Hydroxyapatite surface modified by L-lactic acid and its subsequent grafting polymerization of L-lactide

A new method of surface modification of hydroxyapatite nanoparticles (n-HA) by surface grafting reaction of l-lactic acid and ring-opening polymerization of l-lactide (LLA) was developed. Two modified HA nanoparticles were obtained: HA modified by l-lactic acid (l-HA) and HA grafting with poly(l-lactide) (PLLA; p-HA). The modified surface of n-HA was attested by Fourier transformation infrared, (31)P MAS NMR, and thermal gravimetric analysis. The results showed that l-lactic acid could be easily grafted onto the n-HA surface by forming a Ca carboxylate bond and initiated by the hydroxyl group of the grafted l-lactic acid and LLA could be graft-polymerized onto the n-HA surface in the presence of stannous octanoate. The highest grafting amounts of l-lactic acid and PLLA were about 33 and 22 wt %, respectively. The modified HA/PLLA composites showed good mechanical properties and uniform microstructure. The tensile strength and modulus of the p-HA/PLLA composite containing 15 wt % of p-HA were 67 MPa and 2.1 GPa, respectively, while those of the n-HA/PLLA composites were 45 MPa and 1.7 GPa, respectively. The elongation at the break of the l-HA/PLLA composite containing 15 wt % l-HA could reach 44%, in comparison with 6.5% of the n-HA/PLLA composites containing 15 wt % n-HA.     

Enzymatic preparation of novel thermoplastic di-block copolyesters containing poly[(R)-3-hydroxybutyrate] and poly(epsilon-caprolactone) blocks via ring-opening polymerization

Enzymatic modification of a microbial polyester was achieved by the ring-opening polymerization of epsilon-caprolactone (CL) with low-molecular weight telechelic hydroxylated poly[( R)-3-hydroxybutyrate] (PHB-diol) as initiator and Novozym 435 (immobilized Candida antarctica Lipase B) as catalyst in anhydrous 1,4-dioxane or toluene. The ring-opening polymerization was investigated at different conditions with two different types of PHB-diols: PHB-diol(P), containing a primary OH and a secondary OH end groups, and PHB-diol(M), consisting of 91% PHB-diol(P) and 9% PHB-diol containing two secondary OH end groups. The reactions were followed by GPC analyses of the resulting polymers at different time points, and the optimal conditions were established to be 70 degrees C at a weight ratio of CL/enzyme/solvent of 8:1:24. The ring-opening polymerization of CL with PHB-diol(M) (Mn of 2380, NMR) at the molar ratio of 50:1 under the optimal conditions in 1,4-dioxane gave the corresponding poly[HB(56 wt %)-co-CL(44 wt %)] with Mn (NMR) of 3900 in 66% yield. Polymerization of CL and PHB-diol(P) ( Mn of 2010, NMR) at the same condition in toluene gave the corresponding poly[HB(28 wt %)-co-CL(72 wt %)] with Mn (NMR) of 7100 in 86% yield. Both polymers were characterized by (1)H and (13)C NMR and IR analyses as di-block copolyesters containing a PHB block with a secondary OH end group and a poly(epsilon-caprolactone) (PCL) block with a primary OH end group. NMR analyses and control experiments suggested no formation of random copolymers and no change of the PHB block during the reaction. The enzymatic ring-opening polymerization was selectively initiated by the primary OH group of PHB-diol, whereas the secondary OH group remained as an end group in the final polymers. The thermal properties of the di-block poly(HB-co-CL)s were analyzed by DSC, with excellent T g values for the elastomer domain: poly[HB(56 wt %)- co-CL(44 wt %)] with M n (NMR) of 3900 demonstrated a T g of -57 degrees C, Tm of 145, 123, and 53 degrees C; and poly[HB(28wt%)-co-CL(72wt%)] with Mn (NMR) of 7100 gave a Tg of -60 degrees C, Tm of 147 and 50 degrees C. Thus, the selective enzymatic ring-opening polymerization with PHB-diol as macro-initiator provides a new method for the preparation of PHB-based block copolymers as biomaterials with good thermoplastic properties and novel structures containing functional end groups.     

Purification and characterization of PrbA,a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens)

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine.     

Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.     

Linear copolymeric poly(thia-alkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'-thiodipropionic acid and its dimethyl ester with alpha,omega-alkanediols

Linear copolymeric polyesters (polyoxoesters) containing thioether functions [poly(3,3'-thiodipropionic acid-co-alpha,omega-alkanediols)] were formed in good yield by esterification of an equimolar mixture of 3,3'-thiodipropionic acid (4-thiaheptane-1,7-dioic acid) and 1,6-hexanediol (weight average molecular mass, M(W) >600 Da: approximately 81% after 6 h) or 1,12-dodecanediol (M(W) > 900 Da: approximately 90% after 6 h) catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) for up to 336 h in moderate vacuo without a solvent or drying reagent in the reaction mixture. Poly (3,3'-thiodipropionic acid-co-1,6-hexanediol) and poly (3,3'-thiodipropionic acid-co-1,12-dodecanediol) were extracted from the reaction mixtures using tetrahydrofurane and precipitated from tetrahydrofurane-iso-hexane (1:1, v/v) at approximately 0 degrees C. The precipitate of poly(3,3'-thiodipropionic acid-co-1,6-hexanediol) showed a maximum molecular weight of 6 x 10(5) Da corresponding to a M(W) of approximately 24,200 Da and a degree of polymerization of up to 2,150 monomer units. The precipitated poly(3,3'-thiodipropionic acid-co-1,12-dodecanediol) showed a maximum molecular weight of 8 x 10(5) Da corresponding to a M(W) of approximately 27,200 Da and a maximum degree of polymerization of up to 2,200 monomer units. The chemical structures of both polyesters containing thioether functions were confirmed by chemical derivatization and NMR spectrometry. The chemical structures of various low-molecular weight reaction intermediates of the esterification of 3,3'-thiodipropionic acid with 1,6-hexanediol were elucidated by GC-MS.     

Purification and characterization of a new xylanase from Acrophialophora nainiana

A new xylanase activity (XynII) was isolated from liquid state cultures of Acrophialophora nainiana containing birchwood xylan as carbon source. XynII was purified to apparent homogeneity by gel filtration and ion exchange chromatographies. The enzyme was optimally active at 55 degrees C and pH 7.0. XynII had molecular mass of 22630+/-3.0 and 22165 Da, as determined by mass spectrometry and SDS-PAGE, respectively. The purified enzyme was able to act only on xylan as substrate. The apparent K(m) values on soluble and insoluble birchwood xylans were 40.9 and 16.1 mg ml(-1), respectively. The enzyme showed good thermal stability with half lives of 44 h at 55 degrees C and ca. 1 h at 60 degrees C The N-terminal sequence of XynII showed homology with a xylanase grouped in family G/11. The enzyme did not show amino acid composition similarity with xylanases from some fungi and Bacillus amyloliquefaciens.     

Biodegradation of polyvinyl alcohol by a mixed microbial culture

A mixed culture capable of degrading 1 g l⁻¹ polyvinyl alcohol (PVA) completely was screened from sludge samples at Pacific Textile Factory, Wuxi, China. This mixed culture had stronger capability of degrading PVA with low polymerization and high saponification than degrading PVA with high polymerization and low saponification. Inorganic nitrogen source was more suitable for the mixed culture to grow and degrade PVA than organic nitrogen source. Microorganisms and relative abundance of this mixed culture were explored by terminal restriction fragment length polymorphism (T-RFLP). Small PVA molecules were detected in cell extracts of the mixed culture. This indicated that PVA degradation in the mixed culture was in fact a combined action of extracellular and intracellular enzymes. Two strains producing extracellular PVA-degrading enzyme were isolated from the mixed culture. They could individually degrade PVA1799 with polymerization of 1700 from initial average molecular weight 112,981 to 98,827 Da and 84,803 Da, respectively. However, only small amount of PVA124 in polymerization of 400 could be degraded by these two strains.     

Thermophilic esterase from the archaeon Archaeoglobus fulgidus physically immobilized on hydrophobic macroporous resin: A novel biocatalyst for polyester synthesis

This paper reviews the immobilization of a thermophilic esterase, AFEST from the archaeon Archaeoglobus fulgidus, on a hydrophobic macroporous resin and its application in polyester synthesis using the ring-opening polymerization of ?-caprolactone as a model. Using the physical adsorption technique, the AFEST loading concentration after 24 h was 152 mg AFEST per g of support. Particle size and surface morphology of the immobilized enzyme were investigated using laser scattering analysis and scanning electron microscopy. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically investigated. Through the optimization of reaction parameters, poly(?-caprolactone) was obtained at an almost 100% monomer conversion rate and with a low average molecular weight (< 1,100 g/mol). Finally, the immobilized enzyme exhibited good operational stability, with a monomer conversion value of more than 55% after four batch reactions.     

Reversible immobilization of glutaryl acylase on sepabeads coated with polyethyleneimine

The immobilizaton of the enzyme glutaryl-7-aminocephalosporanic acid acylase (GA) was performed via ionic adsorption onto several supports: a new anionic exchange resin, based on the coating of Sepabeads internal surfaces with polyethyleneimine (PEI) of different molecular weights, and conventional EC-Q1A-Sepabeads and DEAE-agarose. Immobilization occurred very rapidly in all cases, but the adsorption strength was much higher in the case of PEI-Sepabeads than in the other supports at pH 7 (e.g., at 150 mM NaCl, 90% of the enzyme was eluted from the DEAE agarose and 15% was eluted from the EC-Q1A-Sepabeads, whereas no desorption was detected with the best PEI-Sepabeads). Interestingly, the adsorption strength of the GA was increased when it was immobilized on PEI-Sepabeads with higher molecular weights. For instance, enzyme desorption was detected from 75 mM NaCl for the derivative prepared onto Sepabeads coated with PEI 700 Da, whereas in the derivative prepared with the highest molecular weight PEI (600 000 Da) no enzyme desorption was detected below 150 mM NaCl. Optimal PEI-Sepabeads (prepared with PEI of 600 000 Da) was even much more thermostable than the covalent derivative prepared onto cyanogen bromide agarose. Moreover, this derivative presented a half-life 26-fold higher than that of the soluble enzyme at 45 degrees C, and the support could be reused 10 times after the full desorption of the enzyme without decreasing loading capacity.     

Enzymatic conversion of averufin to hydroxyversicolorone and elucidation of a novel metabolic grid involved in aflatoxin biosynthesis

The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from Aspergillus parasiticus. When (1'S,5'S)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed. When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed. However, (1'R,5'R)-AVR did not serve as the substrate. In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later. Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed. When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos. The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE. Separate feeding experiments performed with A. parasiticus NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins. Remarkably, we found that VONE and VOROL had ring-opened structures. Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported. These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1'S,5'S)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA. A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered.     

Characterization of O-acetyl-L-serine sulfhydrylase purified from an alkaliphilic bacterium

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) activity was shown to be very high compared with O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity and L-cystathionine cleaving activities, in an extract of cells of an alkaliphilic bacterium grown in a synthetic medium. The synthesis of the first enzyme was repressed by approximately 55% by both L-cystine and L-djenkolic acid added to the medium at a concentration of 0.5 mM, but L-methionine (1 mM) and S-adenosyl-L-methionine (0.5 mM) affected it to lesser extents. Its enzyme activity was inhibited by 25% and 12% by methionine (10 mM) and S-adenosylmethionine (5 mM), respectively. The enzyme was purified from the extract through ammonium sulfate fractionation, heat treatment, and chromatography on columns of DEAE-cellulose, Sephacryl S-300, and Octyl Sepharose CL-4B with a recovery of 21%. Polyacrylamide gel electrophoresis with sodium dodecylsulfate of the preparation obtained finally showed its homogeneity and the molecular mass of 37,000 Da for dissociated subunits. Gel filtration of the enzyme on a Sephacryl S-300 column showed an approximate molecular mass of 72,000 Da, suggesting that the enzyme was comprised of two identical subunits. The enzyme catalyzed the beta-replacement reaction with O-acetylserine as a substrate, and showed no reactivity to other O-substituted amino acids tested. The reaction proceeded best at 40 degrees C (when tested at pH 7.5), and at pH 6.5 (at 40 degrees C). The enzyme kept 90% its activity after incubation at 65 degrees C (at pH 7.5) for 30 min, and more than 90% after 30 min incubation at pHs 7-12 at 30 degrees C. The enzyme had a Km of 4 mM for O-acetyl-L-serine and a Vmax of 37.0 micromol/min/mg of protein, a very low value compared with those of other organisms. However, the content of the enzyme in the extract was calculated to be approximately 3.5% total protein. Sensitivity of the enzyme to carbonyl reagents was very low, although it was shown to have pyridoxal 5'-phosphate as a cofactor by examination of its absorption spectrum. Sulfhydryl reagents tested showed no inhibition. The novelty of this enzyme among analogous sulfhydrylases purified from other organisms was discussed.     

Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala

The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850 Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519 Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer.     

Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate

Objective of this study is to realize appropriate enzyme immobilization onto a suitable support material and to develop a model which enables reactions catalyzed with different enzymes arranged in order. Thence, this model was potential for developing a multi-enzyme system. The reactions need more than one enzyme can be realized using immobilized form of them and the enzymes will be in one support at wanted activities. In this study, sodium alginate was used as immobilization material and glycidyl methacrylate was grafted onto sodium alginate. Thus reactive epoxy groups were added to sodium alginate which also has carboxyl groups. Average molecular weight of sodium alginate was determined using Ubbelohde viscosimetri. The molecular mass of sodium alginate was calculated as 15,900 Da. Graft polymerization was made in two steps. Firstly, sodium alginate was activated with benzophenone using UV-light at 254 nm. Secondly, glycidyl methacrylate was grafted under UV-light at 365 nm onto activated sodium alginate. Grafted glycidyl methacrylate was determined gravimetric and titrimetric. Additional groups after grafting were showed with FT-IR spectrum. 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide was used for immobilization urease from carboxyl groups at pH 5.0. Suitable 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide/–COOH ratio was found 1/10 and immobilized product activity was 197 U/g support. Reaction medium pH was 8.0 for immobilization from epoxy group. Optimum immobilization reaction time was found as 2 h and immobilized product activity was 285 U/g support. Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate was made from –COOH and epoxy groups, respectively.     

Use of a batch-stirred reactor to rationally tailor biocatalytic polytransesterification

Despite favorable thermodynamics, high-molecular weight and low-dispersity polyesters are difficult to synthesize biocatalytically in organic solvents. We have reported previously that the elimination of solvent can improve the kinetics and apparent equilibrium significantly (Chaudhary et al., 1997a). We now present the design and use of a batch-stirred enzyme reactor to control the biocatalytic polymerization. Using the reactor, polyester having a molecular weight of 23,400 Da and a polydispersity of 1.69 was synthesized in only 1 h at 60 degrees C. Additional factors like enzyme-deactivation kinetics, enzyme specificity, and initial exothermicity were investigated to develop a better understanding of this complex reaction system.     

Exploring mild enzymatic sustainable routes for the synthesis of bio‐degradable aromatic‐aliphatic oligoesters

The application of Candida antarctica lipase B in enzyme‐catalyzed synthesis of aromatic‐aliphatic oligoesters is here reported. The aim of the present study is to systematically investigate the most favorable conditions for the enzyme catalyzed synthesis of aromatic‐aliphatic oligomers using commercially available monomers. Reaction conditions and enzyme selectivity for polymerization of various commercially available monomers were considered using different inactivated/activated aromatic monomers combined with linear polyols ranging from C₂ to C₁₂. The effect of various reaction solvents in enzymatic polymerization was assessed and toluene allowed to achieve the highest conversions for the reaction of dimethyl isophthalate with 1,4‐butanediol and with 1,10‐decanediol (88 and 87% monomer conversion respectively). M_w as high as 1512 Da was obtained from the reaction of dimethyl isophthalate with 1,10‐decanediol. The obtained oligomers have potential applications as raw materials in personal and home care formulations, for the production of aliphatic‐aromatic block co‐polymers or can be further functionalized with various moieties for a subsequent photo‐ or radical polymerization.     

Hydroxysteroid dehydrogenase transformations of 5β-scymnol and identification of oxoscymnol transformation products by liquid chromatography-tandem mass spectroscopy

A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS(2)) detection was developed to identify for the first time the oxidation products of 5β-scymnol [(24R)-(+)-5β-cholestan-3α,7α,12α,24,26,27-hexol] catalysed by bacterial hydroxysteroid dehydrogenase (HSD) reactions in vitro. The authentic scymnol (MW 468) standard yielded a protonated molecular ion [M+H](+) at m/z 469 Da, and higher mass adduct ions attributed to [M+NH(4)](+) (m/z 486), [M+H+CH(3)OH](+) (m/z 501) and [M+H+CH(3)COOH](+) (m/z 530). (24R)-(+)-5β-Cholestan-3-one-7α,12α,24,26,27-pentol (3-oxoscymnol, m/z 467 Da, relative retention time (RRT)=0.89) was identified as the principle molecular species of scymnol in the reaction with 3α-HSD pure enzyme. [S](0.5) for the reaction of 3α-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5β-cholestan-7-one-3α,12α,24,26,27-pentol (7-oxoscymnol, m/z 467 Da, RRT=0.79) and (24R)-(+)-5β-cholestan-12-one-3α,7α,24,26,27-pentol (12-oxoscymnol, m/z 467 Da, RRT=0.81) were similarly identified as principle molecular species in the respective 7α-HSD and 12α-HSD reactions. Polarity of the oxoscymnol species was established as 7-oxoscymnol>12-oxoscymnol>3-oxoscymnol>scymnol (in order from most polar to least polar). Confirmation that 5β-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated liquid chromatography-mass spectrometry (LC-MS) techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound.     

Biosynthesis of dextrans with different molecular weights by selecting the concentration of Leuconostoc mesenteroides B-512FMC dextransucrase, the sucrose concentration, and the temperature

Leuconostoc mesenteroides B-512FMC dextransucrase was found to synthesize dextrans of varying molecular weights by selecting the concentrations of dextransucrase and sucrose, as well as the temperature. Four enzyme concentrations (50, 10, 1.0, and 0.1 U/mL), five sucrose concentrations (20, 50, 100, 200 and 1000 mM), and two temperatures (20 °C and 30 °C) were studied. The highest amount of enzyme (50 U/mL), with the lowest concentration of sucrose (20 mM), and the lower temperature of 20 °C gave the lowest number-average molecular weight (MW_n) of 20,630 Da, respectively. As the sucrose concentration was increased, 50 mM, 100 mM, and 200 mM, the MW_n was 49,240 Da, 63,350 Da, and 126,720 Da, respectively. The next enzyme concentration (10 U/mL) gave a similar upward trend, starting at 73,130 Da and ending at 237,870 Da at 20 °C and 130,040 Da and ending at 415,770 Da at 30 °C. The upward trend continued for the 1.0 and 0.1 U/mL enzyme concentrations. An increase in the temperature had the overall effect of increasing the MW_n for each decreasing concentration of enzyme and increasing concentration of sucrose. For 0.1 U/mL and 1000 mM sucrose at 30 °C, the MW_n was 1,645,700 Da. The results of the study show that the molecular weights of the synthesized dextrans were inversely proportional to the concentration of the enzyme and directly proportional to the concentration of sucrose and the temperature.     

Modeling enzymatic kinetic pathways for ring-opening lactone polymerization

A unified kinetic pathway for the enzyme-catalyzed polymerization and degradation of poly(ε-caprolactone) was developed. This model tracks the complete distribution of individual chain lengths, both enzyme-bound and in solution, and successfully predicts monomer conversion and the molecular mass distribution as a function of reaction time. As compared to reported experimental data for polymerization reactions, modeled kinetics generate similar trends, with ring-opening rates and water concentration as key factors to controlling molecular mass distributions. Water is critically important by dictating the number of linear chains in solution, shifting the molecular mass distribution at which propagation and degradation equilibrate. For the enzymatic degradation of poly(ε-caprolactone), the final reaction product is also consistent with the equilibrium dictated by the propagation and degradation rates. When the modeling framework described here is used, further experiments can be designed to isolate key reaction steps and provide methods for improving the efficiency of enzyme polymerization.     

The Hfq-like protein NrfA of the phototrophic purple bacterium Rhodobacter capsulatus controls nitrogen fixation via regulation of nifA and anfA expression

A novel esterase catalyzing regioselective hydrolysis was purified from the membrane fraction of Microbacterium sp. 7-1W, and characterized. The enzyme was solubilized with Brij 58 and purified 13.8-fold to apparent homogeneity with 2.58% overall recovery. The relative molecular mass of the native enzyme as estimated by gel filtration was more than 600,000 Da, and the subunit molecular mass was 62,000 Da. The enzyme catalyzed cleavage of the terminal ester bonds of cetraxate esters and pantothenate esters. The K(m) and V(max) values for methyl cetraxate were 0.380 mM and 7.76 micromole min(-1) mg(-1) protein, respectively. The enzyme was inhibited by serine hydrolase inhibitors.     

Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila.

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6 X 10(6) Da, compared with 20 proteins totalling 0.35 X 10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15 X 10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87 X 10(6) Da mass); a smaller difference. 0.15 X 10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8 X 10(6) and 1.65 X 10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.