Biocatalytic surface modification of knitted fabrics made of poly (ethylene terephthalate) with hydrolytic enzymes from Thermobifida fusca KW3b

Knitted fabrics made from poly(ethylene terephthalate) fibres were treated with an enzyme preparation from Thermobifida fusca KW3b showing hydrolytic activity on p-nitrophenyl butyrate. The fabrics were also treated with NaOH and the results were compared. Both enzyme- and alkaline-treated fabrics showed an increase in reactive dye uptake, vertical wicking height and water absorption capacity of the fabrics indicating an increased surface hydrophilicity. However, X-ray photoelectron (XPS) and energy-dispersive spectroscopy (EDS) did not give conclusive results on the presence of newly introduced hydrophilic groups on the surface of the fibres. Analysis of the enzyme-treated poly(ethylene terephthalate) fibres by scanning electron microscopy (SEM) and by atomic force microscopy (AFM) indicated an increase in surface roughness of the fibres, which may contribute to the observed increased hydrophilicity of the PET fabrics. However, much longer treatment times (24 h) were required to obtain these effects with the enzymes compared to the chemical treatment (1 h).     

Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca

Bacterial cutinases are promising catalysts for the modification and degradation of the widely used plastic polyethylene terephthalate (PET). The improvement of the enzyme for industrial purposes is limited due to the lack of structural information for cutinases of bacterial origin. We have crystallized and structurally characterized a cutinase from Thermobifida fusca KW3 (TfCut2) in free as well as in inhibitor-bound form. Together with our analysis of the thermal stability and modelling studies, we suggest possible reasons for the outstanding thermostability in comparison to the less thermostable homolog from Thermobifida alba AHK119 and propose a model for the binding of the enzyme towards its polymeric substrate. The TfCut2 structure is the basis for the rational design of catalytically more efficient enzyme variants for the hydrolysis of PET and other synthetic polyesters.     

聚对苯二甲酸乙二醇酯水解酶研究进展

塑料自20世纪首次合成以来给人类生活带来了极大的便利。然而,塑料稳定的高分子结构导致了塑料废弃物的持续堆积,对生态环境和人类健康均造成严重威胁。聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]是产量最高的一种聚酯类塑料,近年来PET水解酶的相关研究展现出生物酶法对塑料进行降解、回收的巨大潜力,也为塑料生物降解机制研究建立了参考范例。本文综述了不同微生物来源的PET水解酶及其PET降解能力,阐述了最具代表性的PET水解酶—IsPETase降解PET的催化机理,并总结了近年来通过酶工程改造而获得的高效降解酶,为未来的PET降解机制研究、PET高效降解酶的进一步挖掘和改造提供参考。     

Enzymatic surface modification of poly(ethylene terephthalate)

This study unambiguously confirms hydrolysis using cutinase of the persistent synthetic polymer poly(ethylene terephthalate), the most important synthetic fiber in the textile industry by direct measurement and identification of the different hydrolysis products. In this aqueous heterogeneous system, dissolved cutinase from Fusarium solani pisi acts on different solid poly(ethylene terephthalate) substrates. The extent of hydrolysis was detected by measuring the amount of (soluble) degradation products in solution using reversed-phase HPLC. Crystallinity greatly affects the capability of the enzyme to hydrolyze the ester bonds, displaying relatively high activity towards an amorphous polyester film and little activity on a highly crystalline substrate. The enzyme is sufficiently stable, hydrolysis rate on the amorphous substrate maintained at sufficient high level over a long period of time of at least five days. From an industrial point of view it is highly recommended to increase the hydrolysis rates.     

Functionalization of catalase for a photochemical immobilization on poly(ethylene terephthalate)

The enzyme catalase (EC 1.11.1.6) was covalently immobilized on textile carrier fabrics made of poly(ethylene terephthalate) (PET) by a novel combined wet chemical and photochemical process. The functionalization of catalase with allyl groups succeeds in a wet chemical treatment of the enzyme with allylglycidylether. This modified enzyme was bonded covalently to the textile material by a photochemical immobilization using a monochromatic excimer UV lamp (222 nm). Using this two-step procedure nearly 60 mg enzyme/g carrier could be fixed durably. The efficiency of the immobilization products was investigated by measuring the enzymatic decomposition of hydrogen peroxide in comparison to the free enzyme. The relative activity of the catalase after the immobilization was nearly 5% compared to the free, not fixed enzyme; however, even after 30 reuses, the modified and immobilized catalase still showed a distinct activity.     

Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Upcycling of poly(ethylene terephthalate) to produce high-value bio-products

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Solvent-free vapor-phase photografting of acrylamide onto poly(ethylene terephthalate)

Poly(ethylene terphthalate) (PET) films were photografted under reduced pressure in a solvent-free vapor of acrylamide and a co-initiator, benzophenone. Characterization of grafted samples by ESCA and contact angles showed that the grafting increased with grafting time and temperature. The amide groups obtained by the acrylamide grafting were converted into amine groups by the Hofmann rearrangement to be used in coupling reactions. The amine groups were confirmed by reaction with pentafluorobenzoyl chloride, which provides a fluorine label for ESCA. Surface grafting of polymeric substrates in the vapor phase induced by plasma or high energy and UV irradiation is reviewed.     

RGD peptides micro-patterning on poly(ethylene terephthalate) surfaces

The aim of this study was to evaluate the impact of RGD micro-patterned poly(ethylene terephthalate) (PET) on human osteoblast progenitor (HOP) cells attachment. Biomimetic modifications were performed by means of a four-step reaction: surface hydrolysis, oxidation in order to create COOH functions, coupling agent grafting (EDC, NHS) and finally immobilization of peptides. In addition to homogeneous or statistically distribution of peptides, micro-patterns of RGD were generated by: optical photolithography and UV excimer laser ablation. Modification steps were validated by physico-chemical techniques: XPS was used to prove covalent grafting at each stage of the surface functionalization, toluidine blue assay and high resolution µ-imager (using [3H]-Lys) to evaluate peptide densities and validate micro-patterns formation. Finally, the efficiency of this biomodification of PET was demonstrated onto homogeneous surfaces by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC.     

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His₆ tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L⁻¹ could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.     

Cloning and heterologous expression of a beta-D-mannosidase (EC 3.2.1.25)-encoding gene from Thermobifida fusca TM51

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.     

Characterization of a new extracellular hydrolase from Thermobifida fusca degrading aliphatic-aromatic copolyesters

The paper describes the purification, biochemical characterization, sequence determination, and classification of a novel thermophilic hydrolase from Thermobifida fusca (TfH) which is highly active in hydrolyzing aliphatic-aromatic copolyesters. The secretion of the extracellular enzyme is induced by the presence of aliphatic-aromatic copolyesters but also by adding several other esters to the medium. The hydrophobic enzyme could be purified applying a combination of (NH(4))SO(4)-precipitation, cation-exchange chromatography, and hydrophobic interaction chromatography. The 28 kDa enzyme exhibits a temperature maximum of activity between 65 and 70 degrees C and a pH maximum between pH 6 and 7 depending on the ion strength of the solution. According to the amino sequence determination, the enzyme consists of 261 amino acids and was classified as a serine hydrolase showing high sequence similarity to a triacylglycerol lipase from Streptomyces albus G and triacylglycerol-aclyhydrolase from Streptomyces sp. M11. The comparison with other lipases and esterases revealed the TfH exhibits a catalytic behavior between a lipase and an esterase. Such enzymes often are named as cutinases. However, the results obtained here show, that classifying enzymes as cutinases seems to be generally questionable.     

Functionalization of poly(ethylene terephthalate) fibers by photografting of a carbohydrate derivatized with a phenyl azide group

Grafting of a new carbohydrate UV-reactive molecule, an azidophenyl lactamine (AzPhLac), was achieved on fibers of three different diameters: 12, 18, and 32 microm. Adsorption of AzPhLac on fibers was obtained by using the dip-coating method in solution. The effect of the solution concentration on surface density and yield of grafted AzPhLac was investigated. Surface densities in the range 3-67 nmol/cm2 were obtained without marked difference related to the diameter of the fiber. Quantitative grafting was obtained with a surface of fiber of 1 cm2 and the lowest concentration (0.5 mM) of AzPhLac solution. The surface density and grafting yield decreased with the available surface of the fibers. This phenomenon could be attributed to a masking core-shell effect with outer fibers in the shell preventing the UV grafting of the fibers located in the core of the fibers' bundles. Scanning electron (SEM) and atomic force (AFM) microscopic observations suggested that homogeneous grafting might be obtained.     

Production of butyric acid by a cellulolytic actinobacterium Thermobifida fusca on cellulose

Thermobifida fusca not only produces cellulases, hemicellulases and xylanases, but also excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.     

Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca

An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm(-1) and form 2 with ν(FeC) and ν(CO) at 518 and 1920 cm(-1), respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.     

Drug release from and hydrolytic degradation of a poly(ethylene glycol) grafted poly(3-hydroxyoctanoate)

Monoacrylate-poly(ethylene glycol)-grafted poly(3-hydroxyoctanoate) (PEGMA-g-PHO) copolymers were synthesized to develop a swelling-controlled release delivery system for ibuprofen as a model drug. The in vitro hydrolytic degradation of and the drug release from a film made of the PEGMA-g-PHO copolymer were carried out in a phosphate buffer saline (pH 7.4) medium. The hydrolytic degradation of the copolymer was strongly dependent on the degree of grafting (DG) of the PEGMA group. The degradation rate of the copolymer films in vitro increased with increasing DG of the PEGMA group on the PHO chain. The copolymer films showed a controlled delivery of ibuprofen to the medium in periods of time that depend on the composition, hydrophilic/hydrophobic characteristics, initial drug loading amount and film thickness of the graft copolymer support. The drug release rate from the grafted copolymer films was faster than the rate of weight loss of the films themselves. In particular, a combination of the low DG of the PEGMA group in the PHO chains with the low ibuprofen solubility in water led to long-term constant release from these matrices in vitro.     

RGD peptides grafting onto poly(ethylene terephthalate) with well controlled densities

The aim of this study was to graft RGD peptides with well controlled densities onto poly(ethylene terephthalate) (PET) film surfaces. Biomimetic modifications were performed by means of a four-step reaction procedure: surface modification in order to create -COOH groups onto polymer surface, coupling agent grafting and finally immobilization of peptides. The originality of this work is to evaluate several grafted densities peptides. Toluidine blue and high-resolution mu-imager (using [(3)H]-Lys) were used to evaluate densities. Moreover, mu-imager has exhibited the stability of peptides grafted onto the surface when treated under harsh conditions. Benefits of the as-proposed method were related to the different concentrations of peptides grafted onto the surface as well as the capacity of RGD peptide to interact with integrin receptors.     

Mapping the substrate binding site of phenylacetone monooxygenase from Thermobifida fusca by mutational analysis

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.     

Production of thermostable xylanases in batch and continuous culture by Thermomonospora fusca KW 3

Summary The actinomycete Thermomonospora fusca KW 3 produced novel thermostable xylanases in batch and continuous cultures in media containing insoluble xylan. The production of xylanases could be induced with oat spelt or beech xylan. Very low activities were detected when the strain was grown on glucose or xylose. In continuous cultivations, mycelial wall growth could be prevented using a stirrer speed controller. Homogeneous mixing of the insoluble substrate was obtained by vibrating the flexible tubes. T. fusca KW 3 could be grown on insoluble xylan at growth rates as high as 0.23 h^–1, equivalent to a doubling time of 3 h. Xylanase activity decreased from maximum levels of 2.5 units (U) ml^–1 with increasing dilution rate and was nearly constant at a level of 0.5 U ml^–1 with dilution rates greater than 0.1 h^–1. Correspondence to: P. Röthlisberger