Synthesis of novel photoresponsive molecularly imprinted polymer microspheres with special binding properties

An azobenzene‐containing molecularly imprinting polymer microsphere with photoresponsive binding properties toward 2,4‐dichlorophenoxyacetic acid (2,4‐D) was successful prepared via silica surface polymerization. The number‐average diameters of silica and imprinting polymer microsphere are 0.5 and 0.7 μm, respectively. The static adsorption, binding and selectivity experiments were performed to investigate the adsorption properties and recognition characteristics of the polymers for 2,4‐D. The equilibrium adsorptive experiments indicated that 2,4‐D‐SMIP(surface molecularly imprinted polymers) has significantly higher adsorption capacity for 2,4‐D than its nonimprinted polymers (SNIP).The binding constant K_d and apparent maximum number Q_max of the imprinted polymer were determined by Scatchard analysis as 0.054 mmol L^?1 and 0.167 mmol g^?1_, respectively. The result of photoregulated release and uptake of 2,4‐D experiment demonstrated that azo‐containing SMIP can make use of light and change it into mechanical properties to release and take up the template molecules. It means that the SMIP can be controlled by light. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 869‐876, 2013     

Precipitation polymerization of molecularly imprinted polymers for recognition of melamine molecule

Molecular imprinted polymer microspheres (MIPs) were prepared by precipitation polymerization using melamine as template molecule, methacrylic acid (MAA) as functional monomer, trimethylol‐propane trimethacrylate (TRIM) as crosslinking agent, acetonitrile as solvent and dispersion medium. Release of the template was performed by continuous extraction with methanol containing 10% acetic acid. The microspheres were observed by scanning electron microscopy (SEM). The perfect microspheres were produced when the addition of crosslinker was 7.48 mmol. The binding capacity of MIPs was examined, Q_max = 68.36 μmol g^?1, and the dissociation constant at binding site of MIPs, K_d = 0.761, was estimated. Compared with the performance of conventional imprinted polymer, the imprinted microspheres showed high selectivity in special binding to template molecule. The imprinted microspheres could be used as the stationary phase in HPLC or SPE for selective extraction of melamine in daily products. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Water‐compatible imprinted polymers based on CS@SiO2 particles for selective recognition of naringin

Molecularly imprinted polymers are being proposed for the development of novel biorecognition elements for active components. In this study, an imprinted chitosan coated silica nanoparticles (I‐CS@SiO₂) polymer was prepared by a simple procedure, in which, naringin (NG) with antioxidant activity, acted as a template, silica as a matrix and CS as a functional polymer. The binding properties were discussed by the equilibrium binding experiment method. Experiments show that the adsorption characteristics of I‐CS@SiO₂ are better than that of nonimprinted polymer. It exhibited high selectivity for NG when compared with the nonimprinted polymer, with an imprinting factor α of 1.74. Scatchard analysis of the I‐CS@SiO₂ indicated that there was a class of binding sites during the I‐CS@SiO₂ recognizing NG: The dissociation constant of K_D is 0.016 mmol L^?1, the maximum apparent binding capacity of B_max is 6.56 μmol g^?1. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40491.     

Biosorption of phenolic compounds by the brown alga Sargassum muticum

Phenol, 2‐chlorophenol (2‐CP), and 4‐chlorophenol (4‐CP) biosorption on Sargassum muticum, an invasive macroalga in Europe, has been investigated. The efficiency of this biosorbent was studied measuring the equilibrium uptake using the batch technique. A chemical pre‐treatment with CaCl₂ has been employed in this study in order to improve the stability as well as the sorption capacity of the algal biomass. The influence of pH on the equilibrium binding and the effect of the algal dose were evaluated. The experimental data at pH = 1 have been analysed using Langmuir and Freundlich isotherms. It was found that the maximum sorption capacity of chlorophenols, q_max = 251 mg g^?1 for 4‐CP and q_max = 79 mg g^?1 for 2‐CP, as well as that of a binary mixture of both chlorophenols, q_max = 108 mg g^?1, is much higher than that of phenol, q_max = 4.6 mg g^?1. Moreover, sorption kinetics have been performed and it was observed that the equilibrium was reached in less than 10 h. Kinetic data have been fitted to the first order Lagergren model, from which the rate constant and the sorption capacity were determined. Finally, biosorption of the phenolic compounds examined in the present study on Sargassum muticum biomass was observed to be correlated with the octanol‐water partitioning coefficients of the phenols. This result allows us to postulate that hydrophobic interactions are the main responsible for the sorption equilibrium binding. Copyright © 2006 Society of Chemical Industry     

Caffeine Molecular Imprinted Microgel Spheres by Precipitation Polymerization

Imprinted uniform microgel spheres were prepared by precipitation polymerization. Acetonitrile was used as the dilute solvent with MAA as the monomer, EDMA as the crosslinker and caffeine as the print molecule. Comparison of caffeine adsorption on molecular imprinted and blank microgel spheres was made. Langmuir model was used to fit the adsorption data. It was found that the caffeine imprinted microgel spheres show specific binding sites to the target molecules. A binding study of caffeine on imprinted microgel spheres was made by Scatchard analysis; the dissociation constants (K_D) and the maximum binding capacity were K_D= 1.84×10⁻⁴mol/L,Q _max = 16.98 μmol/g for high affinity binding site and K_D=1.33×l0⁻³ mol/L, Q_max=46.84 μmol/g for lower affinity binding site, respectively This microgel spheres can be useful affinity adsorbents in further applications.     

Preparation of pH and temperature dual‐sensitive molecularly imprinted polymers based on chitosan and N‐isopropylacrylamide for recognition of bovine serum albumin

pH and temperature dual‐sensitive protein imprinted microspheres with high absorption capacity have been successfully synthesized on the surface of SiO₂ using chitosan grafted N‐isopropylacrylamide (CS‐g‐NIPAM) as the pH and temperature sensitive monomer, with acrylamide as comonomer, N,N′‐methylenebisacrylamide as the crosslinking agent and bovine serum albumin (BSA) as the template protein. The pH and temperature dual‐sensitivity was also investigated. The results showed that the adsorption capacity and imprinting factor improved slowly with increasing incubation pH from 4.6 to 7.0, and then decreased sharply in alkaline conditions due to the reduction of non‐specific binding from electrostatic and hydrogen bonding interactions. Fourier transform infrared spectroscopy, thermogravimetric analysis and transmission electron microscopy were used to characterize the polymers. The as‐prepared SiO₂@BSA molecularly imprinted polymers were also found to have high adsorption capacity (119.88 mg g^?1) within 2 h, an excellent imprinting factor (α = 2.25), specific selectivity and good reusability. © 2019 Society of Chemical Industry     

Evaluation of a new water‐compatible hybrid molecularly imprinted polymer combined with restricted access for the selective recognition of folic acid in binding assays

This article deals with the synthesis of an organic?inorganic hybrid molecularly imprinted poly(methacrylic acid‐trimethylolpropane trimethacrylate)/SiO₂ combined with restricted access. Fourier Transform Infrared Spectroscopy, Transmission Electron Microscopy, Thermogravimetric Analysis, and textural data were used to characterize the structure of material. Higher selectivity was obtained for the molecularly imprinted polymer with restricted access when compared to nonimprinted polymer with restricted access, confirming the imprinting effect. The folic acid sorption for the polymers molecularly imprinted with restricted access, nonimprinted with restricted access, and molecularly imprinted, exhibited the following values of 5.6, 4.5, and 4.8 mg g^?1, respectively. Kinetic experimental data were very well adjusted to Elovich and pseudo‐second‐order models, which suggest that the sorption of folic acid takes place in binding sites with different energies. The obtained values of enthalpy, entropy and Gibbs free energy of ?12.79 kJ mol^?1, ?36.38 K^?1 J mol^?1, and ?1.76 kJ mol^?1, respectively, showed that the sorption of folic acid onto molecularly imprinted polymer combined with restricted access occurs spontaneously, is of both exothermic and of physical nature with an increase of ordering at the solid?solution interface. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43463.     

Adsorptive removal of technetium‐99 using macroporous poly(GMA‐co‐EGDMA) modified with diethylene triamine

The possibility of sorption of technetium‐99 in the form of pertechnetate anion (TcO₄^?) and the sorption kinetics for removing TcO₄^? from aqueous solution by chelating polymers based on glycidyl methacrylate (GMA) were investigated. Two samples of macroporous crosslinked poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) (PGME), with different amount of the crosslinker (ethylene glycol dimethacrylate, EGDMA), were synthesized by suspension copolymerization and functionalized with diethylene triamine (deta). We propose that nonspecific sorption of pertechnetate anion via electrostatic interactions takes place at the protonated amino groups of macroporous crosslinked copolymer. The results of batch experiments performed at pH 1–14 showed fast sorption kinetics for removing TcO₄^? by amino‐functionalized PGME‐deta in a wide range of pH, that is, from 1.0 to 9.0. Almost complete removal of TcO₄^? (91–98%) was reached within 180 min in the stated pH range (1.0–9.0), with the sorption half‐times of under 25 min. The partitioning coefficients of linear adsorption isotherms, with 180‐min equilibrium time, reach the high values of 2130 mL g^?1 and 1698 mL g^?1 for the two samples of synthesized PGME‐deta. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Characterization of a chelating resin functionalized via azo spacer and its analytical applicability for the determination of trace metal ions in real matrices

A selective flame atomic absorption spectrometric method has been developed for the determination of trace amount of metal ions after preconcentration on salicylic acid (SA) loaded Amberlite XAD‐4 resin (AXAD‐4) at suitable pH. The chelating resin AXAD‐4‐SA was characterized based on FTIR, thermal and chemical stability, and hydrogen ion capacity. The sorption capacity was found to be 245.0, 156.2, 155.0, 145.0, 125.0, 122.5, and 70 μmol g^?1 for Cu(II), Cr(III), Zn(II), Cd(II), Mn(II), Ni(II), and Co(II), respectively with t_1/2 <15 min. All the metals could be eluted by 5 mL of 4 mol L^?1 HCl/HNO₃ resulting in high preconcentration factor of 200–360 up to a low preconcentration limit of 5.5–10 μg L^?1. The accuracy and precision of the developed method was checked by analyzing standard reference materials. The experimental values were not statistically significant from the certified values with <5% RSD. The detection limits were found to be 0.42, 0.57, 0.63, 0.77, 0.94, 0.96, and 1.41 μg L^?1, respectively. The analytical utility of the AXAD‐4‐SA for preconcentration and determination of metal ions was explored by analyzing river, canal, sewage, and tap water by direct as well as standard addition method. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Molecularly imprinted polyethersulfone microfibers for the binding and recognition of bisphenol A

Bisphenol A (BPA)‐imprinted polyethersulfone (PES) microfibers for the recognition of BPA were prepared by means of wet‐spinning method based on the liquid–liquid phase separation technique. Two kinds of the BPA‐imprinted microfibers were fabricated with the diameters of about 85 μm and 265 μm, respectively. Extraction of BPA from the microfibers was carried out by immersing the imprinted microfibers into ethanol. In the recognition experiments, the binding amounts were about 24.0, 43.0, and 70.0 μmol/g for the nonimprinted microfibers, whereas those were about 50.0, 87.0, 120.0 μmol/g for the imprinted PES microfibers with the diameter of about 85 μm in 100, 200, and 400 μM BPA solution, respectively. Both of the binding amounts and the adsorption rate for the imprinted microfibers with smaller diameter were higher than that for the microfibers with larger diameters. The selectivity results indicated that the BPA‐imprinted microfibers showed higher recognition coefficient (α) for BPA than phenol. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

In situ synthesis of monolithic molecularly imprinted stationary phases for liquid chromatographic enantioseparation of dibenzoyl tartaric acid enantiomers

A monolithic molecularly imprinted polymer (monolithic MIP) for dibenzoyl-D-tartaric acid (D-DBTA) was prepared in a stainless-steel chromatographic column tube (50?mm?×?4.6?mm I.D.) as HPLC stationary phase through in situ polymerization. By optimizing polymeric and chromatographic conditions, the chiral separation of DBTA enantiomers was successfully achieved in the obtained MIP in less than 25?min with a resolution Rs?=?1.25, whereas no enantioseparation effect was found on the monolithic non-imprinted polymer (NIP). Thermodynamic data of the enantioseparation were calculated. The results revealed that two different thermodynamic processes existed within the temperature range investigated, moreover, just at the transition temperature (50?°C) of the two processes, separation factor ?? reached its maximum. Scathcard analysis indicated that only one class of binding sites existed in the obtained MIP, with its K _d and Q _max estimated to be 5.457?×?10^?4?mol?L^?1 and 229.6???mol?g^?1, respectively. Nitrogen adsorption experiment proved that the prepared MIP had a large specific surface area of 105?m²?g^?1. Scanning electron microscopy showed that large flow-through pores were present in the prepared monolith. As a consequence, the column backpressure was only 1.2?MPa with acetonitrile as mobile phase at a flow rate of 1.0?mL?min^?1.     

Influence of synthesis conditions on the production of molecularly imprinted polymers for the selective recovery of isovaleric acid from anaerobic effluents

Two molecularly imprinted polymers (MIPs) – poly(methacrylic acid‐co‐TRIM) (TRIM, trimethylolpropanetrimethacrylate) and poly(acylamide‐co‐TRIM) – were synthesized in different solvents for the selective recovery of isovaleric acid (template) generated during the anaerobic digestion process. The chemical and structural characterizations of the synthetic adsorbent were carried out by Fourier transform infrared spectroscopy, TGA and porosimetry through N₂ adsorption–desorption isotherms. The selective and adsorptive performances of the imprinted polymers were evaluated by kinetic, isothermal, thermodynamic and selectivity studies and by adsorbent reuse experiments. The poly(methacrylic acid‐co‐TRIM) synthesized with dimethyl sulfoxide:chloroform presented higher selectivity and adsorption capacity for isovaleric acid in the presence of six volatile fatty acids. The kinetic results were well adjusted to the pseudo‐nth order and intraparticle diffusion models, leading to k values of 10^?4 and 6 × 10^?5 for the best synthesis of MIPs and not‐imprinted polymers, respectively. Moreover, the Sips model best described the adsorption isotherm and generated a maximum adsorption capacity of ca 209 mg g^?1 (at 25 °C). Cycles of MIP use–desorption–reuse indicated that the selective adsorbent performed better than commercial adsorbents, losing less than 3% of adsorption capacity after three cycles. © 2018 Society of Chemical Industry     

Preparation of a surface molecular‐imprinted adsorbent for Ni2+ based on Penicillium chrysogenum

A new chitosan molecular‐imprinted adsorbent was prepared from the mycelium of waste biomass. The results showed that an adsorbent using Penicillium chrysogenum mycelium as the core material was better than one derived from peanut coat. The adsorption capacity of the surface‐imprinted adsorbent for Ni²⁺ was enhanced by increasing the chitosan concentration in the imprinting process. Epichlorohydrin was better than glutaraldehyde as a cross‐linking agent; the optimal imprinted Ni²⁺ concentration for preparing the surface‐imprinted adsorbent was 2 mg (Ni²⁺) g^?1 of mycelium. The adsorption capacity of the surface‐imprinted adsorbent was 42 mg g^?1 (at 200 mg dm^?3 initial metal ions concentration) and twice that of the mycelium adsorbent. The surface‐imprinted adsorbent can be reused for up to 15 cycles without loss of adsorption capacity. Copyright © 2005 Society of Chemical Industry     

Fe2+-modified Vermiculite for the Removal of Chromium (VI) from Aqueous Solution

A novel adsorbent: Fe²⁺-modified vermiculite was prepared in a two-step reaction. Adsorption experiments were carried out as a function of pH, contact time, and concentration of Cr(VI). It was found that Fe²⁺-modified vermiculite was particularly effective for the removal of Cr(VI) at pH 1.0. The adsorption of Cr(VI) reached equilibrium within 60 min, and the pseudo-second-order kinetic model best described the adsorption kinetics. The adsorption data follow the Langmuir model more than the Freundlich model. At pH 1.0, the maximum Cr(VI) sorption capacity (Q _max ) was 87.72 mg · g^?1. Desorption of Cr(VI) from Fe²⁺-modified vermiculite using NaOH treatment exhibited a higher desorption efficiency by more than 80%. The sorption mechanisms including electrostatic interaction and reduction were involved in the Cr (VI) removal. The results showed that Fe²⁺-modified vermiculite can be used as a new adsorbent for Cr(VI) removal which has a higher adsorption capacity and a faster adsorption rate.     

Selective molecularly imprinted polymer nanofiber sorbent for the extraction of bisphenol A in a water sample

Novel molecularly imprinted polymer nanofibers (MIP‐NFs) were prepared for the adsorption of bisphenol A (BPA) in a water sample using the sol–gel process and the electrospinning technique. The effects of a number of synthesis parameters on the adsorption efficiency were investigated. The successful removal of BPA from MIP‐NFs was studied using UV–visible spectroscopy. The prepared MIP‐NFs were characterized by Fourier transform infrared, field emission SEM, TEM and energy dispersive X‐ray analysis. The results showed that the required molar ratio of 3‐aminopropyltriethoxysilane (APTES) to BPA was 15:1, which indicates a good performance in the rebinding test. Likewise, the molar ratio of APTES:acid:water was 1:2:9. The nylon 6 polymer solution, with a concentration of 12 wt%, showed a maximum adsorption capacity for BPA due to a decrease in the nanofiber diameter and an increase in the accessible sites. Furthermore, the maximum adsorption capacity of BPA was achieved at pH 7. Concerning the binding of BPA on MIP‐NFs, the experimental data matched well with the pseudo‐second‐order kinetics data and the Sips isotherm model. The saturated binding capacity for MIP‐NFs was predicted to be 115.1 mg g^?1, which was more than twice as high as that for non‐imprinted polymer nanofibers (46.82 mg g^?1). The results obtained in this study confirmed that the prepared MIP‐NFs showed considerable binding specificity for BPA in comparison with similar structural compounds such as phenol, naphthol and Naphthol AS, in aqueous solution. The binding capacity of MIP‐NFs remained almost constant after five cycles of reuse. The real sample analysis indicated that MIP‐NFs could be utilized as a useful sorbent material for the extraction of BPA from a water sample.     

Double‐functional characteristics of a surface molecular imprinted adsorbent with immobilization of nano‐TiO2

A new chitosan molecular imprinted adsorbent obtained by immobilization of nano‐TiO₂ on the adsorbent surface (surface‐imprinted adsorbent with nano‐TiO₂) was prepared. Based on photocatalytic reaction and the surface molecular imprinting technology, this new kind of surface‐imprinted adsorbent with immobilization of nano‐TiO₂ can not only adsorb template metal ions but can also degrade organic pollutants. The results showed that, after the nano‐TiO₂ was immobilized on the adsorbent surface, the adsorption ability for the imprinted ion (Ni²⁺) of this new imprinted adsorbent immobilized with nano‐TiO₂ was not affected, but the degradation ability for p‐nitrophenol (PNP) of the surface‐imprinted adsorbent with nano‐TiO₂ increased three‐fold compared with that of the surface‐imprinted adsorbent without nano‐TiO₂, from 23.8 to 76.1% (at an initial PNP concentration of 20 mg·dm^?3). The optimal TiO₂ concentration in the adsorbent preparation was 0.025 g·TiO₂ g^?1 adsorbent. The removal capacity for PNP reached 60.25 mg·g^?1 (at 400 mg·dm^?3 initial PNP concentration) under UV irradiation. The surface‐imprinted adsorbent with nano‐TiO₂ can be reused for at least five cycles without decreasing the removal ability for PNP and the imprinted ion (Ni²⁺). Copyright © 2006 Society of Chemical Industry     

Using polystyrene‐co‐maleic acid for molecularly imprinted membranes prepared in supercritical carbon dioxide

Supercritical CO₂ (ScCO₂) has been used to prepare molecularly imprinted polymers of poly(styrene‐co‐maleic acid) (PSMA) for targeted uracil (URA). The condition of ScCO₂ was on 16 MPa between 35 and 50°C. The resultant imprinted membranes prepared at 35 and 50°C bound URA with 9.2 ± 0.10 and 12.6 ± 0.06 μmol g⁻¹, respectively. Competitive binding studies were undertaken in binary substrate solution containing each of URA/DMURA, URA/Thymine, and URA/Cytosine with 2 μM. The URA imprinted membrane showed high separation factor (α) with 17 for both URA/DMURA and URA/Thymine and for URA/Cytosine, α = 13. Results strongly suggested that the URA imprinted membrane had effective selectivity hydrogen bonding to separately bind in the binary components to the template. Effect of organic solvents on the URA imprinting in ScCO₂ was also studied, in addition to comparison of properties with those obtained in both ScCO₂ and water. Evidence presented that ScCO₂ medium was effective to prepare the URA imprinted membrane. We discussed that ScCO₂ fluid was efficient to fix the shape of URA template into the PSMA membrane through hydrogen bonding. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Synthesis of amine‐functionalized block copolymers for nanopollutant removal from water

Polyamines are rare in literature owing to increased reactivity, sensitivity to air and moisture, low stability, and processing difficulties. Here, we report the synthesis and characterization of highly processable polyamines and use them for the removal of dissolved metallic nanoparticles from water. Three amphiphilic block polyamines such as poly(N‐aminoethyl acrylamide‐b‐styrene), poly(N‐aminopropyl acrylamide‐b‐styrene), and poly(N‐aminoxylyl acrylamide‐b‐styrene) have been synthesized using atom transfer radical polymerization of ethyl acrylate and styrene followed by aminolysis of the acrylic block. The polymerization and properties of the polymers are studied using different physicochemical techniques. Surface morphology of films prepared from these block copolymers by dissolving in different solvents such as chloroform, tetrahydrofuran and N,N‐dimethylformamide, and drop‐casting polymers on a glass substrate show interesting porous films and spherical nanostructures. In addition, the amine‐functionalized block copolymers have been used for the removal of nanoparticles from water and show high extraction efficiency toward silver (Ag) and gold (Au) nanoparticles. All three amine‐functionalized block copolymers show higher extraction capacities (Q_e) toward Au NPs (50–109 mg g^?1) and Ag NPs (99–117 mg g^?1). Our approach allows us to make amine‐functionalized block copolymers which are stable in air and can be easily processed in nonpolar solvents. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40943.     

3,3′‐Diindolylmethane imprinted polymers: synthesis,characterization and analytical procedure for template isolation from biological matrix

A novel sorbent for the separation of 3,3′‐diindolylmethane (DIM) has been developed. DIM was used as a template molecule to prepare twelve imprinted polymers from seven different functional monomers in six various porogens in the presence of ethylene glycol dimethacrylate as a crosslinker. The binding capacity showed that the highest binding specificity was achieved when allylamine as the monomer and carbon tetrachloride as the porogen were used to form bulk polymers. Binding capacities were 130.4 ± 2.8 µg g^?1 for imprinted MIP7_a polymer versus 49.03 ± 0.44 µg g^?1 for non‐imprinted NIP7_a polymer. An imprinting factor of 3.12 reflected the high specificity of MIP7_a. A solid‐phase extraction protocol was fully optimized. Two methanol–water systems were selected as the most effective for loading and washing steps, while acetonitrile was chosen as the most efficient eluent suitable for recovery of 91.7 ± 3.8% of DIM. Optimal concentration of DIM and volume of loaded sample were also examined. A loaded volume of 2.5 mL was the most appropriate for 0.625 and 1.25 mg L^?1. The use of MIP7_a for the separations of DIM from spiked bovine serum albumin was evaluated, showing a total recovery of 87.7 ± 3.5%. The commercial sorbent C18 was not suitable for such an application. © 2013 Society of Chemical Industry     

Catalysis of an Essential Step in Vitamin B2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione 5′‐phosphate ( 6 ), was purified about 800‐fold from a riboflavin‐producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (v_max, 12 μmol mg^?1 min^?1; K_M, 54 μm ) and of FMN (v_max, 25 μmol mg^?1 min^?1; K_M, 135 μm ). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.