Further studies on the bioconversion of penicillin G into deacetoxycephalosporin G by resting cells of Streptomyces clavuligerus NP-1

Resting cells of Streptomyces clavuligerus NP-1, which posses deacetoxy-cephalosporin C synthase activity, have been shown previously to perform oxidative ring expansion of penicillin G in the presence of iron, ascorbic acid, and α-ketoglutaric acid to form deacetoxycephalosporin G. Further studies on this bioconversion indicated that use of MOPS or HEPES buffer at pH 6.5 more than doubled the extent of the reaction observed with the previously used Tris-HCl at pH 7.4. Levels of bioconversion as high as 16.5% were achieved at low penicillin G concentrations. Previously, conversion yields were <1%.     

Optimization of yield in kinetically controlled synthesis of ampicillin with immobilized penicillin acylase in organic media

Immobilized penicillin acylase is a moderately priced versatile enzyme, that is able to catalyze the synthesis of derived penicillins and cephalosporins from the corresponding β-lactam nuclei and proper side-chain precursors. Kinetically controlled synthesis is a better strategy when product yield is a key issue. Yield should increase at reduced water activity by depressing the competing hydrolytic reactions in favor of synthesis; therefore, organic cosolvents can be a suitable reaction media for synthesis. Using response surface methodology and product yield as objective function, temperature and pH were optimized in the kinetically controlled synthesis of ampicillin using previously screened cosolvents and reaction conditions. Optimum pH was 6.0 for ethylene glycol (EG) and glycerol (GL) and 6.6 for 1–2 propanediol (PD); optimum temperature was 30°C for GL and for EG and PD was in the lower extreme of the range studied, optimum lying below 26°C. Maximum molar yields predicted by the model were 58,51, and 46% for EG, GL, and PD, respectively, which were experimentally validated. Highest yield in aqueous buffer was always <40%. Molar yields about 60% compare favorably with values reported for the kinetically and thermodynamically controlled synthesis of ampicillin and other derived penicillins.     

Polymeric adsorbents with peptide pendants as artificial receptors for β-lactam antibiotics by mimicking the binding sites of β-lactamases

A series of polymeric adsorbents with peptide pendants were designed as the artificial receptors of β-lactam antibiotics by mimicking the structures of binding site in β-lactamases. Crosslinked poly(N, N-dimethyl acrylamide) gel as a carrier was prepared by suspension copolymerization of N, N-dimethyl acrylamide and N. Ń-bisacryl-diaminoethane and then functionalized with ethylenediamine after partial hydrolysis. Using solid-phase peptide synthesis with symmetrical anhydride of protected amino acid step by step, various peptide pendants were respectively anchored onto the functionalized carrier. The adsorption properties of these peptide-containing adsorbents for β-lactam antibiotics such as ampicillin and cefotaxime were then studied. The results showed that only those adsorbents in which peptide chains contained more than one lysine residues could obviously adsorb both β-lactams and that static interaction as well as hydrogen bond played an important role during the adsorption. Project supported by the National Natural Science Foundation of China (No. 59493207).     

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase

Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca²⁺-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a “ trickle-bed ” reactor.     

The Potential of Immobilized Biocatalysts for Production of Industrial Chemicals

The successful translation from conception to practice of processes based on immobilized biocatalyst technology has been slower than anticipated. There are severe barriers, both technical and economic, limiting the introduction of immobilized biocatalyst technology to replace conventional processing procedures and processes for the production of chemicals by synthetic or fermentative routes. A small number of immobilized enzyme processes are in operation commercially, the most noteworthy being in food-related processes and in the pharmaceutical industry, where they are used for carbohydrate conversions and antibiotic transformations, respectively. There does not, as yet, appear to be any large-scale industrial application of immobilized cell technology. Examples from our laboratory—immobilized yeast for ethanol production andAspergillus niger for citric acid synthesis—illustrate the problems that have to be overcome.     

Pyrogallol Immobilized Amberlite XAD-2: A Newly Designed Collector for Enrichment of Metal Ions Prior to their Determination by Flame Atomic Absorption Spectrometry

 Pyrogallol is covalently linked with the benzene ring of Amberlite XAD-2, through an azo (–N=N–) spacer group and the resulting new polymer characterized by elemental analyses, thermogravimetric analysis (TGA) and infrared (IR) spectra. It has been used for separation and preconcentration of Cu(II), Cd(II), Co(II), Ni(II), Pb(II), Zn(II), Mn(II), Fe(III) and U(VI) prior to their determination by flame atomic absorption spectrometry (FAAS) or fluorimetry (for U(VI) only). The pH ranges for quantitative sorption are 5.5–6.5, 5.5–7.5, 5.5–7.0, 5.5–7.0, 5.5–6.5, 5.5–6.5, 5.5–8.0, 5.5–6.2 and 5.5–6.2, respectively, for the nine metal ions, which can be desorbed (recovery 90–99%) with 4 mol L⁻¹ HNO₃ or HCl. The sorption capacity of the resin has been found to be in the range 4.10 to 6.71 mg of metal g⁻¹ of dry resin. The loading half time (t_1/2) was ≤3.3 min for all the metal ions. The effects of NaF, NaCl, NaNO₃, Na₂SO₄, and Na₃PO₄ on the sorption of these metal ions (0.2 μg mL⁻¹) are reported. The Ca(II) and Mg(II) are tolerable up to a concentration level of 40–400 and 25–240 μg mL⁻¹, respectively. The enrichment factor for Cu(II), Cd(II), Co(II), Ni(II), Pb(II), Zn(II), Mn(II), Fe(III) and U(VI) has been found to be 65, 40, 65, 120, 25, 160, 120, 140, and 70 (concentration level 2–25 ng mL⁻¹), respectively. The limit of detection for these nine metal ions is 5.0, 1.3, 5.0, 4.0, 25.0, 0.5, 1.0, 2.0 and 1.0 ng mL⁻¹, respectively. After enrichment on the present matrix, the flame AAS method has been applied to determine these metal ions (except U) in river water samples (RSD ≤ 7%) and well water (RSD ≤ 8%). Uranium present in well water samples has been enriched on the present matrix and determined by a fluorimetric method (RSD ≤ 6%). The cobalt present in pharmaceutical vitamin tablets was also preconcentrated with the aid of the present chelating resin and determined by FAAS to be 1.93 μg g⁻¹ (RSD ∼4%). Received May 16, 2000. Revision April 3, 2001.     

Molecular imprinting of β-cyclodextrin/cholesterol template into a silica polymer for cholesterol separation

The molecular assembly formed by the inclusion complex of cholesterol in β-cyclodextrin (β-CD:chol) was used as a template for the molecular imprinting of a sol–gel polymer (MIP/β-CD:chol), produced with tetraethoxysilane (TEOS) as precursor. The MIP/β-CD:chol and pure silica matrix (PSM) were tested for the efficiency of cholesterol removal from solutions at different cholesterol concentrations (1–10 mg/mL). The adsorption tests were run at 25°C using 1% (w/v) solid/liquid suspensions during 24 h. The MIP/β-CD:chol data on cholesterol adsorption was fitted by the Langmüir isotherm model, giving a maximum adsorption capacity of 76.5 mg cholesterol/g-adsorbent. The PSM data did conform to the Langmüir model. The maximum cholesterol adsorption achieved with the PSM was higher, 251 mg/g, probably due to multilayer adsorption. The hydrophobic silica matrix, imprinted with the inclusion complex of β-CD and a target molecule, has the potential of being used as an adsorbent for other organic molecules.     

Evaluation and validation of an HPLC procedure for the determination of the positional isomeric impurity in 2-[4-(1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl)-phenyl]-2-methylpropionic acid

A high-performance liquid chromatographic (HPLC) procedure was evaluated for the determination of a positional isomeric impurity in bulk 2-[4-(1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl)-phenyl]-2-methylpropionic acid HCl drug substance. The use of a β-cyclodextrin bonded-phase column with a mobile phase of 20/80 (v/v) acetonitrile/water containing an ammonium acetate buffer at apparent pH 4.0 and a flow rate of 0.45 mL/min resulted in an excellent separation of the isomers. Ultraviolet detection was used at 220 nm. A recovery study of known spike levels (0.1 to 1.5% w/w) showed that the procedure was accurate. A two-day, two-column repeatability study showed consistent results with the test batch of the bulk compound. The level of impurity in the tested lot of the compound had a mean level of 0.32% (w/w) and a standard deviation of 0.038% (w/w, n = 5). The text was submitted by the author in English.     

Quartz Crystal Microbalance for Selenite Sensing Based on Growth of Cadmium Selenite Crystals Immobilized on a Monolayer of Phosphorylated 11-Mercapto-1-Undecanol

 A quartz crystal microbalance (QCM) sensor for selenite ions in aqueous solution was constructed based on crystal formation of cadmium selenite, immobilized with a self-assembly monolayer (SAM) of phosphorylated 11-mercapto-1-undecanol (MUD) on a QCM gold electrode surface. The mass change caused by the selective adsorption of selenite ions on the cadmium selenite crystals at the solid/solution interface was detected by the QCM. The response (−ΔF) of the modified QCM oscillator increased with increasing selenite ion concentrations in sample solutions, ranging from 9.7×10⁻⁵ to 9.0×10⁻⁴ M at pH 7.4. The synthetic process of anchoring cadmium selenite crystals on the phosphorylated MUD organic film was also followed by using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The atomic concentrations measured by XPS confirmed the crystal growth of cadmium selenite on the phosphorylated MUD SAM at the QCM gold electrode surface. From the AFM images, changes in surface topographic features were followed: the MUD SAM and phosphorylated MUD on the QCM gold electrode had similar surface roughness; however the difference for the cadmium selenite film on the phosphorylated MUD SAM was clearly seen. The observed QCM frequency change of the modified QCM oscillator per unit time was found to be proportional to the square of the supersaturation of cadmium selenite, indicating the crystal growth of cadmium selenite at the solid/solution interface. The modified QCM oscillator exhibited selectively strong QCM response to SeO₃ ²⁻ ion. In contrast, the responses to tested interfering anions were almost negligible. The order of anion selectivities of the present modified QCM sensor was SeO₃ ²⁻≫CO₃ ²⁻>SeO²⁻ ₄, SO₄ ²⁻, Br⁻, I⁻, NO₃ ⁻. These selectivities were basically attributable to the differences in solubility products and solubilities for the salts of each anion with cadmium (II) ion. Received May 12, 1998. Revision December 29, 1998.     

Preparation of a set of selectively protected disaccharides for modular synthesis of heparan sulfate fragments: toward the synthesis of several <Emphasis Type="Italic">O</Emphasis>-sulfonated [β-D-GlcUA-(1→4)-β-D-GlcNAc]OPr types

A concise method for a stereocontrolled synthesis of a set of selectively protected disaccharides is reported. Coupling of the donor 11 onto acceptors 23 and 24, promoted by trimethylsilyl triflate-N-iodosuccinimide (TMSOTf-NIS), generated the disaccharides 25 and 26. Under typical conditions, condensation of the fully protected donor 12 onto acceptors 23 and 24 produced the disaccharides 27 and 28. The building blocks 25–28 were prepared in moderate yields having exclusive β-stereoselectivity. A unique pattern of protecting groups distinguished clearly between positions to be sulfated and functional groups remaining as free hydroxyl groups. Acetyl and/or levulinoyl esters temporarily protected the positions to be sulfated, while benzyl ethers were used for permanent protection. The anomeric positions were protected as allyl ethers, whereas the 4′-positions were masked as p-methoxybenzyl (PMB) ethers. The orthogonality of the PMB and allyl groups can then be used for further elongation of the chain by recurrent deprotection and activation steps. The hydroxyl group, OH-6, of glucosamine moieties was protected as a TBDPS ether to avoid oxidation. A five-step deprotection/sulfonation sequence was applied to the disaccharide 27 to generate the corresponding sulfated [β-D-GlcUA-2-OSO₃Na-(1→4)-β-D-Glc pNAc]-(1→O-Pro) 34.     

Selection and microencapsulation of an “NADH-oxidizing” bacterium and its use for NAD regeneration

An alternative approach to the regeneration of coenzymes is described here using immobilized microorganisms possessing “NADH-oxidase” function. Bacteria containing NADH-oxidase activity are immobilized by microencapsulation within artificial cells. In this form, the microencapsulated bacteria can recycle NADH back to NAD in the presence of molecular oxygen as an electron acceptor. The only byproduct of the recycling reaction is water. In order to perform the biological regeneration of NAD, the activity of NADH-oxidase was investigated in 13 strains of aerobic bacteria and yeast. The NADH-oxidizing bacteriaLeuconostoc mesenteroides exhibited the highest activity among the microorganisms tested. The permeabilized bacteria showed 10% of their initial activity after microencapsulation. Light and electron microscopy studies of bacteria loaded microcapsules have been done. Enzymatic properties of microcapsule-immobilized bacteria were investigated in comparison with those of the free enzyme complex.Leuconostoc mesenteroides, containing NADH-oxidase, has been microencapsulated together with 3α-hydroxysteroid dehydrogenase (3α-HSDH) for stereospecific steroid oxidation. In a batch reactor, 2 mg of NAD, with recycling, allowed the same substrate consumption as 4.4 mg of NAD without recycling. The microencapsulated system can be used repeatedly. The system is functional for 10 h, during which time each molecule of NAD has been used 7.6 times.