Distribution of Tyrosine Phenol Lyase in Microorganisms

The distribution of tyrosine phenol lyase activity in microorganisms was studied with intact cells in a synthetic reaction mixture containing l-serine and phenol or pyrocatechol. This activity was found in various bacteria, most of which belonged to the Enterobacteriaceae; especially to the genera Escherichia, Proteus and Erwinia. Cells of Erwinia herbicola ATCC 21434 were selected as a promising source of enzyme.

Intact cells of Erwinia herbicola ATCC 21434 prepared from a broth cultured for 24 hr contained markedly high enzymic activity and catalyzed the synthetic reaction of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from l-serine and phenol or pyrocatechol in significantly high yields.

Results of the isolation and identification of the products showed that the amino acid synthesized by this enzymatic method was identical with l-tyrosine or l-dopa.     

Purification and Characterization of an Esterase Involved in Poly(vinyl alcohol) Degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45°C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K _m and V _max of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 μM, and 6.52 and 12.6 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.     

Preparation of single or double-network chitosan/poly(vinyl alcohol) gel films through selectively cross-linking method

A selectively cross-linking method, which is based on the “di–diol” interaction between poly(vinyl alcohol) and borate and the strong electrostatic interaction between chitosan and tripolyphosphate, was developed. Chitosan/poly(vinyl alcohol) films cross-linked separately with borate, tripolyphosphate and borate/tripolyphosphate were then prepared in terms of this method. Water vapor permeation, mechanical strength, surface morphology and molecular interactions of the films were studied by water permeation test, texture test, atomic force microscopy and ATR-FTIR spectroscopy. With the introduction of cross-linking structure, there is a large improvement in elastic modulus from 271 ± 14.2 to 551 ± 14.7 MPa and a large decrease in water vapor permeability from (5.41 ± 0.21) × 10⁻⁷ g/m h Pa to (3.12 ± 0.24) × 10⁻⁷ g/m h Pa of chitosan/poly(vinyl alcohol) films. The surface morphology of the cross-linked films exhibits a nanoparticle aggregation structure. The size and aggregation behavior of these nanoparticles are strongly related to the type of cross-linker. Furthermore, ATR-FTIR results indicate that strong interaction between polymer matrix and cross-linker exists in our system. This work provides a simple and efficient way to prepare chitosan/poly(vinyl alcohol) films with controllable network structure.     

Immobilization of hog pancreas lipase in macroporous poly(vinyl alcohol)-cryogel carrier for the biocatalysis in water-poor media

Hog pancreas lipase was covalently attached to the beads of poly(vinyl alcohol)-cryogel – a macroporous hydrogel prepared by means of freeze-thaw technique. The immobilized biocatalyst thus obtained was examined in the reaction of enantioselective hydrolysis of the ethyl ester of N-benzylidene derivative of DL-phehylalanine in the medium of acetonitrile (contained 5 vol.% of water without any buffers). Eighty-three %-enantiomeric excess of the l-amino acid was reached after 144 h. Virtually the same result was obtained in the repeated use of the same immobilized biocatalyst after its 6-months-storing in a refrigerator.     

Research on the Long Time Swelling Properties of Poly (vinyl alcohol)/Hydroxylapatite Composite Hydrogel

Poly(vinyl alcohol)/hydroxylapatite(PVA/HA)composite hydrogel was prepared by repeated freezing and thawing.Thewater loss properties of the resultant hydrogel were investigated by using optical microscope.Long time immersion tests ofPVA/HA composite hydrogel were carried out in the diluted calf serum solution to study the change laws of swelling propertieswith the freezing-thawing cycles and HA content.The micro-morphologies of PVA/HA composite hydrogel after long timeimmersion were observed by means of the high-accuracy 3D profiler.The results show that the swelling process of PVA/HAcomposite hydrogel is the converse process of its water loss.Long time swelling ratio curves of PVA/HA composite hydrogel inthe calf serum solution are manifested as four stages of quick increase,decrease,slow decrease and stable balance,and itsequilibrium swelling ratio decreases with the increase of freezing-thawing cycles and HA content.It is revealed that the networkstructure of the composite hydrogel immersed for a long period is significantly improved with the increase of HA content.Perfect network structures of PVA/HA composite hydrogel as well as full and equilibrium tissues after swelling equilibrium areobtained when the HA content is 3% and the number of freezing-thawing cycles is 7.     

Bovine Serum Albumin Binding and Drug Delivery Studies with PVA-Ferrofluid

This paper describes a single-step method for the biomimetic synthesis of stably suspended magnetite nanoparticles in poly(vinyl alcohol) termed ferrofluids. The challenge is to synthesize water based stable magnetic colloids with a control over the particle size and morphology for biomedical applications. The polymer possibly plays a dual role of a surfactant and a functionalizing agent. Transmission electron microscopy, infrared spectroscopy and vibrating sample magnetometry were used to investigate the properties of the synthesized ferrofluids. It has a strong affinity towards the tryptophan residues in bovine serum albumin protein as determined from the fluorescence emission studies. For in vivo applications this could indirectly mean a resistance to immune response and thus ensure long-term circulation. The ability of the synthesized ferrofluid to bind a che-motherapeutic drug ceftriaxone and its ionic release was observed. The polymer hydroxyl group allows drug-binding and the magnetic property allows targeting to specific sites. Magnetic hybrid fluids with combined advantages of magnetism and polymer open up new perspectives for applications.     

Activity and Stability of Native and Modified Subtilisins in Various Media

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.     

流动注射复合酶电极法测定麦芽糖的研究

利用聚乙烯醇(PVA)包埋法共固定糖化酶和葡萄氧化酶(GOD)制成酶膜,与氧电极结合成为复合酶电极;该电极可以用于流动注射分析系统中测定溶液中麦芽糖.对酶电极的pH效应和温度效应作了研究.酶电极的线性范围为0.5-35mmol/L.响应周期小于2min.变异系数(CV)为1.8%.在半连续使用状态下,酶电极可以使用10d以上.糖化酶保持活力为60%.对延长酶电极寿命和克服共存葡萄糖的干扰的方法作了探讨.     

Ion-Conductive Poly(vinyl alcohoi)-Based IPMCs

Partially crosslinked and sulfonated poly(vinyl alcohol) (s-PVA) membranes were prepared as ion-conductive matrices of Ionic Polymer-Metal Composite (IPMC) and a new IPMC based on the s-PVA membrane was fabricated via an electroless plating procedure of platinum. PVA was reacted with sulfosuccinic acid (SSA) as a crosslinking agent with a sulfonic group and 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS) as a side chain with a sulfonic group. The crosslinked s-PVA membranes were characterized using a FT-IR spectroscope and a scanning electron microscope-combined energy-dispersive X-ray spectrometer and were assessed in terms of water absorption, proton conductivity, and the feasibility of electroless plating, Among the prepared ionomers, the s-PVA membrane obtained at 20 wt.% SSA and 10 wt.% EPPS (S20E10 membrane) registered the highest proton conductivity of 2.9 × 10~(-2) S·m~(-1), which corresponds to one third of that of Nafion series, and only the S20E10 membrane was successfully plated via the electroless plating method without any crack and broken part. The s-PVA-based IPMC showed the one-directional displacement with 1-minute-long time-lapse comparable to typical Nafion-based IPMCs. However, the displacement under an AC potential was very limited due to its slow deformation response and the actuation performance was severely varied with actuation time including the short service life of several minutes in air. The short and variable actuation of the s-PVA-based IPMC was attributed to its large variation of surface and ionic resistances during air-operation, which is induced by the low ratio of bound to free water.     

Rheological characterisation of a novel thermosensitive chitosan/poly(vinyl alcohol) blend hydrogel

Thermosensitive hydrogels that are triggered by changes in environmental temperature thus resulting in in situ hydrogel formation have recently attracted the attention of many investigators for biomedical applications. In the current work, the thermosensitive hydrogel was prepared through the mixture of chitosan (CS), poly(vinyl alcohol) (PVA) and sodium bicarbonate. The mixture was liquid aqueous solutions at low temperature (about 4 °C), but a gel under physiological conditions. The hydrogel was characterized by FTIR, swelling and rheological analysis. The effect of hydrogel composition and temperature on both the gel process and the gel strength was investigated from which possible hydrogel formation mechanisms were inferred. In addition, the hydrogel interior morphology as well as porosity of structure was evaluated by scanning electron microscopy (SEM). The potential of the hydrogels as vehicles for delivering bovine serum albumin (BSA) were also examined. In this study, the physically crosslinked chitosan/PVA gel was prepared under mild conditions without organic solvent, high temperature or harsh pH. The viscoelastic properties, as investigated rheologically, indicate that the gel had good mechanical strength. The gel formed implants in situ in response to temperature change, from low temperature (about 4 °C) to body temperature, which was very suitable for local and sustained delivery of proteins, cell encapsulation and tissue engineering.     

Prevalent Mutations of Human Prion Protein: A Molecular Modeling and Molecular Dynamics Study

Abstract

Point mutations in the human prion protein gene, leading to amino acid substitutions in the human prion protein contribute to conversion of PrP^C to PrP^Sc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia. We have investigated impressions of prevalent mutations including Q217R, D202N, F198S, on the human prion protein and compared the mutant models with wild types. Structural analyses of models were performed with molecular modeling and molecular dynamics simulation methods. According to our results, frequently occurred mutations are observed in conserved and fully conserved sequences of human prion protein and the most fluctuation values occur in the Helix 1 around residues 144–152 and C-terminal end of the Helix 2. Our analysis of results obtained from MD simulation clearly shows that this long-range effect plays an important role in the conformational fluctuations in mutant structures of human prion protein. Results obtained from molecular modeling such as creation or elimination of some hydrogen bonds, increase or decrease of the accessible surface area and molecular surface, loss or accumulation of negative or positive charges on specific positions, and altering the polarity and pKa values, show that amino acid point mutations, though not urgently change the stability of PrP, might have some local impacts on the protein interactions which are required for oligomerization into fibrillar species.     

Synthesis and properties of carrageenan grafted copolymer with poly(vinyl alcohol)

The aim of this work was to prepare a carrageenan-g-poly(vinyl alcohol) (CG-g-PVA) polymer using potassium persulphate as an initiator. The effect of different ratios of the polymer blends on the parameters of the grafted polymer was investigated. The grafting ratio decreased with an increase of the CG content in the graft copolymer. The resulting CG-g-PVA was characterized by ATR-FTIR, tensile strength, elongation at break, swelling ratio, contact angle and biodegradation in soil. From the ATR-FTIR the 3,6-anhydride-galactose of the CG showed a peak at 927 cm⁻¹ that was absent in the CG-g-PVA and the ether linkage of PVA-g-CG between the hydroxyl group of PVA and the 3,6-anhydride-galactose of CG showed a peak at 1089 cm⁻¹ in the graft copolymer. The tensile strength and elongation at break decreased with an increase of the CG due to its phase separation. The highest tensile strength was observed at 2:8 CG/PVA. In addition, the swelling ratio decreased and the contact angle increased as a function of the increase of the CG in the grafted copolymer. The best ratio of CG-g-PVA was 2:8 CG/PVA. This graft copolymer was easily biodegraded in natural soil.     

Amyloid fibrils as functionalizable components of nanocomposite materials

Amyloid fibrils are a form of protein nanofiber that show promise as components of multifunctional bionanomaterials. In this work, native bovine insulin and bovine insulin that had been previously converted into amyloid fibrils were combined with poly(vinyl alcohol) (PVOH) via solution casting to determine the effect of fibrillization on the thermomechanical properties of the resulting composite. The synthesis method was found to preserve the amyloid fibril structure and properties of the resulting fibril-PVOH composite were investigated. At a filling level of 0.6 wt %, the fibril-reinforced PVOH was 15% stiffer than the PVOH control. Various properties of the films, including the glass transition temperature, degradation temperature, microstructure, and film morphology were characterized. Although more work is required to optimize the properties of the composites, this study provides proof of principle that incorporation of amyloid fibrils into a polymeric material can impart useful changes to the mechanical and morphological properties of the films.     

Contribution of proline to the pre-structuring tendency of transient helical secondary structure elements in intrinsically disordered proteins

Background

IDPs function without relying on three-dimensional structures. No clear rationale for such a behavior is available yet. PreSMos are transient secondary structures observed in the target-free IDPs and serve as the target-binding “active” motifs in IDPs. Prolines are frequently found in the flanking regions of PreSMos. Contribution of prolines to the conformational stability of the helical PreSMos in IDPs is investigated.

Methods

MD simulations are performed for several IDP segments containing a helical PreSMo and the flanking prolines. To measure the influence of flanking-prolines on the structural content of a helical PreSMo calculations were done for wild type as well as for mutant segments with Pro → Asp, His, Lys, or Ala. The change in the helicity due to removal of a proline was measured both for the PreSMo region and for the flanking regions.

Results

The α-helical content in ~ 70% of the helical PreSMos at the early stage of simulation decreases due to replacement of an N-terminal flanking proline by other residues whereas the helix content in nearly all PreSMos increases when the same replacements occur at the C-terminal flanking region. The helix destabilizing/terminating role of the C-terminal flanking prolines is more pronounced than the helix promoting effect of the N-terminal flanking prolines.

General significance

This work represents a novel example demonstrating that a proline is encoded in an IDP with a defined purpose. The helical PreSMos presage their target-bound conformations. As they most likely mediate IDP-target binding via conformational selection their helical content can be an important feature for IDP function.     

A new strain, Streptomyces venezuelae GY1, producing a poly(vinyl alcohol)-degrading enzyme

Summary An actinomycete strain, which could produce an extracellular poly(vinyl alcohol) (PVA)-degrading enzyme, was isolated from a PVA-contaminated soil sample using PVA as the sole carbon source. The strain was identified as Streptomyces venezuelae according to the whole-nucleotide-sequence analysis of 16S rDNA, the morphological and the physiological characteristics. The strain produced 120 U/l extracellular PVA-degrading enzyme when PVA was used as the sole carbon source. When glucose was used as the sole carbon source, however, the extracellular enzyme activity was very low (12 U/l). This is the first report showing that an actinomycete strain can produce a PVA-degrading enzyme.     

Poly(vinyl alcohol) acetoacetate-based tissue adhesives are non-cytotoxic and non-inflammatory

Polymer-based tissue adhesives composed of poly(vinyl alcohol) acetoacetate (PVOH acac) and cross-linking amines were investigated for their effects on cell survival and inflammatory cell activation using in vitro mouse cell cultures. Cytotoxicity of tissue adhesives was evaluated by placing adhesives in direct contact with 3T3 fibroblast cells. Tissue adhesives formulated from PVOH acac and 3-aminopropyltrialkoxysilane (APS) were non-cytotoxic to fibroblasts; adhesives formulated from PVOH acac and aminated poly(vinyl alcohol) (PVOH amine) were also non-cytotoxic to fibroblasts. In contrast, a commercial adhesive composed of 2-octyl cyanoacrylate was highly cytotoxic to fibroblasts. The inflammatory potential of tissue adhesives was evaluated by exposing J774 macrophage cells to adhesives, and measuring TNF-α release from macrophages. PVOH acac-based tissue adhesives did not elicit inflammatory TNF-α release from macrophages. These results suggest that PVOH acac-based tissue adhesives are non-cytotoxic and non-inflammatory. Such tissue adhesives represent a promising technology for a variety of medical applications, including surgical wound closure and tissue engineering, and the results are also significant in the design of in vitro cell culture systems to study biomaterials.