus Agrocybe aegerita (AaeAPO=Agrocybe aegerita aromatic peroxygenase) has been isolated and characterized. AaeAPO specifically converts aromatic hydrocarbons into the corresponding phenols and has recently been classified as an “unspecific peroxygenase” in the E.C. nomenclature (E.C.1.11.2.1). Herein we report that AaeAPO is an active and versatile catalyst for enantiospecific hydroxylation and epoxidation reactions. Under nonoptimized reaction conditions AaeAPO performance (in terms of turnover number) exceeds that reported for CPO by at least two orders of magnitude. Furthermore, we demonstrate that AaeAPO, in contrast to CPO, can also hydroxylate nonactivated C H bonds. Since AaeAPO, like all heme-dependent enzymes, is easily inactivated by H2O2, strict control over the in situ Scheme 1. Simplified phylogenetic tree of the heme/thiolate superfamily. The names marked in bold refer to fungal species whose heme/ thiolate enzymes have been purified and characterized. The other terms belong to taxonomic units of the fungal kingdom with putative CPOor APO-like proteins found in databases.

Over-expression of chloroperoxidase in Caldariomyces fumago

The filamentous fungus Caldariomyces fumago secretes a chloroperoxidase (CPO). To increase its production, we integrated a CPO-expression cassette into the non-transcribed spacer regions of the rDNA in C. fumago. One strain was obtained that had twice the CPO activity when grown in shake-flask and bioreactor compared to the wild-type. The highest CPO activity from the bioreactor cultivation was 3,236 U ml(-1). This is the highest value reported so far.

A biocatalytic route towards rose oxide using chloroperoxidase

The chiral monoterpene alcohol citronellol was converted to the corresponding bromohydrin by the haem-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago in the presence of hydrogen peroxide and bromide ions. A conversion rate of 51% could be achieved under adapted reaction conditions, which easily yield product in the gramme per litre range while only needing catalytic amounts of enzyme. The bromohydroxylation was shown to be highly regioselective yielding 6-bromo-3,7-dimethyloctane-1,7-diol as the sole product. Product identity was confirmed by GC–MS, 1H- and 13C-NMR spectroscopy and the synthesis of reference compounds. However, the reaction was shown to be non-stereospecific because enantiopure (R)- and (S)-citronellol, respectively, gave 1:1-diasteromeric mixtures of the corresponding bromohydrins. A racemic mixture of (R/S)-citronellol was bromohydroxylated without any detectable enantiodiscrimination. The total lack of stereospecificity and enantiodiscrimination points to a reaction mechanism where the oxidised bromide intermediate is not a ligand to the Fe(III)-haem at the distal site but is released from the enzyme active site. The final bromide transfer occurs probably outside the active site via a diffusible oxidised bromide species and the demonstrated regioselectivity is purely chemically controlled. The generated bromohydrins can be straightforward converted via two reactions steps into rose oxide which is a highly valuable flavour and fragrance substance.

Biotransformation of cyclohexene to value-added oxygenated derivative catalyzed by chloroperoxidase in the presence of small quantities of quaternary ammonium salts

Biotransformation of cyclohexene catalyzed by chloroperoxidase (CPO) from Caldariomyces fumago was employed to prepare value-added oxygenated derivative: trans-1,2-cyclohexanediol (CHD) using H 2O 2 as oxidants. The conversion of substrate was enhanced to 89.2% in the presence of small quantities of quaternary ammonium salts (QAS) compared to that of only 48.1% in aqueous phosphate buffer after 150 min. The enhancement was dependent on the concentration and alkyl group length of QAS. QAS were found playing multiple functions in reaction media: phase transfer catalysis and tuning the composition of product. UV–vis, fluorescence and circular dichroism (CD) spectral assay were employed to investigate the effect of QAS on micro-environment around active center and structure of protein. The strengthening of α-helix structure of CPO and more exposure heme for easily access of substrate was found responsible for the improvement of catalytic performance of CPO. Moreover, the determined kinetic parameters indicated the affinity and selectivity of CPO to substrate was improved according to the observation of a decrease of Michaelis constant K m while an increase of the second order rate constants k cat/ K m. The strategy reported in this work is environment-friendly, straightforward and applicable to large scale preparation.

Reduced‐oxidized difference spectral analysis and chemiluminescence‐based Scatchard analysis demonstrate selective binding of myeloperoxidase to microbes

Myeloperoxidase (MPO), a microbicidal haloperoxidase of neutrophil leukocytes, was observed to selectively bind to bacteria. Binding was quantified by dithionite-reduced minus oxidized (R-O) difference spectral analysis. Escherichia coli and Pseudomonas aeruginosa showed large MPO binding by R-O difference spectral analysis, whereas Streptococcus sanguinis did not. For increased sensitivity, free and microbe-bound MPO and chloroperoxidase (CPO) activities were quantified by acid-optimum haloperoxidase-dependent chemiluminescence (CL) measurements, and these data were used for Scatchard analysis. The MPO bound/free (B/F) CL ratio was 49.5 for P. aeruginosa, 14.6 for Staphylococcus aureus, 2.8 for E. coli, 0.7 for Candida albicans and 0.4 for S. sanguinis. By comparison, the CPO B/F CL ratio was 0.03 for P. aeruginosa, 0.09 for S. aureus, 0.31 for E. coli, 0.18 for C. albicans and 0.16 for S. sanguinis. As a member of the lactic acid family of bacteria and a viridans streptococcus, S. sanguinis does not synthesize cytochromes and is catalase-negative. The metabolic products of S. sanguinis, i.e. lactic acid and hydrogen peroxide, provide optimal acidity and substrate for MPO oxidation of chloride to hypochlorite. Hypochlorite can react with organic substrates to yield dehydrogenated or chlorinated products, but when peroxide is not limiting, hypochlorite reacts with peroxide yielding singlet oxygen. The reactivity of hypochlorite is dependent on substrate availability. The microsecond half-life of electronically excited singlet oxygen restricts reactivity to within a radius of <0.25 µm; i.e. the reactivity of singlet oxygen is both substrate and half-life dependent. Poor MPO binding provides protection and possibly competitive advantage to viridans streptococci.

Molecularly imprinted catalytic polymers with biomimetic chloroperoxidase activity

A method for the rational design of molecularly imprinted polymers with catalytic activity (MICs) that mimic hemeproteins is described. Density Functional Theory calculations applied to the pre-polymerization adduct and to a possible reaction intermediate allow to choose the best functional monomer that builds up the active sites. This functional monomer should stabilize both relevant stages, the first one in an adequate porogenic solvent and the second one in the aqueous reaction medium. In this work, a MIC was synthesized to catalyze the oxidation of 2,4,6-trichlorophenol (TCP), the substrate and template molecule. A hemin molecule was used as the catalytic centre and four functional monomers and six solvents were tested. According to the theoretical predictions, methacrylamide and 4-vinylpyridine MIPs successfully mimic the recently discovered ability of Chloroperoxidase (CPO) to catalyze the oxidative dechlorination of TCP in batch analysis. They exhibit good catalytic properties and substrate selectivity.

Enhancing oxidation activity and stability of iso-1-cytochrome c and chloroperoxidase by immobilization in nanostructured supports

The immobilization of enzymes in inorganic materials has been widely used because it can produce an enhancement of the catalytic stability and enzymatic activity. In this article, the effect of the immobilization of iso-1-cytochrome c (CYC-Sc) from Saccharomyces cerevisiae and chloroperoxidase (CPO) from Caldariomyces fumago on the enzyme stability and catalytic oxidation of styrene was studied. The immobilization was carried out in three silica nanostructured supports with different pore size MCM-41 (3.3 nm), SBA-15 (6.4 nm) and MCF (12.1 nm). The adsorption parameters and leaching degree of immobilized enzymes were determined. Catalytic parameters of immobilized and free enzymes were determined at different temperatures (20–60 °C) and in different acetonitrile/water mixtures (15–85% of acetonitrile). The results show that there is low leaching of the enzymes in the three supports assayed and the adsorption capacity (qmax) was higher as the pore size of the support increased. The pore size also produces the enhancement of peroxidase activities on the styrene oxidation. Thus, CPO adsorption into SBA-15 and MCF showed remarkable thermal and solvent stabilities at 40 °C showing a total turnover numbers of 48,000 and 54,000 times higher than free CPO, respectively. The enhancement of activity and stability doubtless is interesting for the potential industrial use of peroxidases.

ChemInform Abstract: Bienzymatic Synthesis of Benzothia/(Oxa)zoles in Aqueous Medium.

Abstract A series of 2-arylbenzothiazoles and 2-arylbenzoxazoles were synthesized by the reactions of aldehydes and 2-aminothiophenol and 2-hydroxythiophenol, respectively, using glucose oxidase (GOX)–chloroperoxidase (CPO) catalytic system under oxygen atmosphere.

Identification of a New Source of Contamination of Quercus sp. Oak Wood by 2,4,6-Trichloroanisole and Its Impact on the Contamination of Barrel-Aged Wines

Thanks to practical experience in various wineries in recent years, it is now clear that, similarly to the well-known phenomenon in corks, there are several sources of unpredictable contamination of oak wood by 2,4,6-trichloroanisole (TCA). TCA affects staves in the same barrel very sporadically, with extremely limited contaminated areas on the surface that may reach several millimeters in depth. The precise origin of the TCP and TCA in oak wood is not known at this stage. Available data indicate that the phase where stavewood is naturally dried and seasoned is the source of these undesirable organochlorine contaminants. The strictly chemical formation of 2,4,6-trichlorophenol (TCP), derived from organochlorine biocides, was demonstrated to be impossible under traditional cooperage conditions, and its accumulation remained highly improbable. Similarly to previous discoveries in corks, all the analyses of oak wood suggested that the TCP was of biochemical origin. The capacity to biomethylate chlorophenols is well-known and relatively widespread among the usual microflora in stavewood, but the precise origin of the intermediary leading to TCP formation is still unknown. One probable hypothesis is that this reaction involves chloroperoxidase (CPO). Several ideas have been proposed, but the microorganisms responsible for the formation of the TCA precursor in oak wood have not yet been identified. The extent of this problem is still severely underestimated by coopers and barrel-users, due to the extremely unpredictable, localized contamination of the staves.

Catalytic performance and thermostability of chloroperoxidase in reverse micelle: achievement of a catalytically favorable enzyme conformation

The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40 °C, CPO essentially lost all its activity after 5 h incubation, while 58-76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50 °C, about 44-75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (V(pentanol)/V(isooctane)), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (k(cat)) was increased in reverse micelle. Moreover, the lower K(m) and higher k(cat)/K(m) demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV-vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.

Immobilization of chloroperoxidase onto highly hydrophilic polyethylene chains via bio-conjugation: Catalytic properties and stabilities

Chloroperoxidase (CPO) was covalently immobilized on poly(hydroxypropyl methacrylate-co-polyethyleneglycole-methacrylate) membranes, which were characterized, by swelling test, FT-IR spectroscopy, scanning electron microscopy, and contact angle measurement. The Km and Vmax values for free and immobilized CPO were found to be 34.6 and 47.2 μM, and 287.5 and 245.2 U/mg protein, respectively. The optimum pH for both the free and immobilized enzyme was observed at 3.0. The immobilized enzyme showed wide pH and temperature profiles. Most importantly, the increased thermal, storage and operational stability of immobilized CPO should depend on the creation of a comfortable strong hydrophilic microenvironment on the designed support to the host enzyme molecule.

Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach

The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid–base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25 °C. The activity constants ( k cat) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25 °C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70 °C. The rate of catalysis at temperatures above ~ 70 °C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~ 5. The results have been discussed in the light of understanding the structure–function relationship of the peroxidase properties of these thermostable heme proteins.

Low temperature photo-oxidation of chloroperoxidase Compound II

Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as − 50 °C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene. EPR spectra at 77 K gave the amount of ferric protein at each stage in the reaction sequence. The PN oxidation step gave a 6:1 mixture of Compound II and ferric CPO, the photolysis step gave an approximate 1:1 mixture of active oxidant and ferric CPO, and the final mixture after reaction with excess styrene contained ferric CPO in 80% yield. In single turnover reactions at − 50 °C, styrene was oxidized to styrene oxide in high yield. Kinetic studies of styrene oxidation at − 50 °C displayed saturation kinetics with an equilibrium constant for formation of the complex of K bind = 3.8 × 10 4 M − 1 and an oxidation rate constant of k ox = 0.30 s − 1 . UV–Visible spectra of mixtures formed in the photo-oxidation sequence at ca. − 50 °C did not contain the signature Q-band absorbance at 690 nm ascribed to CPO Compound I prepared by chemical oxidation of the enzyme, indicating that different species were formed in the chemical oxidation and the photo-oxidation sequence.

Analysis of Phosphorothionate Pesticides Using a Chloroperoxidase Pretreatment and Acetylcholinesterase Biosensor Detection

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the nervous system. It is inhibited by organophosphate and carbamate pesticides. However, this enzyme is only slightly inhibited by organophosphorothionates, which makes the detection of these pesticides analytically very difficult. A new enzymatic method for the activation and detection of phosphorothionates was developed with the capability to be used directly in food samples without the need of laborious solvent extraction steps. Chloroperoxidase (CPO) from Caldariomyces fumago was combined with tert-butyl hydroperoxide and two halides. Chlorpyrifos and triazophos were completely oxidized. Fenitrothion, methidathion and parathion methyl showed conversion rates between 54 and 61%. Furthermore, the oxidized solution was submitted to an AChE biosensor assay. Chlorpyrifos spiked in organic orange juice was oxidized, where its oxon product was detected in concentrations down to 5 microg/L (final concentration food sample: 25 microg/L). The complete duration of the method takes about 2 h.

The improvement of chloroperoxidase activities in the presence of ammonium salts and cationic surfactants

The catalytic activities of chloroperoxidase (CPO) including halogenation, oxidation, and peroxidation were investigated in the presence of ammonium salts: tetramethylammonium bromide (TMABr), tetraethylammonium bromide (TEABr), tetrapropylammonium bromide (TPABr), tetrabutylammonium bromide (TBABr), and cationic surfactants: dodecyltrimethylammonium bromide (DTABr) and cetyltrimethylammonium bromide (CTABr). All the mentioned activities were promoted in most cases. The highest modified activity (ABTS peroxidation) was 18.16 times higher in the presence of TMABr than that in pure buffer. The activity enhancement was strongly dependent on the concentration and the hydrophobic chain length of additives, and the structure of substrates. The kinetic parameters showed that the activation was mainly attributed to an increase in k(cat) due probably to a catalytically favorable conformation of CPO induced by the additives. Moreover, a lower K(m) and higher ratio of k(cat)/K(m) (specificity constant) was obtained, indicating that both the affinity and specificity of CPO to substrates were improved in the presence of additives.

Bienzymatic synthesis of benzothia/(oxa)zoles in aqueous medium

A series of 2-arylbenzothiazoles and 2-arylbenzoxazoles were synthesized by the reactions of aldehydes and 2-aminothiophenol and 2-hydroxythiophenol, respectively, using glucose oxidase (GOX)–chloroperoxidase (CPO) catalytic system under oxygen atmosphere.

ChemInform Abstract: Asymmetric Sulfoxidation of a β-Carbonyl Sulfide Series by Chloroperoxidase.

Abstract The chloroperoxidase (CPO)-catalyzed oxidation of a series of β-carbonyl sulfides to sulfoxides has been studied at room temperature in aqueous citrate buffer. For dialkyl β-carbonyl sulfides, the products with methyl and ethyl substituents are obtained in ca. 100% yield. However when the alkyl group is n- propyl or i -propyl the yield drops dramatically (25%). An aryl sulfide derivative afforded product in very low yield (4%), but when the phenyl group bears a carbonyl, and the sulfur substituents are methyl or ethyl, the oxidation occurs with high yields (91–95%). Steric control of the sulfoxidation reaction is also confirmed with cyclohexanone derivatives, where a low product yield is observed even at high enzyme concentrations. Noteworthy are the yields obtained with cyclopentanone sulfide (65%) and an unexpected quantitative yield obtained with the γ-butyrolactone sulfide.

New and classic families of secreted fungal heme peroxidases

Heme-containing peroxidases secreted by fungi are a fascinating group of biocatalysts with various ecological and biotechnological implications. For example, they are involved in the biodegradation of lignocelluloses and lignins and participate in the bioconversion of other diverse recalcitrant compounds as well as in the natural turnover of humic substances and organohalogens. The current review focuses on the most recently discovered and novel types of heme-dependent peroxidases, aromatic peroxygenases (APOs), and dye-decolorizing peroxidases (DyPs), which catalyze remarkable reactions such as peroxide-driven oxygen transfer and cleavage of anthraquinone derivatives, respectively, and represent own separate peroxidase superfamilies. Furthermore, several aspects of the "classic" fungal heme-containing peroxidases, i.e., lignin, manganese, and versatile peroxidases (LiP, MnP, and VP), phenol-oxidizing peroxidases as well as chloroperoxidase (CPO), are discussed against the background of recent scientific developments.

Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment

Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the “steady state kinetics” of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of “the product of interest” in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of “substrate concentration at maximum observed rate”. The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.

Covalent anchoring of chloroperoxidase and glucose oxidase on the mesoporous molecular sieve SBA-15.

Functionalization of porous solids plays an important role in many areas, including heterogeneous catalysis and enzyme immobilization. In this study, large-pore ordered mesoporous SBA-15 molecular sieves were synthesized with tetraethyl orthosilicate (TEOS) in the presence of the non-ionic triblock co-polymer Pluronic P123 under acidic conditions. These materials were grafted with 3-aminopropyltrimethoxysilane (ATS), 3-glycidoxypropyltrimethoxysilane (GTS) and with 3-aminopropyltrimethoxysilane and glutaraldehyde (GA-ATS) in order to provide covalent anchoring points for enzymes. The samples were characterized by nitrogen adsorption, powder X-ray diffraction, solid-state NMR spectroscopy, elemental analysis, diffuse reflectance fourier transform infrared spectroscopy and diffuse reflectance UV/Vis spectroscopy. The obtained grafted materials were then used for the immobilization of chloroperoxidase (CPO) and glucose oxidase (GOx) and the resulting biocatalysts were tested in the oxidation of indole. It is found that enzymes anchored to the mesoporous host by the organic moieties can be stored for weeks without losing their activity. Furthermore, the covalently linked enzymes are shown to be less prone to leaching than the physically adsorbed enzymes, as tested in a fixed-bed reactor under continuous operation conditions.