Journal of Chemical Technology & Biotechnology

The pyrimido[1,2-alpha]benzimidazole derivatives compounds 1-8 were synthesized through cyclocondensation of 2-aminobenzimidazole with the appropriate benzsubstituted benzoylacetone by fusion at 150-170 degrees C for 5 h. Quaternary salts compounds 9-22 were obtained by quaternization of compounds 1-8 with dimethyl or diethyl sulfate and subsequent isolation as the relatively insoluble perchlorate salts. Assignment and confirmation of the structures of the newly synthesized compounds were based upon elemental microanalyses and other spectral evidence.

The photochemical dehalogenation of 1,2,4-tribromobenzene, 1,2,3,5-tetrabromobenzene and pentachlorobenzene in open-air solutions of acetonitrile using natural and artificial sunlight as the irradiation source has been investigated. The regiochemistry of mono-dehalogenation has been determined for 1,2,4-tribromobenzene and 1,2,3,5-tetrabromobenzene. Pentachlorobenzene did not react. 1,2,4-Tribromobenzene yielded three products, 1,4-dibromobenzene, 1,3-dibromobenzene and 1,2-dibromobenzene with isomer percentages of 52 +/- 1%, 39 +/- 2% and 9 +/- 1%, respectively. 1,2,3,5-Tetrabromobenzene yielded 1,3,5-tribromobenzene, 1,2,4-tribromobenzene and 1,2,3-tribromobenzene with relative product percentages of 60 +/- 2%, 29 +/- 2%, 11 +/- 1%.

Some novel 1,3,4-oxadiazoline and 1,3,4-oxadiazole derivatives have been synthesized from 3-hydroxy-5,6-diphenyl-1,2,4-triazine. Illucidation of the structures of the isolated products has been proved in the light of their elemental analysis, spectroscopic data and unambiguous synthesis in certain cases. Some derivatives have shown promising anti-inflammatory activity in comparison to phenylbutazone.

Thermal inactivation kinetics of native and glutaraldehyde cross-linked forms of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105 were studied. Apparent activation energies for thermal inactivation of both native and cross-linked forms of enzyme were calculated to be [57.71 +/- 8.46] and [67.11 +/- 13.83] kcal mol-1 respectively. This slight increase in activation energy suggested that glutaraldehyde cross-linking did not markedly protect against thermal activation. Cross-linked enzyme did, however, have a significantly improved half-life at temperatures between 40 degrees C and 50 degrees C.

Highly purified penicillin G acylase from a mutant derivative of Escherichia coli ATCC 11105 was immobilized on oxirane-acrylic beads by covalent binding via oxirane groups. The highest specific activity, (322 U g-1 dry matrix at 40 degrees C and at pH 8.0) was obtained by using an enzyme solution having 13.8 U mg-1 specific activity and 72.73 mg total protein. The efficiency of immobilization was 95.8%. Kinetic parameters of immobilized penicillin G acylase were determined at the same pH and temperature by a preparation having 8.1 mg bound protein. The Km value and substrate inhibition constant of the enzyme were found to be 11.36 mmol dm-3 and 680 mmol dm-3 penicillin G respectively. Phenylacetic acid and 6-aminopenicillanic acid were estimated as the competitive and non-competitive inhibitors of the enzyme and their inhibition constants were found to be 90 mmol dm-3 phenylacetic acid and 76.1 mmol dm-3 for 6-aminopenicillanic acid. The activation energy of the hydrolytic reaction was calculated to be 7.75 kcal mol-1. The immobilized enzyme showed highest activity at pH 8.0 and at 55 degrees C. The enzyme was stable when incubated at 4 degrees C for one day at a pH range of 5.0 to 9.0. Thermal stability (over 30 min) was observed up to 40 degrees C but decreased at higher temperatures and was almost absent at 60 degrees C. A 95% conversion rate was observed at 28 degrees C and at 40 degrees C with 60 and 30 min operation times respectively. Operational stability of the enzyme was improved further with dithiothreitol treatment. Activity loss was around 5% following 20 cycles of repeated use of the enzyme at 40 degrees C. No significant loss of activity was observed at 28 degrees C when the enzyme was used for 20 cycles. 6-Aminopenicillanic acid in the reaction mixture was observed to be stable during conversion reactions which were carried out at both temperatures.

DNA manipulation techniques have allowed the isolation and identification of many genes which cause disease in man. The same techniques are now being automated, and are on the threshold of application to more common and more complex genetic predispositions to diseases such as diabetes and schizophrenia. This article outlines the technology behind this development, and discusses some of the social, legal and ethical difficulties that widespread screening for genetic traits will raise. It is argued that these issues must be considered now, so that considered social responses to this technology are in place in the five years it is likely to take to develop. The techniques of DNA manipulation that have led to recombinant proteins and biopharmaceuticals have also led to an unprecedented increase in understanding of human genetics, and a corresponding increase in the practical side of human genetics—medical genetic diagnosis. As a result, medical genetics is moving from a speciality of statisticians and cytologists into the mainstream of medicine, and will soon be the concern of every hospital, and probably of every GP. This offers huge benefits to patients, but also threatens misunderstanding and even the potential of abuse if this sophisticated understanding of our fundamental natures is used without precision.

The 2-chloro-3-formyl quinoline derivatives (1a-e) on treatment with acetic anhydride and sodium acetate, afforded the corresponding novel 2-oxopyrano(2,3-b) quinoline derivatives (2a-e), and these were subjected to ammonia treatment to yield the corresponding naphthyridine derivatives (3a-e). The prepared compounds (2a-e) were tested for their antimalarial, diuretic, clastogenic and antimicrobial properties. Not all the compounds showed a diuretic effect and the significant increase in the frequency of micronuclei shows that they are non-clastogens, whereas the 7-chloro derivative (2e) was a very effective antimalarial agent against the mosquito species. All the compounds were found to have optimum antimicrobial activity against Staphylococcus aureus, Escherichia coli and Salmonella typhi. Compounds 2d and 2e were found to be most active against the bacteria tested.

Three types of organic polymers and bead-shape silica gels were activated by graft polymerization of 2,3-epoxypropyl methacrylate; in some cases, epoxide groups on the support surface were modified to NH2 groups. Eight active matrices so obtained were assessed as supports for immobilized enzymes using peroxidase, glucoamylase and urease. The immobilization yield of protein and specific activities of enzymes were better with supports containing NH2 groups than with those containing epoxide spacer arms. Maximum enzyme immobilization and storage stabilities were obtained with silica-gel beads activated by graft polymerization of 2,3-epoxypropyl methacrylate. With all eight matrices tested, the immobilized enzymes showed good stability with not less than 82% of the original activity persisting after 28 days. The developed matrices have potential for use in process-scale biotechnological operations.

Reactive extraction of L-phenylalanine from alkaline aqueous solution into xylene in the presence of tri-octyl-methyl-ammonium chloride (ALIQUAT 336) as complexing agent was studied using a stirred transfer cell. The study investigated the effects of carrier concentration and temperature on mass transfer rates. Transfer rate across the interface in the presence of surfactant molecules was also studied. A two-film model was proposed by considering film mass transfer resistances at the aqueous and organic phases. The model predicted adequately the experimental time-concentration data at different carrier concentrations and temperatures. The model was modified to take into account the presence of surfactant in the organic phase.

The objective of this work was to study the effect of magnetic fields on the rate of phenol biodegradation using immobilized activated sludge. A recirculation flow bioreactor employing immobilized bacterial beads was used with phenol as the substrate to study the biodegradation process. This study was conducted by applying separately the north pole and the south pole magnetic fields to the bioreactor. Rate of dissolved oxygen consumption, phenol concentration and extracellular protein concentration were the parameters monitored during the process. It was observed that by applying a magnetic south pole to the process, biodegradation in the form of biological oxidation was enhanced. A 30% increase in biodegradation rate was obtained by applying a magnetic south pole of strength of 0.45 Tesla to the bioreactor with immobilized microbial beads as compared to the control. Magnetic north pole irradiation inhibited this type of biooxidation. This process has potential for biological treatment of organic wastes.

Macroporous weak cation-exchange methacrylate polymers were synthesized for the immobilization of penicillin G (Pen G) acylase. The role of certain factors such as pore-generating solvent, cross-linking agent, cross-linking density, and comonomer, in enzyme adsorption and expression was studied. Kerosene was a superior pore-generating solvent to paraffin oil. Ethylene glycol dimethacrylate and acrylic acid served as the best cross-linking agent and comonomer, respectively, in the systems studied. 80.3% of the activity of the enzyme adsorbed onto polymer beads prepared with 0.05 mol of acrylic acid (polymer PM-39) was expressed. Properties of the Pen G acylase, immobilized on PM-39 by adsorption and cross-linking with glutaraldehyde (IME-PM-39) were studied. The optimum pH, optimum temperature and Km of Pen G acylase shifted from 8.0 to 7.5-7.8, 50 degrees C to 55 degrees C and 0.038 mol dm-3 to 2.4-3.0 mol dm-3, respectively, as a result of immobilization on PM-39. IME-PM-39 was used repeatedly for 15 cycles in the production of 6-amino penicillanic acid (6-APA).

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Vitamins and related biofactors belong to those few chemicals with a direct positive appeal to people. There is indeed a large need for extra vitamins, other than those derived from plant and animal food sources, due to unbalanced food habits or processing, food shortage or disease. Added vitamins are now either prepared chemically or biotechnologically via fermentation or bioconversion processes. Several vitamins and related biofactors are now only or mainly produced chemically (vitamin A, cholecalciferol (D3), tocopherol (E), vitamin K2, thiamine (B1), niacin (PP or B3), pantothenic acid (B5), pyridoxine (B6), biotin (H or B8), folic acid (B9) or via extraction processes (beta-carotene or provitamin A, provitamin D3, tocopherol, vitamin F-group). However, for several of these compounds microbiological or algal methods also exist or are rapidly emerging. Others are produced practically exclusively via fermentation (ergosterol or provitamin D2, riboflavin (B2), cyanocobalamin (B12), orotic acid (B13), vitamin F-group, ATP, nucleosides, coenzymes, etc. or via microalgal culture (beta-carotene, E, F). Both chemical and microbial processes are run industrially for vitamin B2 while vitamin C (ascorbic acid) is produced via a combination of chemical reactions and fermentation processes. A survey is given here of the current state of vitamin production, with emphasis on developments and strategies for improved biotechnological production and its significance, as compared to existing chemical processes. The screening or construction of vitamin hyperproducing microbial strains is a difficult task; pathway elucidation and metabolic (de)regulation need further study; r-DNA technology has only recently been introduced; improved fermentation processes and immobilised biocatalysts bioconversions for the synthesis of chiral vitamin compounds or intermediates or derivatives are gaining importance; the recovery and purification of these vitamin compounds from their fermentation broths remains equally complex.

Results of preliminary studies on concentration by ultrafiltration and reuse of a Ru(III)EDTA complex in homogeneous catalysis are reported. A method of preparation and selection of conditions of making cellulose acetate membranes for concentrating Ru(III)EDTA is described. At an operating pressure of 75 psig (517 kPa), the above catalyst can be concentrated to seven to eight times its initial concentration. The membrane used exhibits a separation of about 97.5% for the complex with a permeate flow rate of about 3-6 gallons per sq. ft per day. The catalyst activities were determined for two reactions, viz. hydroformylation of 1-hexene and carbonylation of ammonia for five cycles of concentration followed by reuse. The experimental data indicated that the catalyst concentration by ultrafiltration for subsequent reuse is simple and attractive and can provide potentially energy saving alternative to the cumbersome conventional separation processes associated with the recovery of catalysts in homogeneous catalysis.

4-Hydroxyacetophenone (1) was reacted with cinnamonitrile derivatives (2-6) to give 3-cyano-4-(substituted phenyl)-6-(p-hydroxyphenyl)-pyridines (7-11). Interaction of compounds 7-9 with 4'-substituted heterocyclo-benzenesulphonyl diazonium chloride gave the corresponding 3-cyano-4-(substituted phenyl)-6-(3'-azobenzene sulphonamido-4'-hydroxyphenyl) pyridines (12-29). The corresponding iron (III) copper (II) and mercury (II) chelates were also prepared in a 1:2 metal-to-ligand ratio. All the synthesized compounds were characterized on the basis of microanalysis, IR and 1H-NMR spectrometry.

A solvent mixture of 1-hexanol and butyl acetate has been successfully used for the extraction of penicillin G. This solvent mixture may not only synergistically and effectively extract penicillin G, but also inhibit emulsion formation during the extraction of penicillin G from the filtrate of the fermentation broth. The equilibrium constants of the extraction, the molar ratios of 1-hexanol and penicillin G in the extracted species and the synergistic extraction of penicillin G have been studied by chemical analysis and by monitoring the absorbency shift in the FT-IR spectra of the functional groups in the solvents.

The named compound was reacted with thiosemicarbazide and/or semicarbazide to produce the corresponding condensation products II and V respectively. Reaction of II with chloroacetic acid in ethanol containing anhydrous sodium acetate yielded III. Condensation of III with aromatic aldehydes yielded the corresponding arylidene derivatives (IV). Oxidation of the semicarbazone V with selenium dioxide gave 2-(1,2,3-selenadiazole-4-yl)benzimidazole (VIa, b) while with thionyl chloride it gave 2-(1,2,3-thiadiazole-4-yl)benzimidazole (VIIa, b). The chalcones of 2-acetyl and/or 1-methyl-2-acetylbenzimidazole were condensed with hydrazine hydrate, phenylhydrazine and/or hydroxylamine to produce 2-(5-aryl-1(H)-pyrazolin-3-yl)-, 2-(5-aryl-1-phenyl-2-pyrazolin-3-yl)- and 2-(5-aryl-2-isoxazolin-3-yl)benzimidazole (IX, X, XI) respectively.

5-Aryl (or alkyl)-8-hydroxyquinoline-7-sulphonic acids have been prepared by the Mannich reaction of 8-hydroxyquinoline-7-sulphonic acid with primary and secondary amines. Their bactericidal activities have been determined.

The adsorption of phenylalanine, aspartic acid, asparagine and aspartame from phosphate-buffered aqueous solutions with modified divinyl-benzene-polystyrene resins has been investigated using high pressure liquid chromatography (HPLC). The pH studied was 2.8, the temperature range was 293-313 K and the ionic strength was maintained at 1.0 mol dm-3. Over the range of variables investigated, the adsorption isotherms are linear and may be characterized by temperature and pH-dependent apparent adsorption equilibrium constants, characteristic of the resin-adsorbate system. By studying the dependence on temperature of this adsorption constant, heats of adsorption and entropy of adsorption have been estimated. In terms of the heat liberated on adsorption, the amino acids and a dipeptide can be ranked thus: aspartame > phenylalanine > aspartic acid > asparagine.

Copolymers of butyl acrylate and ethylene glycol dimethacrylate with various degree of crosslinking and aminolysis were used for penicillin acylase immobilization. The amount and activity of the bound protein decreased with the decrease of crosslinking when copolymers having nearly constant degrees of aminolysis were compared. The increase of the carrier amino group content increased the ability to bind the protein (by glutaraldehyde method) without a proportional rise of enzyme activity. The comparative studies of immobilization effect and performance of the derivatives showed 40% crosslinked EGDMA, having an amino group content of 0.67 mmol g-3, to be the carrier of choice. This selected carrier was tested for active protein immobilization by use of crude acylase preparation. It was shown that up to a protein concentration of 9 mg cm-3 in the immobilization mixture the active protein was preferentially bound to the polymer matrix. It was indicated also that the derivative stability was mostly affected by multipoint attachment of the enzyme to the carrier surface.

The effects of glutaraldehyde, enzyme concentrations and reactants volumes, ionic strength, pH value and carrier particle diameter on immobilization of penicillin acylase onto acrylic carriers were studied. The activity of immobilized enzyme preparations was also studied over a range of pH values and temperatures and thermal and pH stabilities were determined. The use of the immobilized preparation for penicillin G hydrolysis in a batch reactor was investigated. The immobilized enzyme gave a significant reduction in hydrolysis time compared to hydrolysis by the native enzyme.

Alcohol dehydrogenase (ADH) from horse liver (EC 1.1.1.1), cross-linked through the bifunctional reactive glutaraldehyde, onto nylon tubing was immobilized (35 micrograms cm-2 internal surface of nylon tubing). ADH inactivation kinetics of the immobilized enzyme are of first order (t1/2 = 84.3 h, k = 8.2 x 10(-3) h-1 at 5 degrees C; t1/2 = 2.6 h, k = 0.26 h-1 at 50 degrees C). The activity versus pH profile points to a smaller effect of pH on the activity of the enzyme, which is the case of ADH in solution, explicable on the basis of limitations to proton diffusion towards/from the support. A limiting effect to free external diffusion of the substrate (products) towards/from the support was observed; this effect seems to determine the effective kinetic behaviour of immobilized ADH.

Alpha-amylase from Aspergillus oryzae has been immobilized onto corn grits and porous silica (specific areas 180 and 440 m2 g−1). Kinetic parameters of immobilized enzyme have been determined. Immobilization of alpha-amylase results in the formation of less polymerized products resulting in an apparent decrease in the number of transglycosylation reactions, for both maltotetraose and starch as substrates, when compared with free enzyme. Diffusional limitations for substrate and products have been quantified in the case of the three supports used. External diffusional resistances were important in all cases for the reaction products, whilst they became negligible for the substrate in the case of silica supports. Moreover, internal transfer limitations were identified with silica 180 m2g−1 support. It was demonstrated that diffusional resistances were in direct relation to the apparent modification of the enzyme action pattern after immobilization.

The effects of various commercial and model surfactants of different structure and hydrophilicity were studied on water-in-oil (w/o) emulsion stability, potassium cation leakage and permeation of 6-nitro-3-phenylacetamide benzoic acid in a model system using Penicillin acylase (EC 3.5.1.11) immobilized in a liquid membrane. Both emulsion stability, potassium leakage and permeation of organic substances depend upon hydrophilicity of surfactants. Hydrophilic surfactants may be used to stabilize emulsions only in mixtures with hydrophobic emulsifiers. Additions of small quantities of hydrophilic surfactants to the system in which permeation occurs together within an enzymatic process may be advantageous. Both the rate of permeation and potassium transfer significantly increase when hydrophilic surfactants are present. There was no relationship observed between potassium cation transfer from the internal phase and emulsion stability in the storage test.

A complete characterization of a penicillin acylase biocatalyst is presented, including the determination of physicochemical and kinetic parameters. Stability studies are detailed in terms of both storage temperature and pH as well as operational stability after 150 batch reactions of two hours duration each. An Arrhenius-type model was used to simulate the effect of pH on biocatalyst stability. A kinetic model is proposed to describe batch and continuous stirred tank reactors and to predict the long-term behavior of the process.