Crystalline/amorphous phase structure and molecular mobility of biodegradable poly(butylene adipate-co-butylene terephthalate) and related polyesters

Differential scanning calorimetry (DSC), atomic force microscopy (AFM), wide-angle X-ray scattering (WAXD), and solid-state (13)C NMR have been used to investigate the crystalline/amorphous structure and molecular mobility of biodegradable poly(butylene adipate-co-44 mol % butylene terephthalate) [P(BA-co-44 mol % BT)] copolyester sample crystallized from the melt. The DSC endothermic peak, which is ascribed to the melting of the crystalline region, was broad relative to those reported for conventional partially crystalline polyesters. In AFM observation, spherulitic morphology was not observed while small particles with a size of about 100 nm were detected. The WAXD pattern of the sample was very broad. These results have indicated that a melt-crystallized P(BA-co-44 mol % BT) sample contains small crystals with a wide distribution in size. A solid-state (13)C NMR technique was also used to perform molecular-level and selective analyses for both butylene terephthalate and butylene adipate units. For the butylene terephthalate units, the existence of two components with different microstructure and molecular mobility was detected: one component was assigned to the alpha-form crystal of poly(butylene terephthalate) homopolymer (PBT) and the other was in amorphous regions. In contrast, all of butylene adipate units were located in amorphous regions. Solid-state NMR data have suggested that sizes of crystalline regions are less than 3 nm.     

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.     

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

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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.     

Molecular Dynamics Simulation of Collective Motions in Binary Liquids

Collective dynamic properties of different kind of binary liquid mixtures have been investigated by molecular dynamics simulation. The study includes both the longitudinal and the transverse current spectra in simple liquid alloys, 1:1 molten salts and liquid binary mixtures of neutral particles with an ionic-like structure. These systems were chosen as representative of binary liquids with different static structures in order to analyse the effects of structural ordering on the mechanisms of dynamic collective properties. The effect of the mass asymmetry between the two species in the mixture has been also discussed from the results for two different mass ratios for each kind of structure. Two length scales have been considered. On the one hand, the hydrodynamic scale (low wave numbers), where the modes for the partial currents of the two species are characterised by very close frequencies. On the other hand, the molecular scale (higher wave numbers), where the characteristic frequencies for the two species show noticeable differences. Vibrational concentration current modes (optic modes) have been found in neutral mixtures though their influence is rather weak, being the collective dynamic properties of this kind of systems dominated by the mass current modes (acoustic modes). On the contrary, in mixtures of charged particles such as molten salts the contribution of the concentration (charge) currents to the collective dynamics is important and optic modes can be characterised by a well-defined frequency for a wide range of wave numbers. It has been observed that heavy particles have a more relevant role on the mass current correlations whereas light particles play a dominant role on the concentration current correlations. The overall results for the three kinds of liquid mixtures analysed in this paper show that both the longitudinal and transverse current spectra are little dependent on the static structure of the system whereas marked differences are revealed when the particles in the system are either neutral or carry an electric charge.     

Molecular Dynamics Simulation of Hexamine and Suberic Acid

In order to perform a molecular dynamics (MD) simulation of the incommensurate crystalline structure hexamethylenetetramine suberate (C 6 H 12 N 4 )(HOOC-(CH 2 ) 6 -COOH), we present in a first step the separate simulations of the crystalline structure of each of the two pure components, hexamethylenetetramine (HMT) and suberic acid. The domain decomposition parallel MD program ddgmq is used for this purpose. A second-generation consistent force field (CFF91) is employed to describe the interactions between atoms. Starting from experimental crystal structures, both pure components were heated from low to high temperatures. Our MD results show that the HMT system can be well represented by CFF91. In the case of suberic acid the layered structure of the crystal is largely preserved although deviations in the unit cell lengths from the experimental values are ～10%. Rather than attempt a complex re-parametrisation of CFF91 we chose to impose a fixed compensating external pressure tensor to correct for the deficiencies of the chosen force field. After optimising this compensating external pressure tensor at one temperature we find that experimental lattice constants and angles can be well reproduced over a range of temperatures.     

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     

评论：塑料结合模块促进聚对苯二甲酸乙二醇酯的酶法降解

塑料的大量生产和无节制的使用已造成严重的环境污染。为了减少塑料废物对环境的影响,近年来塑料酶法降解已成为国内外研究者关注的热点。例如,通过蛋白质工程策略提高塑料降解酶催化活性和热稳定性,进一步提高酶法降解的效率。另外,通过融合酶策略将塑料结合模块与塑料降解酶融合,也可以促进塑料降解。近期发表在期刊Chem Catalysis的一项研究表明,采用碳水化合物结合模块融合策略可以在低浓度(<10 wt%)的底物聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]中提高塑料降解酶的活性。但是在高浓度底物(10 wt%−20 wt%)中,该策略无法提高PET的酶法降解。该项研究对于采用塑料结合模块促进酶法降解塑料具有重要的指导意义。     

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.     

Determination of the interparticular effective diffusion coefficient for CO(2) and O(2) in solid state fermentation

A simple experimental diffusion controlled fermentor (DCF), coupled with the use of a mathematical model based on mass balance, is proposed to measure the variation of the gas (CO(2) and O(2)) diffusion coefficients in solid state fermentation. The DCF was packed with an ion-exchange resin impregnated with a nutritive medium and inoculated with Aspergillus niger. The growth conditions in the DCF were very similar to those found in equipment operated with convective oxygen supply. The diffusion coefficient was shown to be very dependent on the biomass concentration within the solid state fermentor, and attained values of less than 5% of the molecular diffusion in air when the biomass in the fermentor reached 27 mg dry/g dry support.     

Molecular Dynamics Simulation of Limiting Conductance for Li + Ion in Supercritical Water using Polarizable Models

We report results of molecular dynamics simulations of the limiting conductance of Li + ion in ambient water and in supercritical water using polarizable models for water and Li + . The limiting conductances of Li + in ambient water calculated from mean square displacement (MSD) using four points transferable intermolecular potential model (TIP4P), extended simple point charge model (SPC/E), and revised polarizable model 1 (RPOL1) are larger than the experimental value. The behavior of the limiting conductance of Li + in supercritical water using the RPOL models results in good agreement with experimental results for the limiting conductance of LiCl. The agreement of the RPOL1 model with the experimental results is much better than the RPOL2 model in the higher-density regime, whereas that of the RPOL2 model is much better than the RPOL1 model in the lower-density regime. Using the RPOL models (in contrast to the SPC/E model), the number of hydration water molecules around Li + is the dominating contributor to the limiting conductance in the higher-density regime. In agreement with the SPC/E model, the interaction strength between Li + and the hydration water molecules is a non-factor in the lower-density region since the potential energy per hydration water molecule decreases with decreasing water density at the lowest water densities.     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Micellization phenomena of amphiphilic block copolymers based on methoxy poly(ethylene glycol) and either crystalline or amorphous poly(caprolactone-b-lactide)

This study focuses on the aggregation behavior of the biodegradable amphiphilic block copolymers based on methoxy poly(ethylene glycol) (mPEG) as a hydrophilic block and either crystalline poly(caprolactone-b-l-lactide) (P(CL-LLA)) or amorphous poly(caprolactone-b-D,L-lactide) (P(CL-DLLA)) as a hydrophobic block. These block copolymers have a strong tendency to form micelles in aqueous medium, with very low critical micelle concentrations (CMCs). The CMC of P(CL-LLA)-b-mPEG is higher than that of P(CL-DLLA)-b-mPEG when the mPEG block has the same molecular weight. Furthermore, the partition equilibrium coefficient (K(v)) of pyrene in the micellar solution of P(CL-LLA)-b-mPEG copolymer was lower than that of P(CL-DLLA)-b-mPEG copolymer when the mPEG block was the same length. These differences were believed to be related to the physical state of the core-forming blocks, i.e., the crystalline P(CL-LLA) block and the amorphous P(CL-DLLA) block. The TEM images showed that micelles formed by P(CL-LLA)-b-mPEG assembled in a cylindrical morphology, whereas those formed by P(CL-DLLA)-b-mPEG took a classical spherical shape. In addition, with differential scanning calorimetry (DSC) and wide-angle X-ray diffraction (WAXD) analyses, it is believed that the crystallization tendency of the core-forming blocks is the main factor governing the morphology of micelles in water. A possible mechanism for the cylindrical assembly morphology was discussed.     

Structural and Dynamical Studies of Humanin in Water and TFE/Water Mixture: A Molecular Dynamics Simulation

Summary

Proline-rich peptides are known to adopt preferentially the extended polyproline II (PPII) helical conformation, which is involved in several protein-protein recognition events. By resorting to molecular modelling techniques, we wished to investigate the extent to which PPII helices could be used for the formation of isohelical peptide-DNA complexes leading to the selective recognition of the major groove of B-DNA. For that purpose, we have grafted to a cationic intercalator, 9-amino-acridine, an oligopeptide having the sequence: Pro-Arg-Pro-Pro-Arg-Pro-Pro-Arg-Pro-Pro-Asp-Pro-Pro. Each residue in the sequence was set in the D configuration, to prevent enzymatic hydrolysis, and each Arg residue was designed to target O6/N7 of a guanine base following the intercalation site. The Asp residue was designed to target a cytosine base, whilst simultaneously forming a bidentate complex with the Arg three residues upstream. Energy-minimization, using the JUMNA procedure, led to the following conclusions: 1) major groove binding is favoured over minor groove or exclusive binding to the phosphates by large energy differences, of over 50 and 90 kcal/mole, respectively; 2) the two best bound sequences are those having three successive guanine bases on the same DNA strand, immediately adjacent to the intercalation site. Sequence d(CGGGC G), encountered in the Primer Binding Site of the HIV retrovirus, thus ranks amongst the best-bound sequences; 3) replacement of an individual guanine amongst the three ones upstream of the intercalation site, by an adenine base, weakens by > 6 kcal/mole the binding energetics; 4) the conformational rigidity of the DNA-bound PPII helix should enable for a modulation of the base sequence selectivity, by appropriate replacements of the Arg and Asp residues. Thus sequence CGGCAAG, also encountered in the HIV genome, could be targeted by an oligopeptide having the sequence Pro-Arg-Pro-Pro-Asp-Pro-Pro- Asn-Pro-Pro-Asn-Pro-Pro-Arg-Ala.     

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources

Poly(ethylene terephthalate) (PET) is one of the most consumed plastics in the world. The development of efficient technologies for its depolymerization for monomers reuse is highly encouraged, since current recycling rates are still very low. In this study, 16 commercial lipases and cutinases were evaluated for their abilities to catalyze the hydrolysis of two PET samples. Humicola insolens cutinase showed the best performance and was then used in reactions on other PET sources, solely or in combination with the efficient mono(hydroxyethyl terephthalate)-converting lipase from Candida antarctica. Synergy degrees of the final titers of up to 2.2 (i.e., more than double of the concentration when both enzymes were used, as compared to their use alone) were found, with increased terephthalic acid formation rates, reaching a maximum of 59,989 µmol/L (9.36 g/L). These findings open up new possibilities for the conversion of post-consumer PET packages into their minimal monomers, which can be used as drop in at existing industrial facilities.     

Classical Molecular Dynamics Simulation of Kappa Squared Factor in Resonance Energy Transfer for Linear Dipole Models

Molecular dynamics simulations of linear models interacting through a dipolar Kihara intermolecular potential are presented. Molecular orientation correlations are used to calculate the orientational factor kappa squared in the resonance energy transfer (RET) as a function of the intermolecular separation. The distance, R 0 (2/3), at which the simulated systems show an isotropic behavior is calculated and an analysis of the dependence of R 0 (2/3) on microscopic properties (molecular aspect ratio and dipole moment) as well on thermodynamics (temperature and density) is presented. An explanation of the use of metallic cations as probes in RET is given and some relations of our models with biological molecules are pointed out.     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.     

Photoreduction of O(2) Primes and Replaces CO(2) Assimilation

A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the first 20 seconds of illumination after a dark period, when no net O₂ evolution or CO₂ uptake was observed, O₂ evolution was normal but completely compensated by O₂ uptake. Similarly, when CO₂ uptake was totally or partially inhibited, O₂ evolution proceeded at a high (near maximal) rate. Under all conditions, O₂ uptake balanced that fraction of the O₂ evolution which could not be accounted for by CO₂ uptake.