Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules

A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.     

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).     

重组角质酶的发酵制备及其对涤纶纤维的表面改性

对大肠杆菌表达嗜热子囊菌Thermobifida fusca角质酶的摇瓶诱导条件及3 L发酵罐扩大培养进行了研究,并探讨了角质酶对涤纶纤维的改性作用。结果表明,在摇瓶培养中,采用工业级TB培养基,用2 g/L乳糖诱导,菌体培养至对数生长前期添加0.5％甘氨酸,角质酶产量可达到128 U/mL。在3 L发酵罐扩大培养中,补料培养生物量 (OD600) 最大达到35,角质酶酶活最高达506 U/mL,是迄今国内外报道细菌来源角质酶的最高水平。紫外分光光度法分析初步表明涤纶纤维经角质酶水解产生了对苯二甲酸类物质     

角质酶在生物可降解聚酯聚己二酸/对苯二甲酸丁二醇酯降解中的应用

随着废弃塑料带来的环境污染越来越严重,生物可降解聚酯已成为大众关注的焦点。聚己二酸/对苯二甲酸丁二醇酯[poly(butylene adipate-co-terephthalate),PBAT]是脂肪族和芳香族共聚形成的生物可降解聚酯,兼具两者的优异性能。针对PBAT在自然条件下对降解环境要求严格且降解周期长的不足之处,本研究探究了角质酶在PBAT降解中的应用和对苯二甲酸-丁二醇酯(butylene terephthalate,BT)含量对PBAT生物降解性的影响,以实现对PBAT降解速率的提升。选取5种不同来源的聚酯降解酶对PBAT进行降解应用并比较出降解效果最优的酶,并测定了含有不同BT含量的PBAT聚酯的降解效率。结果表明,角质酶ICCG为降解效果最好的酶,且BT含量越高PBAT的降解率越低。此外,还确定了角质酶ICCG对高BT含量的PBAT(H)降解的最适温度、最适缓冲液类型、最适pH、最适E/S(enzyme to substrate)和最适底物浓度比分别为75℃、Tris-HCl、9.0、0.4%和1.0%。本研究结果可为角质酶在PBAT降解中的应用提供一定的理论依据和实验...     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability

Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (T_g) of PET, that is, 70°C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6°C improvement in the thermal unfolding temperature (T_m) and a 10‐fold increase in the half‐life of the enzyme activity at 60°C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the T_m of the global structure. Proteins 2016; 84:60–72. © 2015 Wiley Periodicals, Inc.     

Synthesis of multifunctional chitosan with galactose as a targeting ligand for glycoprotein receptor

Chitosan-O-PEG-galactose was synthesized through hydroxyl groups of chitosan, which followed several steps including protection of amino group of chitosan, pegylation of chitosan, galactosylation of pegylated chitosan, and final removal of protection to obtain chitosan-O-PEG-galactose. The synthesized intermediates and final product were characterized and confirmed by ¹H NMR and FTIR, and the amounts of PEG and galactose conjugated with chitosan were measured. The pegylated chitosan possesses amphiphilic property in terms of soluble in both neutral aqueous (e.g., water) and organic solvents (e.g., DMF, dichloromethane). The corresponding critical micelle concentration is measured to be 0.56 mg/mL, and the size of micelles is 294.5 ± 2.3 nm with polydispersity 0.123 ± 0.021. The contents of PEG and galactose conjugated in chitosan-O-PEG-galactose are 98.09 ± 4.63% w/w and 3.06 ± 0.54% w/w, respectively. In terms of the degree of O-substitution of chitosan by PEG (DS_PEG) and the degree of substitution of PEG by galactose (DS_g) are 177.69% and 86.7%, respectively. Exclusively high DS_PEG indicates both C6–OH and C3–OH of chitosan are conjugated with PEG polymer chains. Further prominent attachment of galactose onto hydroxyl end group of PEG allows chitosan-O-PEG-galactose to possess sufficient quantity of targeting moieties for asialoglycoprotein receptor on hepatocytes.     

Poly(ethylene terephthalate) (PET) degradation by Yarrowia lipolytica: Investigations on cell growth,enzyme production and monomers consumption

The development of technologies for poly(ethylene terephthalate) (PET) depolymerization, such as biocatalysis, has been pointed as a very promising alternative to chemical hydrolysis processes. This work aims to understand the behavior of Yarrowia lipolytica, a robust yeast for diverse applications, in the presence of molecules from the PET production chain such as monoethylene glycol (MEG), terephthalic acid (TPA), bis (2-hydroxyethyl) terephthalate (BHET), PET oligomers, amorphous PET and post-consumer PET. The yeast was cultivated in rich media with and without glucose addition, in order to compare monomers release or consumption. TPA and MEG were consumed more intensely in the absence of glucose. The addition of the diester BHET yielded a 3-fold increased lipase production both at 160 rpm (118 U/L) and 250 rpm (385 U/L). In addition, 250 rpm agitation also provided a higher consumption of TPA (26% increase). The mono(2-hydroxyethyl) terephthalate (MHET) was the main intermediate released during polymer hydrolysis, followed by TPA and BHET. Thus, the use of Yarrowia lipolytica, which is capable of catalyzing the PET hydrolysis is of great potential to reduce the environmental impacts caused by unappropriated disposal of packages.     

VFAs bioproduction from waste activated sludge by coupling pretreatments with Agaricus bisporus substrates conditioning

A novel strategy for improving volatile fatty acids (VFAs) bioproduction from waste activated sludge (WAS) was developed by coupling pretreatments with conditioning (CPC). Agaricus bisporus substrate (ABS) was used as external carbohydrate additive source of conditioning step. Pretreatment was studied in three ways: alkaline, alkaline-thermal and ultrasonic. WAS hydrolysis and protein degradation was distinctly improved by CPC treatments, resulting in a considerable enhancement of VFAs yield. The maximal VFAs yield was 614 ± 71, 712 ± 49 and 598 ± 19 mg COD/g VSS at pre-optimized alkaline, alkaline-thermal and ultrasonic CPC treatments, respectively, with an increase of 35%, 50% and 38% compared to the yields of pretreated WAS fermentation. Fourier transformed infrared spectroscopy and three-dimensional excitation-emission matrix fluorescence spectroscopy indicated that a synergistic effect occurred in co-digesting WAS and ABS. The conditioning of carbohydrate with feasible pretreatment provided large room for the digestibility improvement and the operation cost reduction in the whole WAS treatment system.     

Binding mechanisms for Thermobifida fusca Cel5A,Cel6B,and Cel48A cellulose-binding modules on bacterial microcrystalline cellulose

The family II cellulose-binding modules (CBM) from Thermobifida fusca Cel5A and Cel48A were cloned in the Escherichia coli/Streptomyces shuttle vector pD730, and the plasmids were transformed into Streptomyces lividans TKM31. CBM(Cel5A), and CBM(Cel48A), CBM(Cel6B) were expressed and purified from S. lividans. The molecular masses were determined by mass spectrometry, and the values were 10595 +/- 2, 10915 +/- 2, and 11291 +/- 2 Da for CBM(Cel5A), CBM(Cel6B), and CBM(Cel48A), respectively. Three different binding models (Langmuir, Interstice Penetration, and Interstice Saturation) were tested to describe the binding isotherms of these CBMs on bacterial microcrystalline cellulose (BMCC). The experimental binding isotherms of T. fusca family II CBMs on BMCC are best modeled by the Interstice Saturation model, which includes binding to the constrained interstice surface of BMCC as well as traditional Langmuir binding on the freely accessible surface. The Interstice Saturation model consists of three different steps (Langmuir binding, interstice binding, and interstice saturation). Full reversibility only occurred in the Langmuir region. The irreversibility in the interstice binding and saturation regions probably was caused by interstice entrapment. Temperature shift experiments in different binding regions support the interstice entrapment assumption. There was no systematic difference in binding between the two types of exocellulase CBMs--one that hydrolyzes cellulose from the nonreducing (CBM(Cel6B)) end and one that hydrolyzes cellulose from the reducing end (CBM(Cel48A)).     

Preparation and characterization of poly(ethylene glycol)-g-chitosan with water- and organosolubility

A new scheme was proposed for synthesizing poly(ethylene glycol)-g-chitosan (PEG-g-CS), where methoxy poly(ethylene glycol) iodide (MPEG-I) (M_n 2000) was used for N-substitution of triphenylmethyl chitosan (TPM-CS) in organic medium. The graft copolymers were obtained by subsequent removal of protecting groups with dichloroacetic acid. By varying PEG-I/TPM-CS feed ratio, the grafting levels (GL) of PEG can be adjusted. The chitosan derivatives were characterized by FTIR, ¹H NMR, ¹³C NMR and DSC. All the copolymers were soluble in water over wide pH range. Furthermore, organosolubility of the hybrids in DMF and DMSO was also achieved when the DS value more than 24%. The lysozyme degradation rate of the copolymers in aqueous neutral medium decreased with the increase of GL value.     

Chitosan cross-linked with poly(ethylene glycol)dialdehyde via reductive amination as effective controlled release carriers for oral protein drug delivery

The covalently cross-linked chitosan-poly(ethylene glycol)₁₅₄₀ derivatives have been developed as a controlled release system with potential for the delivery of protein drug. The swelling characteristics of the hydrogels based on these derivatives as the function of different PEG content and the release profiles of a model protein (bovine serum albumin, BSA) from the hydrogels were evaluated in simulated gastric fluid with or without enzyme in order to simulate the gastrointestinal tract conditions. The derivatives cross-linked with difunctional PEG₁₅₄₀-dialdehyde via reductive amination can swell in alkaline pH and remain insoluble in acidic medium. The cumulative release amount of BSA was relatively low in the initial 2 h and increased significantly at pH 7.4 with intestinal lysozyme for additional 12 h. The results proved that the release-and-hold behavior of the cross-linked CS–PEG₁₅₄₀H-CS hydrogel provided a swell and intestinal enzyme controlled release carrier system, which is suitable for oral protein drug delivery.     

Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor

A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics. Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria. One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed. Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing. Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed. The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed. The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon. Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample. The other groups occurred either in the lower or upper part, or in both samples. They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas.     

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.     

1H-NMR Study of the effect of synthetic polymers on the fluidity,transition temperature and fusion of dipalmitoyl phosphatidylcholine small vesicles

The interaction of water-soluble polymers with dipalmitoyl phosphatidylcholine small vesicles and the effect on vesicle fusion were studied by means of ¹H-NMR spectrometry. The motion of dipalmitoyl phosphatidylcholine molecules decreased on interaction with the polymers and was detected as a change in the signal intensity. The interaction behavior of polymers is very sensitive to the chemical structure of the applied polymers. Poly(styrene sulfonic acid) and poly(ethylene glycol) decreased the motion of the choline methyl group, predominantly through coulombic and hydrophobic interaction forces, respectively. For example, in the case of the poly(styrene sulfonic acid)-containing system, the signal intensity of the choline methyl group was decreased about 15% while those of the hydrophobic methylene and terminal methyl groups were scarcely decreased by the addition of polymer to a final concentration of 4.0 · 10^t-2 unit mol/1. These polymers are considered to interact with the surface of the vesicle membrane. On the other hand, poly(l-glutamic acid) and poly($\text{N-}\text{vinyl}\text{-2-}\text{pyrrolidone}$) decreased the signal intensities of not only the choline methyl group, but also those of the hydrophobic methylene and terminal methyl groups. This result suggest that part of these polymers might be incorporated into the hydrophobic region of the vesicle membrane.Addition of the non-ionic polymers inhibited vesicle fusion considerably. This effect was explained by the stabilization of dipalmitoyl phosphatidylcholine vesicles by complexation with these polymers.     

The interaction of phospholipid membranes with poly(ethylene glycol). Vesicle aggregation and lipid exchange

(1) The water soluble polymer, poly(ethylene glycol), causes aggregation of sonicated vesicles of dimyristoylphosphatidylcholine in a manner consistent with a steric exclusion mechanism. (2) Poly(ethylene glycol) promotes the exchange of lipids between multilamellar vesicles of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine when the lipids are in the liquid-crystalline state. (3) ³¹P-NMR studies demonstrate that the bilayer configuration of smectic mesophases of dipalmitoylphosphatidylcholine is substantially maintained in the presence of poly(ethylene glycol).     

The effect of storage and active ingredient properties on the drug release profile of poly(ethylene oxide) matrix tablets

Different molecular weight forms of poly(ethylene oxide) can be used successfully in controlled release drug delivery due to their excellent matrix forming properties. Drug release of these materials follows nearly zero order kinetics, and is mainly governed by polymer swelling and erosion and diffusion of drug molecules. Because of its partly amorphous structure, poly(ethylene oxide) undergoes structural changes caused by elevated temperature and relative humidity of the storage medium resulting in an increased drug release. This physical process can be highly influenced by the structure of different drug molecules, such as polymer-binding ability and hydration tendency. These properties of two basic drugs embedded into poly(ethylene oxide) matrices were characterized by molecular modelling and an attempt was made to reveal their effect on the change of drug release stability, a prerequisite of the marketing authorization of dosage forms. The findings suggest that both the hydration properties of the active ingredient and the molecular weight of the polymer influence the effect of physical ageing of poly(ethylene oxide) on the drug release properties of the matrix.     

Effects of poly(ethylene glycol) on liposomes and erythrocytes: Permeability changes and membrane fusion

Poly(ethylene glycol) 6000 induced a concentration-dependent, time-dependent decrease in the latency of the reaction between Arsenazo III sequestered in liposomes and extraliposomal Ca²⁺. This was mediated by a gross change in liposomal permeability, i.e. by a release of Arsenazo III from liposomes rather than simply by an entry of Ca²⁺. The loss of latency was strongly temperature-dependent, and it was markedly diminished on increasing the cholesterol content of the liposomes. It was apparently not due to an osmotic stress of the polymer. The high activation energy found (63 kJ · mol^?1) is thought to indicate that the loss of latency resulted from local discontinuities in the lipid bilayers, caused by dehydration, rather than from partial or total lysis. Related microscopy experiments indicated that the polymer also caused the liposomes to fuse, and it is suggested that membrane fusion may have occurred at the sites of dehydration-induced discontinuities in adjacent bilayers, in addition the polymer was found to enhance the permeability of hen erythrocytes to Ca²⁺ in a manner that was comparable to its effect on liposomal latency, and it is proposed that cell fusion induced by poly(ethylene glycol) may occur at the sites of similarly induced discontinuities in the phospholipid bilayers of two closely adjacent cells.     

Mercaptoheterocyclic ligands grafted on a poly(ethylene vinyl alcohol) membrane for the purification of immunoglobulin G in a salt independent thiophilic chromatography

In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 microg/cm2 of hollow fiber membrane surface area.     

The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation

Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.