In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were ...successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their k cat and K m values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed k cat and K m values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A 1340/A 1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.
A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and ...thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli. Both fusion enzymes and the native one had comparable k cat values in the range of 311 to 342 s–1 on pNP-butyrate, while the catalytic efficiencies k cat/K m decreased from 0.41 s–1/ μM (native enzyme) to 0.21 and 0.33 s–1/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.
•Morphine is used for chronic pain management and ends up in waste water and hospital effluents.•The opiate morphine is a substrate for laccases.•Free or immobilized laccase is eliminating morphine.
...Pharmaceuticals contaminate the environment for several reasons, including metabolic excretion after intake, industrial waste and improper disposal. The narcotic drug morphine is commonly utilized for chronic pain management, and the distribution of morphine in aqueous systems and in waste waters is of high concern. Here, the removal of morphine by a laccase from Myceliophthora thermophila both in its free form as well as immobilized on Accurel MP1000 beads was investigated. Complete morphine elimination was achieved within 30 min for the free and the immobilized enzyme (70% bound protein) for concentrations between 1 and 1,000 mg L−1 according to LC-TOF mass spectrometry analysis. Higher morphine concentrations up to 60 g L−1 were also tested and total elimination was achieved within 6 h. Therefore, laccases are ideal candidates for removing morphine from aqueous systems.
A new esterase from Thermobifida halotolerans (Thh_Est) was cloned and expressed in E. coli and investigated for surface hydrolysis of polylactic acid (PLA) and polyethylene terephthalate (PET). ...Thh_Est is a member of the serine hydrolases superfamily containing the -GxSxG- motif with 85–87% homology to an esterase from T. alba, to an acetylxylan esterase from T. fusca and to various Thermobifida cutinases. Thh_Est hydrolyzed the PET model substrate bis(benzoyloxyethyl)terephthalate and PET releasing terephthalic acid and mono-(2-hydroxyethyl) terephthalate in comparable amounts (19.8 and 21.5 mmol/mol of enzyme) while no higher oligomers like bis-(2-hydroxyethyl) terephthalate were detected. Similarly, PLA was hydrolyzed as indicated by the release of lactic acid. Enzymatic surface hydrolysis of PET and PLA led to a strong hydrophilicity increase, as quantified with a WCA decrease from 90.8° and 75.5° to 50.4° and to a complete spread of the water drop on the surface, respectively.
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•Fatty acids were covalently attached to lignocellulosic material.•Covalent bonding was demonstrated using lignin model substrates.•Environmentally friendly alternative to the known ...methods of functionalizing wood.
Two enzyme based strategies for hydrophobic functionalization of lignocellulose materials were developed and mechanistically compared using sinapic acid and the dimer syringylglycerol β-guaiacyl ether as models respresenting (hardwood) lignin substructures for coupling fatty acid esters. Coupling of lipase/hydrogen peroxide treated methyl linoleate to sinapic acid indeed resulted in a 1:1 coupling product with an m/z peak at 575.5 measured with HPLC–MS. Using laccase for coupling, oligo/polymerization of sinapic acid seems to prevent a coupling-reaction to methyl linoleate. However, methyl linoleate was successfully coupled by laccase 1:1 onto syringylglycerol β-guaiacyl ether through the ether bond at position four. The efficient enzyme mediated incorporation of fatty acid esters as hydrophobic molecules were further confirmed when triglycerides were used to treat veneers resulting in a water contact angle increase from 58.3° to 93.5°. Thus, this study demonstrates for the first time that both (laccase mediator and lipase-hydrogen peroxide) systems can act as promising strategies for introducing fatty acid esters in wood leading to increased hydrophobization.
Alkyd resins are polyesters containing unsaturated fatty acids that are used as binding agents in paints and coatings. Chemical drying of these polyesters is based on heavy metal catalyzed ...cross-linking of the unsaturated fatty acid moieties. Among the heavy-metal catalysts, cobalt complexes are the most effective, yet they have been proven to be carcinogenic. Therefore, strategies to replace the cobalt-based catalyst by environmentally friendlier and less toxic alternatives are under development. Here, we demonstrate for the first time that a laccase-mediator system can effectively replace the heavy-metal catalyst and cross-link alkyd resins. Interestingly, the biocatalytic reaction does not only work in aqueous media, but also in a solid film, where enzyme diffusion is limited. Within the catalytic cycle, the mediator oxidizes the alkyd resin and is regenerated by the laccase, which is uniformly distributed within the drying film as evidenced by confocal laser scanning microscopy. During gradual build-up of molecular weight, there is a concomitant decrease of the oxygen content in the film. A new optical sensor to follow oxygen consumption during the cross-linking reaction was developed and validated with state of the art techniques. A remarkable feature is the low sample amount required, which allows faster screening of new catalysts.
A laccase-mediator system is used as the drying agent in alkyd resin films and replaces the toxic heavy-metal based catalyst. This approach is a more environmentally friendly and less toxic alternative for the coating industry.
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► Cloning and expression of a polyamidase from Nocardia farcinica. ► Partial enzymatic hydrolysis of polyamide fabrics generates new reactive groups. ► Laccase mediated coupling of ...phenolics to polyamide.
A two step enzymatic process for grafting phenolics onto polyamides (PAs) was developed in order to impart special functionalities to inert PA. Therefore, a polyamidase (NfpolyA) from Nocardia farcinica was overexpressed in Escherichia coli BL21-Gold(DE3) and purified in a single step. With p-nitroacetanilide as a substrate, NfpolyA revealed a specific activity of 20Umg−1 compared to 1.5Umg−1 for the wild-type enzyme. NfpolyA showed a KM value of 0.12±0.01mM and a kcat of 19.1s−1 which were both higher than measured for the wild-type enzyme (kcat=3.5s−1; KM=0.06mM). A laccase from Trametes hirsuta was used to oxidize ferulic acid, used as a phenol model substrate and study the covalent grafting of n-butylamine, as model substrate for PA. According to LC–MS, up to three equivalents of n-butylamine were bound to ferulic acid after laccase oxidation of ferulic acid. Both enzymes were used sequentially in a two step process. In a first step the polyamidase is used to partially hydrolyze the amide bond, leading to a surface with amine and carboxylic acids. In a second step, by using a laccase from T. hirsuta ferulic acid was grafted onto the surface of PA as confirmed with FTIR-ATR analysis.
A new cutinase from Thermobifida alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica ...cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7° to 45.0°. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed Km values of 213 and 1933 μM and kcat values of 2.72 and 6.03 s−1 respectively. The substrate specificity was investigated with bis(benzoyloxyethyl)terephthalate (3PET) and Tha_Cut1 was shown to release primarily 2-hydroxyethyl benzoate. This contrasts with the well-studied Humicula insolens cutinase which preferentially liberates terminal benzoic acid from 3PET.
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