Lipase-Catalyzed Synthesis of Poly(1,4-Butanediol Succinate) in Organic Solvent

During the last decade, lipase has gained interest as a biocatalyst for synthesis in organic solvent systems. The paper describes the lipase catalyzed oligocondensation of bis(2-chloroethyl) succinate and 1,4-butanediol to obtain poly (1,4-butanediol succinate). The reaction was carried out at 37°C in organic solvents without any addition of water. Various lipases and solvents were screened to obtain a maximum degree of polymerization. Based on gel permeation chromatography, the highest average molecular weight of the oligomer obtained was 1570 g/mol with a polydispersity of 1.2 when a mixture of 70% diisopropyl ether and 30% chloroform was used as a solvent. The degree of polymerization was 8 in this case. According to thin-layer chromatography, a trimer (HO(CH₂)₄OCO(CH₂)₂COO(CH₂)₄OH) was formed at an early stage, with a subsequent condensation with bis(2-chloroethyl) succinate to give higher oligomers. The structure of the oligomers was confirmed by ¹³C NMR and IR spectra.     

Structural studies of native Paecilomyces sp. Exopolysaccharide

A polysaccharide separated from Paecilomyces sp. was determined by gel permeation chromatography to be homogeneous. HPLC showed a monosaccharide containing D-glucose and D-fructose at a ratio of about 2:1. The results obtained from IR, 1H NMR, and 13C NMR analyses confirmed the proposed structure.     

荔枝壳多糖特性研究

多糖是荔枝壳的重要活性成分,本研究采用阴离子交换柱和凝胶过滤柱对荔枝壳多糖进行分离纯化。凝胶渗透色谱测定其分子量为14000 Dal。通过气相色谱测定其单糖组成为甘露糖、半乳糖和少量的阿拉伯糖,分子链由1,2键、1,3键和1,6键组成,不含1,4键。红外光谱分析表明甘露糖以β-D-甘露糖形式存在,不含羧基基团。     

Heterobifunctional poly(ethylene oxide) oligomers containing carboxylic acids

Syntheses of vinylsilyl alcohols having one to three vinyl moieties and their use as initiators for ethylene oxide polymerizations are discussed. Poly(ethylene oxide) oligomers with vinylsilanes at one end and a hydroxyl group at the other were prepared in base-catalyzed reactions. Molecular weights determined from 1H NMR and gel permeation chromatography were close to the targeted values. Carboxylic acid functional poly(ethylene oxide) oligomers were prepared from ene-thiol addition reactions of mercaptoacetic acid across the vinylsilane terminus. It is anticipated that these carboxylic acid functional oligomers will complex to magnetite nanoparticles to afford complexes that can be dispersed in aqueous media.     

Structure of bovine casein oligomers

We have characterized the size, molecular weight, and composition of the oligomeric particles produced by dialysis of bovine casein micelles against solutions lacking calcium ion. The particles were stabilized against further dissociation after dialysis by glutaraldehyde fixation. The progress of the dissociation was monitored by Biogel A-15 gel permeation chromatography, sucrose density gradient ultracentrifugation, inelastic laser light scattering, sedimentation velocity and equilibrium, and urea starch gel electrophoresis. The casein oligomers isolated after dialysis against either 0.5 m NaCl/SMUF A/azide or calcium-free SMUF/ azide have a hydrodynamic radius of about 5.5 nm and a molecular weight of about 95,000 corresponding to roughly four casein monomers (SMUF, simulated milk ultrafiltrate). The oligomers are highly hydrated and contain one-third of the calcium ion found in native micelles. During the course of dialysis, the micelles gradually break down into a broad distribution of intermediatesized particles and then into the oligomers described above.     

Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein. I. Purification and characterization

A new thick-filament-associated protein, the 86 kd protein, of chicken pectoralis major muscle was isolated from a crude C-protein preparation by a method similar to that used to purify H-protein from rabbit skeletal muscle. However, the protein with an apparent Mr of 86,000 and 370,000 as estimated by gel electrophoresis and gel permeation, respectively, is not related to C-protein and differs from rabbit H-protein by its elution behaviour from hydroxyapatite columns, by its molecular weight, ultraviolet light spectrum, amino acid composition and localization, and by its amount present in myofibrils. The amino acid composition reveals a high content of proline and gel permeation indicates an either highly asymmetric or polymeric structure of the molecule. Antibodies raised in rabbits against the 86 kd protein were demonstrated by double immunodiffusion and immunoblotting experiments to be specific for this protein. They show no cross-reactivity with any other myofibrillar protein of chicken pectoralis muscle, e.g. myosin, M-band proteins, titin or C-protein, nor did they exhibit a significant cross-reactivity with H-protein from rabbit. The 86 kd protein, which has been purified also by antibody affinity chromatography from a freshly prepared Guba-Straub extract of washed myofibrils, is a specific myofibrillar component located within each half of the A-band.     

Urinary protein profiling by high-performance gel permeation chromatography

We describe a new application of high-performance aqueous gel permeation chromatography for the analysis of human proteinuria. Separations of urinary proteins from normal subjects and patients with renal impairment were performed with TSK G 3000 SW columns. The effects of pH and icnic strength of the eluent on the separation of urinary proteins were investigated. Albumins were selectively separated from urine by affinity chromatography on Blue Sepharose CL-6B. According to the results of clinical investigations, urinary protein pattern derived from gel permeation chromatography revealed a good prediction of the site of renal involvement. Predominant excretion of proteins with lower molecular weight than albumin correlated with tubular damage. Albumin and higher molecular weight protein patterns were associated with glomerular disease. Absorbance measurements of the eluent at 280 nm were used for quantitative determination of total urinary protein. Gel permeation chromatography was compared to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and the resulting protein patterns are in good agreement.     

A modified method for isolating poly(vinyl alcohol)-degrading bacteria and study of their degradation patterns

An addition of catalase or peroxidase into an agar plate containing poly(vinyl alcohol) (PVA), was effective for the isolation of PVA-degrading microorganisms. A Gram-negative bacterium, strain TK-2 (-group of proteobacteria), rapidly degraded a high molecular weight PVA to low molecular weight material after 1 day thereby producing oligomers of PVA as shown by gel permeation chromatography. Conversely, Sphingomonas strain TJ-7 did not produce any PVA oligomers, suggesting that the strain TJ-7 degraded PVA from the terminal ends of the molecules, whereas the strain TK-2 cleaved PVA at random.     

High-performance liquid chromatography of proteins by gel permeation chromatography

The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.     

Low molecular weight polyethylenimine grafted N-maleated chitosan for gene delivery: properties and in vitro transfection studies

To develop chitosan-based efficient gene vectors, chitosans with different molecular weights were chemically modified with low molecular weight polyethylenimine. The molecular weight and composition of polyethylenimine grafted N-maleated chitosan (NMC-g-PEI) copolymers were characterized using gel permeation chromatography (GPC) and (1)H NMR, respectively. Agarose gel electrophoresis assay showed that NMC-g-PEI had good binding ability with DNA, and the particle size of the NMC-g-PEI/DNA complexes was 200-400 nm, as determined by a Zeta sizer. The nanosized complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The NMC-g-PEI copolymers showed low cytotoxicity and good transfection activity, comparable to PEI (25 KDa) in both 293T and HeLa cell lines, except for NMC 50K-g-PEI. The results indicated that the molecular weight of NMC-g-PEI has an important effect on cytotoxicity and transfection activity, and low molecular weight NMC-g-PEI has a good potential as efficient nonviral gene vectors.     

Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane

Different molecular weight, 90% deacetylated chitosans were obtained by ultrasonic degradation on 90% deacetylated chitosan at 80 °C for various times.

Ninety percent deacetylated chitosan was prepared from alkali treatment of chitin that was obtained from red shrimp waste. Number average-, viscosity average- molecular weights were measured by gel permeation chromatography and the viscometric method, respectively. Degree of deacetylation was measured by the titration method. Enthalpy, maximum melting temperature, tensile strength and elongation of the membranes, flow rate of permeates and water are properties measured to elucidate the effect of molecular weight of chitosan on the above thermal, mechanical, and permeation properties, respectively of the prepared membranes. Results show tensile strength, tensile elongation, and enthalpy of the membrane prepared from high molecular weight chitosans were higher than those from low molecular weight. However, the permeability show membranes prepared from high molecular weight chitosans are lower than that from those of low molecular weight.     

Isolation and characterization of nerve growth factor from the venom of Naja naja atra.

Nerve growth factor was isolated from the venom of Naja naja atra by ion exchange and gel permeation chromatography and was found to be homogeneous by disc gel electrophoresis. The molecular weight was estimated to be approximately 20,000 by gel filtration and 22,000 by ultracentrifugation. This protein, which showed an isoelectric point of pH 7.02, probably consists of two subunits of equal molecular weight which are held together or interact with each other noncovalently. The biological activity survives treatment by a number of proteolytic enzymes, such as trypsin [EC 3.4.21.4], chymotrypsin [EC 3.4.21.1], and pepsin [EC 3.4.23.1].     

Poly(3-hydroxyalkanoate)s functionalized with carboxylic acid groups in the side chain

Biodegradable polyesters represent an important class of materials, and one subset of these polymers are the bacterially produced poly(3-hydroxyalkanoate)s (PHA), a bacterially produced material. These polymers are very hydrophobic, and chemical methods to increase their hydrophilicity will ultimately lead to new applications. Many copolymers of PHA are known that contain simple, nonpolar functionality in the side chain, and we explored the conversion of side-chain olefins to carboxylic acids under conditions that minimize molecular weight degradation. With the use of osmium tetraoxide and oxone, the conversion proceeded to completion with little backbone degradation, which was confirmed with NMR, IR, and gel permeation chromatography (GPC). The solubility character of the polymer before and after reaction is very different, and several solvents were explored including acetone, tetrahydrofuran (THF), and water.     

Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone

Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed.     

Physicochemical properties of (1 → 6)-branched (1 → 3)-β-D-glucans. 1. Physical dimensions estimated from hydrodynamic and electron microscopic data

The physical dimensions of several (1 → 6) branched (1 → 3) -β-D -glucan samples obtained from different organisms and their derivatives have been studied by electron microscopy, light scattering measurements, viscometry, and gel permeation chromatography. The electron micrographs indicate that in most samples these biopolymers are adequately described as linear worm-like coils. A sample reconstituted from alkaline media appeared as a blend of the linear, circular, and aggregated polymer morphologies. The average mass per unit length, M_L = M_w/L_w for the macroscopically linear samples, was estimated to be 2100 ± 200 g mol^?1 nm^?1. The parameter m_L was determined from the contour lengths obtained by electron microscopy and the molecular weight by light scattering measurements. The observed M_L was consistent with the triple-helical structure reported from x-ray diffraction studies and observed degree of side-chain substitution. From the molecular snapshots shown in the electron micrographs, the persistence lengths of these β-D -glucans were determined to be 140 ± 30 nm. The experimentally determined intrinsic viscosities were consistent with these estimates of M_L and persistence length. Comparison of the molecular weight distributions obtained from gel permeation chromatography and those deduced from the electron micrographs indicates that number and weight average contour lengths are more reliable than z and z + 1 averages. © 1993 John Wiley & Sons, Inc.     

Altered proteins with triosephosphate isomerase activity in suppressor-containing strains of Bacillus subtilis

Suppressor mutations in Bacillus subtilis cause the synthesis of a new protein with the enzymatic activity of l-leucine dehydrogenase and two groups of new proteins with the activity of triosephosphate isomerase. The new isoenzymes of triosephosphate isomerase are separable by zone electrophoresis and differ among themselves in elution behavior upon gel permeation chromatography. One group has an apparent average molecular weight of 120,000 to 135,000, which is more than twice that of the wild-type enzyme. Another group appears to be even higher in molecular weight. These data are consistent with the working hypothesis that the new isoenzymes are produced by extension of growing polypeptide chains through one or more chain-terminating triplets, although other mechanisms resulting in alteration of shapes, charges, or associations of the enzymes are not excluded.     

A beta-D-glucan from the sclerotia of Pleurotus tuber-regium (Fr.) Sing

An alkali-soluble polysaccharide (Hunai polysaccharide, 1) from the fruit body of Pleurotus tuber-regium (Fr.) Sing was shown to be homogeneous by gel permeation chromatography and its molecular weight was approximately 4.3 x 10(5). Complete acid hydrolysis, periodate oxidation, Smith degradation, methylation, FT-IR and 13C NMR analysis, complex formation with Congo Red, indicated that 1 has a beta-(1 --> 3)-linked D-glucopyranosyl backbone with a single beta-D-glucopyranosyl group at O-6 of every third glucose residue.     

Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin

In solutions containing fibrinogen and fibronectin, factor XIIIa catalyzes the formation of two types of crosslinked polymers: hybrid oligomers consisting of equimolar amounts of fibrinogen and fibronectin, and fibrinogen oligomers. The two types of oligomers are produced in amounts proportional to the starting concentration of fibronectin and fibrinogen in the reaction mixture. Increasing the fibronectin concentration relative to the fibrinogen concentration results in the production of more hybrid and less fibrinogen type oligomers. The lowest molecular weight hybrid oligomer, a dimer, is formed by ligation of one molecule of fibrinogen and fibronectin. The A alpha-chain of fibrinogen and one fibronectin subunit participate in the crosslinking. Larger size hybrid oligomers form by the joining of two hybrid dimers to each other via gamma-chain dimerization in the fibronectin moiety of the dimers. In fibrinogen oligomer formation, fibrinogen molecules are ligated by gamma-chain dimerization in a step-wise fashion producing fibrinogen dimers, trimers, tetramers, etc. without A alpha-chain crosslinking. The hybrid type and the fibrinogen type of oligomer grow in size and eventually become crosslinked to each other yielding large molecular weight complexes that interact to form a gel network.     

Rubber-degrading enzyme from a bacterial culture

Rubber-degrading activity was found in the extracellular culture medium of Xanthomonas sp. strain 35Y which was grown on natural rubber latex. Natural rubber in the latex state was degraded by the crude enzyme, and two fractions were separately observed by gel permeation chromatography of the reaction products. One fraction was of higher molecular weight (HMW) with a very wide MW distribution from 10 to 10, and the other fraction was of lower molecular weight (LMW) with a MW of a few hundred. H-nuclear magnetic resonance spectra of the partially purified fractions were those expected of cis-1,4-polyisoprene mixtures with the structure OHC-CH(2)-(-CH(2)-C(-CH(3)) = CH-CH(2)-)(n)-CH(2)-C(=O)-CH(3), with average values of n of about 113 and 2 for HMW and LMW fractions, respectively. The LMW fraction consisted mostly of one component in gas-liquid chromatography as well as in gel permeation chromatography, and the main component was identified as 12-oxo-4,8-dimethyl trideca-4,8-diene-1-al (acetonyl diprenyl acetoaldehyde, A(l)P(2)A(t)) by C-nuclear magnetic resonance and gas chromatography-mass spectra. Not only the latices of natural and synthetic isoprene rubber, but also some kinds of low-MW polyisoprene compounds of cis-1,4 type, were degraded by the crude enzyme. The rubber-degrading reaction was found to be at least partly oxygenase catalyzed from the incorporation of O into A(l)P(2)A(t) under an O(2) atmosphere.     

Chemical synthesis of (1 → 2)-α-D-mannopyranan

The polymerization of 1,2-anhydro-3,4,6-tri-O-benzyl-β-D -mannopyranose proceeds in the presence of Lewis acids, cationic coordination catalysts, and strong bases. Debenzylation of the products yields oligomeric saccharides or low polymers. Polymerization in toluene by means of potassium alkoxide complexed with crown ethers leads to essentially stereoregular (1 → 2)-α-D -mannopyranan. The original derivatives have been characterized by optical rotation, viscosity, molecular weight, gel permeation chromatography, and spectrometry. The free polysaccharides have been characterized by optical rotation, molecular weight, and ¹H- and ¹³C-nmr spectrometry and compared to yeast mannan hydrolysate oligomers.