Single-step conversion of cephalosporin-C to 7-Aminocephalosporanic acid by free and immobilized cells of <Emphasis Type="Italic">Pseudomonas diminuta</Emphasis>

7-Aminocephalosporanic acid (7-ACA), the starting material for the production of a number of clinically used semisynthetic cephalosporins, is produced by deacylation of cephalosporin-C. The production of 7-ACA was studied in various modes, at the optimal conditions using free and immobilized whole cells of Pseudomonas diminuta.     

Immobilization of glutaryl-7-aminocephalosporanic acid acylase on silica gel and enhancement of its stability

Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase isan enzyme that converts GL-7-ACA to 7-aminocephalosporanic acid, a starting material for semisynthetic cephalosporin antibiotics. In this study, optimal conditions for the immobilization of GL-7-ACA acylase were determined by experimental observations and statistical methods. The optimal conditions were as follows: 1.1 M phosphate buffer (pH 8.3) as buffer solution, immobilization temperature of 20°C, and immobilization time of 120 min. Unreacted aldehydegroups were quenched by reaction with a low-molecular-weight material such as l-lysine, glycine, and ethanolamine after immobilization in order to enhance the activity of immobilized GL-7-ACA acylase. The activities of immobilized GL-7-ACA acylase obtained by using the low-molecular-weight materials were higher than those obtained by immobilized GL-7-ACA acylase not treated with low-molecular-weight materials. In particular, the highest activity of immobilized GL-7-ACA acylase was obtained using 0.4% (v/v) ethanolamine. We also investigated the effect of sodium cyanoborohydride in order to increase the stability of the linkage between the enzyme and the support. The effect on operational stability was obvious: the activity of immobilized GL-7-ACA acylase treated with 4% (w/w) sodium cyanoborohydride remained almost 100% after 20 times of reuse.     

Enzymatic transformation of cephalosporin C to 7-ACA by simultaneous action of immobilized d-amino acid oxidase and glutaryl-7-ACA acylase

The enzymatic transformation of cephalosporin C (CEPH C) to 7-amino-cephalosporanic acid (7-ACA) using D-amino acid oxidase (DAO) and glutaryl-7-ACA acylase (G1-7-ACA) is reported. The enzymes have been immobilized separately on different carriers, in order to maximize the catalytic activity and the stability. The reaction has been carried out in single-step-like conditions, using the two enzymes simultaneously. The effect of catalase contamination, present in the DAO preparations, was balanced by addition of extra hydrogen peroxide. In optimum conditions, the conversion of CEPH C to 7-ACA was higher than 90%, with byproduct formation lower than 4%. The mixture of immobilized enzymes was reused in repeated reaction cycles, showing an appreciable operational stability.     

Purification and stability of glutaryl-7-ACA acylase from pseudomonas sp.

The enzyme glutaryl-7-ACA acylase fromPseudomonas sp. NCIMB 40474, produced by a recombinantEscherichia coli host, was purified to homogeneity. The enzyme is a tetramer composed of two couples of asymmetric dimers, each of them constituted of two subunits of mol wt 18 and 52 kDa, respectively. It was found that glutaric acid, one of the products of the substrate hydrolysis, is an effective acylase inhibitor. Between pH 6.0 and pH 10.0, the enzymatic activity is almost constant, but below pH 6.0 it progressively declines. The acylase activity decreased sharply as a function of guanidine HC1 concentration. The loss is significant even at concentrations of denaturant lower than those causing unfolding, as suggested by UV spectroscopy and fluorescence emission studies. In these conditions (low denaturant concentration and low pH) the inactivation of the enzyme is caused by the tetramer dissociation into dimers. The lability of the quaternary structure of the enzyme is a key feature that must be taken into account for the improvement of the catalyst stability.     

Inoculum studies in production of penicillin g acylase by Bacillus megaterium ATCC 14945

This article reports studies concerning the production of penicillin G acylase (PGA) by Bacillus megaterium. This enzyme has industrial use in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid, an essential intermediate for the production of semisynthetic β-lactam antibiotics. Although most microorganisms produce the enzyme intracellularly, B. megaterium provides extracellular PGA. The enzyme production by microorganisms involves several steps, resulting in a many operational variables to be studied. The study of the inoculum is an important step to be accomplished, before addressing other issues such as culture optimization and downstream processing. In this study, using a standard inoculum as reference, several runs were performed aiming at the definition of operational conditions in the PGA production. Cell concentration and PGA activity in the production medium were measured after 24, 48, and 72 h of the beginning of the production phase. This study encompasses the duration of the inoculum germination phase and the concentration of cells used to startup the germination. Based on these results, PGA productivity during the production phase was maximized. The selected values for these variables were 1.5 × 10⁷ spores/mL of germination medium, germination during 24 h, and 72 h for the production phase.     

The Effect of 2-Mercapto-5-Methyl-1,3,4-Thiadiazole on Enzymatic Synthesis of Cefazolin

The effect of unreacted residual 2-mercapto-5-methyl-l,3,4-thiadiazole (MMTD), the reagent for 3-[5-methyl-l,3,4-thiadiazole-2-yl]-7-aminocephalosporanic acid (M-7-ACA) synthesis, on the enzymatic acylation of M-7-ACA by the methyl ester of 1,2,3,4-tetrazol-1-acetic acid (MeTzAA) to produce cefazolin (CEZ) was studied. In the two-step process of synthesizing CEZ from 7-aminocephalosporanic acid (7-ACA), one of the key parameters controlling the overall CEZ yield was the ratio of MMTD to 7-ACA in M-7-ACA synthesis. The increase of the ratio showed opposing effects by increasing the M-7-ACA yield in the first step, while decreasing CEZ yield in the subsequent enzymatic reaction by the inhibitory effect of the increased content of MMTD as an impurity in the M-7-ACA preparation. It was revealed that the decrease of CEZ yield in the enzymatic reaction was caused by the selective retardation of the rate of CEZ synthesis reaction by a typical competitive inhibition, while not affecting the rate of MeTzAA hydrolysis reaction. The optimum MMTD-to-7-ACA ratio rendering the highest overall CEZ yield over 7-ACA was 1.2:1.     

Expression and Production of Human Interleukin-7 in Insect Cells Using Baculovirus Expression Vector System (BEVS)

Interleukin-7 (IL-7) is a glycoprotein cytokine with significant clinical and biomedical potential, such as cancer therapy and HIV infections. Earlier it has been cloned and expressed in various protein expression systems; however, they are not efficient for large-scale production. To address this inadequacy, we report in this paper the production of recombinant human interleukin-7 (hIL-7) in insect cells. A recombinant bacmid containing hIL-7 was constructed, purified, and characterized. It was used to infect Trichoplusia ni (BT1-TN-5B1/High Five™) insect cells. Result shows that T. ni cells successfully produce hIL-7 in shake flask cultures. A scale up to 2.5-L laboratory batch bioreactor showed the efficacy of this system for large-scale production. Our results offer a highly efficient, inexpensive, and convenient system for the large-scale expression and production of recombinant hIL-7.     

Contributions to the synthesis of some ferrocene-containing antibiotics

This Review discusses the synthesis and characterization by our Group of new antibiotics belonging to the class of penicillins, cephalosporins and rifamycins with ferrocenyl and 1, 1′-ferrocenilene residues in the molecule. As reactants for 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid (7-ACA) the following were used: 1, 1-bis(chlorocarbonyl)ferrocene, ferrocenyl sulfochloride, 1, 1′-ferrocenylenedisulfochloride and thioglycolic acids S-modified with ferrocene. In the synthesis of rifamycins, the hydrazides of the thioglycolic acids, S-modified with ferrocene, were employed as nucleophilic agents. The synthesized intermediates were characterized by elemental analysis, TLC, IR, UV and ¹H NMR spectra. The characterization of new antibiotics was made by TLC, IR and UV spectral analysis. Biological activity was tested on Gram-negative and Gram-positive bacteria. Good activity is reported towards Gram-positive bacteria in the case of derivatives containing residues of thioglycolic acid S-modified with ferrocene, the antibacterial activity being similar to that of amoxicillin, carbenicillin and cephalothin. All compounds are inactive towards Gram-negative bacteria.     

Preparation and characterization of multipoint yeast D-amino acid oxidase mutants with improved stability and activity

D-amino acid oxidase (DAAO) is an FAD-containing oxidoreductase that stereospecifically oxidases D-amino acids to produce α-keto-acids, an ammonium ion, and hydrogen peroxide. The most important biotechnological process involving DAAO is the production of 7-amino cephalospranic acid (7-ACA) from cephalosporin C. The reaction product, 7-ACA, is then used as a precursor for the synthesis of cephalosporin antibiotics of different generations. We previously obtained mutant DAAOs from the yeast Trigonopsis variabilis (TvDAAO). The mutants with point amino acid substitutions were characterized by either an increased thermal stability or improved catalytic properties in the oxidation of cephalosporin C. In the present study, we obtained two new mutant TvDAAOs with two and four amino acid substitutions, respectively. The catalytic constants of these mutant TvDAAOs for the oxidation of cephalosporin C were 1.8 and 4 times higher than the respective parameter of the wild-type enzyme (wt-TvDAAO). The combination of substitutions increased the thermal stabilities of both mutant TvDAAOs by a factor of 2–3 as compared with the wt- TvDAAO.     

Effect of Cultural Conditions and Media Constituents on Production of Penicillin V Acylase and CTAB Treatment to Enhance Whole-Cell Enzyme Activity of <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis> (NCIM 3425)

Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic β-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 °C. Fructose was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170 and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic detergent. Whole-cell activity was found to be doubled (204%) on treatment of 0.01 g dry weight of cells with 50 μg/ml solution of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.     

Immobilization of whole-cell penicillin g acylase by entrapping within polymethacrylamide beads

Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide andN,N’- methylenebisacrylamide. A solution of monomer that was made up from methacrylamide andN,N’-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013–0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45‡C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.     

Stabilization ofd-amino acid oxidase from yeastTrigonopsis variabilis used for production of glutaryl-7-aminocephalosporanic acid from cephalosporin C

The studies to improve the production of glutaryl-7-ACA from cephalosporin C are described in this paper. During the conversion of cephalosporin C to keto-adipyl-7-aminocephalosporonic acid by d-amino acid oxidase (d-AAO), with the simultaneous production of equimolar amount of hydrogen peroxide, an incomplete nonenzymatic conversion of the keto form into the glutaryl form occurs, where cephalosporin C as well asd-AAO are partly destroyed in the presence of hydrogen peroxide. d-AAO was immobilized to different carriers in order to achieve better enzyme stability. The activity of immobilizedd-AAO on manganese oxide remained above 100% during the first 9 h of a semicontinuous conversion of cephalosporin C. The presence of catalase coimmobilized with D-AAO and coupled to CNBr-activated Sepharose 4B improved the operation stability ofd-AAO. An additional approach for the continuous transformation of cephalosporin C used whole cells ofTrigonopsis variabilis, containingd-AAO, immobilized to magnetic iron oxide particles.     

Efficient procedure for c '- 3 substitution and c - 7 n-acylation of 7-aminocepha-losporanic acid (7-aca): Synthesis of cefazolin antibiotic and related compounds

A new preparative method for the C'-3 substitution of 7-aminocepha-losporanic acid (7-ACA), is described. The key feature of our method is based on the protection of the anino group as a Schiff base instead of the usual procedure based on the acylation of the amino group. The relative incapacity of 7-ACA derivatives to produce organic solutions with usual tertiary bases is easily overcome with bicyclic amidines. Catalytic amounts of these bases and N-trimethylsilyl-2-oxazolidinone are used to obtain the silylated products. Activation of sensitive tetrazolylacetic acid by means of N,N-dimethylchloro-sulfitemethaniminium chloride (SOCl₂-DMF) and preparation of cefazolin antibiotic under anhydrous conditions is also described.     

Microglial P2X₇ receptor expression is accompanied by neuronal damage in the cerebral cortex of the APPswe/PS1dE9 mouse model of Alzheimer's disease

The possibility that P2X₇ receptor (P2X₇R) expression in microglia would mediate neuronal damage via reactive oxygen species (ROS) production was examined in the APP_swe/PS1dE9 mouse model of Alzheimer''s disease (AD). P2X₇R was predominantly expressed in CD11b-immunopositive microglia from 3 months of age before Aβ plaque formation. In addition, gp91^phox, a catalytic subunit of NADPH oxidase, and ethidium fluorescence were detected in P2X₇R-positive microglial cells of animals at 6 months of age, indicating that P2X₇R-positive microglia could produce ROS. Postsynaptic density 95-positive dendrites showed significant damage in regions positive for P2X₇R in the cerebral cortex of 6 month-old mice. Taken together, up-regulation of P2X₇R activation and ROS production in microglia are parallel with Aβ increase and correlate with synaptotoxicity in AD.     

Probing the role of N-linked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis

The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that “tuning” enzyme glycosylation for expression from heterologous expression hosts is essential for generating engineered enzymes with optimal stability and activity.     

Enzymatic Fluorimetric Determination of Ursodeoxycholic Acid in Urine Using Clostridium Absonum 7β-Hydroxysteroid Dehydrogenase

Abstract

A simple and rapid enzymatic fluorimetric method for the determination of ursodeoxycholic acid (UDCA) and its glycine (GUDCA) and taurine (TUDCA) conjugates in urine has been developed. Octadecylsilane-bonded silica cartridges (Sep-Pak C₁₈) are used for the solid-phase extraction of bile acids (BA) from urine samples. the method is based on the fluorimetric monitoring of NADPH formed via the reaction of 7β hydroxylated BA (7β-BA) with β-nicotinamide adenine dinucleotide phosphate (β-NADP⁺) catalysed by 7β hydroxysteroid dehydrogenase (7β-HSD). the 7β-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC # 27555) and purified by affinity chromatography.

The method has a limit of detection of 2 μmol/L (initial sample concentration), within-run precision varied from 8.3% to 5.3% and between-run precision varied from 12% to 1.8% for low and high concentrations respectively. the recovery of ursodeoxycholic acid added to urine samples was about 98% (range 88–110%). the method was successfully applied for UDCA determination in urine samples from patients subjected to UDCA therapy. Randomly collected urine samples from patients and controls were used and the results were expressed as ratio of [UDCA]/[creatinine] to correct for variation in urine flow.     

Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.     

Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO₄·5H₂O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.     

Co-cultured production of lignin-modifying enzymes with white-rot fungi

Co-cultivation was a potential strategy in lignocellulolytic biodegradation with producing high activity enzymes due to their synergistic action. The objective of this study was to investigate the rarely understood effects of co-culturing of two white-rot fungi on lignin-modifying enzymes (LMEs) production. Six species, Bjerkandera adusta, Phlebia radiata, Pleurotus ostreatus, Dichomitus squalens, Hypoxylon fragiforme and Pleurotus eryngii, were cultured in pairs to study the production of LMEs. The paired hyphal interaction observed showed that P. eryngii is not suitable for co-growth. The use of agar plates containing dye RBBR showed elevated decolourisation at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. radiata with D. squalens under submerged cultivation; the highest value was measured after 4 days of incubation (120 U mg⁻¹). The improved productions of MnP and LiP were simultaneously observed at the co-culture of P. ostreatus and P. radiata (MnP = 800 nkat L⁻¹ after 4 days of incubation; LiP = 60 nkat L⁻¹ after 7 days of incubation), though it was not a good producer of laccase. P. ostreatus appeared to possess specific potential to be used in co-cultured production of LMEs. The phenotype of LMEs production was not only dependent on the species used but also regulated by different nutritions available in the culture medium. The present data will provide evidence for illustrating the regulatory roles of C/N on LMEs production under the co-cultures’ circumstances.     

Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient

Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia entropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal σ⁷⁰ promotor. The pKS/PHB was the most efficient plasmid for phboperon expression among the three plasmids used: i.e., pKS⁻, pAED4, and pJM9131. It was observed that isopropyl-β-d-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkan cote content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.