Enzymatic synthesis and various properties of poly(catechol)

Poly(catechol) was prepared by using peroxidase as a catalyst in two types of solvent systems: an aqueous dioxane solution and a reverse micellar solution. Peroxidases derived from two sources, horseradish (HRP) and soybean (SBP), were employed as catalyst. The structure of the prepared polymer was elucidated by infrared analysis. Enzymatically prepared film of poly(catechol) at a Pt electrode was subjected to cyclic voltammetric studies in aqueous HCl medium and phosphate buffer pH 6.5. Thermal studies of the polymer were performed by thermogravimetric analysis. The iodine-labelled polymers showed low electrical conductivity in the range of 10⁻⁶ to 10⁻⁹ S cm⁻¹. The magnetic susceptibility and surface morphological property of the polymer were also studied.     

Enzymatic synthesis of poly(hydroxyalkanoates) in ionic liquids

Ring-opening polymerization of five lactones catalyzed by Candida antarctica lipase B in ionic liquids yielded poly(hydroxyalkanoates) of moderate molecular weights up to Mn=13,000. In the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethane)-sulfonimide and with a low weight ratio of enzyme to lactone (1:100) we obtained polymers from beta-propiolactone, delta-valerolactone, and epsilon-caprolactone with degrees of polymerization as high as 170, 25, and 85, respectively; oligomers from beta-butyrolactone and gamma-butyrolactone with degrees of polymerization of 5; and a copolymer of beta-propiolactone and beta-butyrolactone with a degree of polymerization of 180. Water-immiscible ionic liquids were superior to water-miscible ionic liquids. Reducing the water content of the enzyme improved the degree of polymerization by as much as 50% for beta-propiolactone and epsilon-caprolactone.     

Enzymatic synthesis and antioxidant property of gelatin-catechin conjugates

Gelatin-catechin conjugate was synthesized by the laccase-catalyzed oxidation of catechin in the presence of gelatin. The conjugate had a good scavenging activity against superoxide anion radicals. Moreover, the conjugate showed an amplified inhibition effect on human low density lipoprotein oxidation initiated by 2,2'-azobis(2-amidinopropane)dihydrochloride as a radical generator.     

Enzymatic synthesis and properties of uridine-5'-O-(2-thiodiphospho)-N-acetylglucosamine

This paper describes an enzymatic approach to obtain a thio-containing UDP-GlcNAc analog. We use an assay based on capture of the carbohydrate and analysis by mass spectrometry to quantitatively characterize the activity of this unnatural sugar donor in a LgtA-mediated glycosylation reaction.     

Enzymatic degradation of liposome-grafted poly(hydroxyethyl L-glutamine)

Liposomes coated with poly(hydroxyethyl L-glutamine) (PHEG) show prolonged circulation times and biodistribution patterns comparable to PEG-coated liposomes. While PEG is a nondegradable polymer, PHEG is expected to be hydrolyzed by proteases. In this study the enzymatic degradability of PHEG both in its free form and grafted onto liposomes was investigated, using the proteases papain, pronase E, and cathepsin B. Enzymatic action was monitored with a ninhydrin assay, which quantifies amine groups formed due to hydrolysis of amide bonds, and the degradation products were characterized by MALDI-ToF mass spectrometry. PHEG, both in its free form and when grafted onto liposomes, showed degradation into low molecular weight peptides by the enzymes. Thus, we present a polymer-coated long-circulating liposome with an enzymatically degradable coating polymer, avoiding the risk of cellular accumulation.     

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.     

Enzymatic synthesis and surface properties of novel rhamnolipids

New rhamnolipids were obtained via the development of a synthesis procedure consisting of two biocatalyzed steps. In the first step, naringinase was used to introduce a primary alcohol function onto rhamnose by glycosylation of 1,3-propanediol. In the second step, immobilized lipase B from Candida antarctica catalyzed the esterification of the primary hydroxyl group with mono- and di-carboxylic fatty acids of increasing chain length (from C8 to C14). For the monoic acids, the initial rate and 24 h yield decreased with increasing chain length. For the dioic acid, the number of carbon atoms of the acid did not influence these parameters. The new rhamnolipid obtained with tetradecanoic acid showed very good surface properties. At pH 5, it had a very low critical aggregation concentration of 1.70 μM and it diminished water's surface tension to 27.6 mN/m. It was also able to form stable insoluble monolayers. On the other hand, the rhamnolipid formed with tetradecanedioic acid showed far less interesting surface properties.     

Enzymatic synthesis of a conducting complex of polyaniline and poly(2-arcylamido-2-methyl-1-propanesulfonic ACID) using palm tree peroxidase and its properties

An enzymatic method of producing a conducting polyelectrolyte complex of polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) was developed. Acidic stable peroxidase isolated from royal palm tree (Roystonea regia L.) leaves was used as a catalyst in the oxidative polymerization of aniline at pH 2.8. The synthesis procedure was optimized. Spectroscopic and electrochemical characteristics of nanoparticles of obtained PANI/PAMPS complexes at different pH were studied. It was shown that the acidity of the medium affects their properties.     

Enzymatic synthesis of a conducting complex of polyaniline and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) using palm tree peroxidase and its properties

An enzymatic method of producing a conducting polyelectrolyte complex of polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) was developed. Acidic stable peroxidase isolated from royal palm tree (Roystonea regia L.) leaves was used as a catalyst in the oxidative polymerization of aniline at pH 2.8. The synthesis procedure was optimized. Spectroscopic and electrochemical characteristics of nanoparticles of obtained PANI/PAMPS complexes at different pH were studied. It was shown that the acidity of the medium affects their properties.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 283–287.Original Russian Text Copyright © 2005 by Mazhugo, Caramyshev, Shleev, Sakharov, Yaropolov.     

Enzymatic polymerization of tyrosine derivatives. Peroxidase- and protease-catalyzed synthesis of poly(tyrosine)s with different structures

Polymerization of tyrosine derivatives has been carried out by using two enzymes, peroxidase and protease, as catalyst to give poly(tyrosine)s with different structures. Tyrosine ester hydrochlorides were oxidatively polymerized by a peroxidase in a buffer. Using a high buffer concentration produced the polymer in good yields. The resulting polymer was soluble in N,N-dimethylformamide, dimethyl sulfoxide, and methanol but was insoluble in acetone, tetrahydrofuran, and water. The ester moiety of the polymer was subjected to the alkaline hydrolysis, yielding a water-soluble polymer having the amino acid group in the side chain. The peroxidase also catalyzed the oxidative polymerization of N-acetyltyrosine to give the polymer soluble in water. The polymerization of tyrosine ester hydrochlorides proceeded in the presence of papain catalyst to give a polymer of alpha-peptide structure. The polymerization in the buffer of high phosphate concentration efficiently produced the polymer. On the other hand, the polymer formation was not observed in the low buffer concentration. The molecular weight was several thousands and almost constant during the reaction. The morphology of the precipitated polymer was examined. The product of the initial reaction stage was amorphous. After 24 h, the precipitates exhibiting clear birefringence were formed. Scanning electron microscopy observation of the polymer after 72 h showed the formation of a globular crystal in a diameter larger than 50 microm, which was not found by recrystallization of poly(tyrosine).     

PNA conjugated to high-molecular weight poly(ethylene glycol): synthesis and properties

The conjugation of a bioactive, fluorescent PNA sequence to high-molecular weight poly(ethylene glycol) (PEG) is described and the properties of the PEG-PNA conjugate are evaluated.     

Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.     

Enzymatic methylation of chemically alkylated DNA and poly(dG-dC) X poly(dG-dC) in B and Z forms

The enzymatic methylation of chemically alkylated DNA and of poly(dG-dC) X poly(dG-dC) by beef brain DNA(cytosine-5-)-methyltransferase have been tested. The alkylation by dimethylsulfate, which yields mostly 7 methylguanine (m7G) and 3 methyladenine (m3A) do not affect the enzymatic methylation. The dimethylsulfate alkylated poly(dG-dC) X poly(dG-dC) converted into the Z-form in the presence of MgCl2, is just as well methylated as the native or the alkylated polynucleotide in the B-form. The alkylation of DNA or of poly(dG-dC) X poly(dG-dC) by methylnitrosourea yields, in addition to the above base modifications described for dimethylsulfate, methylphosphotriesters and O6-methylguanine. The enzymatic methylation of these substrates modified by methylnitrosourea is decreased. This decrease is proportional to the extent of the chemical alkylation of the substrate.     

8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase

The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.     

Enzymatic synthesis of a catecholic polyphenol product with excellent antioxidant activity

Abstract

Polyphenols, especially catecholic stilbene derivatives, have attracted much attention due to the huge pharmacological effects and promising health benefits. However, their chemical synthesis via regioselective ortho-hydroxylation on aromatic rings is highly challenging. In this study, 3′-hydroxypterostilbene (HPS) is taken as a model product due to its strong potential as an antitumor agent. One-step enzymatic synthesis of HPS from pterostilbene (PS) was explored, with immobilised tyrosinase as catalyst. The impact of solvent, pH, temperature, oxygen and reductant concentration on the reaction was investigated, and the conversion was optimised by employing the response surface methodology (RSM). Finally, a high yield of 77.9% was obtained in 2.7?h. This study demonstrates the first successful use of a biotechnological strategy to synthesise HPS. The antioxidant activities of both PS and HPS were evaluated by using the DPPH assay, demonstrating that HPS is more potent than PS as a radical scavenger.