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PubMed1997

Pharmacokinetics and metabolism of the novel anticonvulsant agent N-(2,6-dimethylphenyl)-5-methyl-3-isoxazolecarboxamide (D2624) in rats and humans.

N-(2,6-dimethylphenyl)-5-methyl-3-isoxazolecarboxamide (D2624) belongs to a new series of experimental anticonvulsants related to lidocaine. This study was undertaken to understand the pharmacokinetics and metabolism of D2624 in rats and humans, with emphasis on the possible formation of 2,6-dimethylaniline (2,6-DMA). After oral administration of stable isotope-labeled parent drug to rats and GC/MS analysis of plasma samples, two metabolites were identified: D3017, which is the primary alcohol, and 2,6-DMA, formed by amide bond hydrolysis of either D2624 or D3017. In urine, three metabolites of D2624 were identified: namely D3017,2,6-DMA, and D3270 (which is the carboxylic acid derivative of D3017). Based on plasma AUC analysis, D3017 and 2,6-DMA accounted for > 90% of the dose of D2624. After oral administration, D2624 was found to be well absorbed (93%), but underwent extensive first-pass metabolism in the rat, thus resulting in 5.3% bioavailability. Rat and human liver microsomal preparations were capable of metabolizing D2624 to D3017 and 2,6-DMA. The formation of D3017 was NADPH-dependent, whereas 2,6-DMA formation was NADPH-independent and probably was catalyzed by amidase(s) enzymes. In a single-dose (25-225 mg) human volunteer study, the parent drug (D2624) was not detected in plasma at any dose, whereas 2,6-DMA was detected only at the two highest doses (150 and 225 mg). D3017 was detected after all doses of parent drug, with approximate dose proportionality in AUC and a half-life of 1.3-2.2 hr. The metabolic behavior observed in humans suggests there is a marked species difference in the oxidative and hydrolytic pathways of D2624.

Aniline Compounds↗

PubMed1996

[Isolation and characteristics of ekamulin--a prothrombin activator from multiscaled viper (Echis multisquamatus) venom].

Ecamulin, a novel prothrombin activating enzyme, has been isolated and purified 63-fold with a 57% yield from the venom of the Middle-Asian sand viper Echis multisquamatus using three-step ion-exchange chromatography. The enzyme was shown to activate prothrombin similarly to Ecarin, a prothrombin-converting enzyme from Echis carinatus venom, however, differing from the latter by structural and physico-chemical properties. The enzyme is a Zn-proteinase: it contains 1 mol Zn per 1 mol of protein. The molecular mass of the enzyme as determined by Sephacryl S-200 chromatography is 93 +/- 2 kDa. Upon SDS-PAAG electrophoresis ecamulin produces two bands with Mr of 67 and 27 kDa under non-reducing conditions, and three bands with Mr of 67, 14 and 13 kDa in the presence of DTT. During native PAGE without SDS, the activator yields one slow mobility band: two bands are observed after addition of DTT or EDTA. Carbohydrates containing N-acetyl-alpha-D-glucosamine residues are localized in the 67 kDa chain. Ecamulin has two isoforms, S2 and S3, that are distinguished by the charge and partial coagulation activities: form S2 has 250 NIH units/mg, while the S3 form has 524 NIH units/mg. The amino acid sequences of the both isoforms are similar but the more active S3 form has 4 times higher content of Gln and 4 times less of Gly than the S2 form. The isoelectric point is 4.3-4.5; E280 of 1% solution is 10.2. Forms S2 and S3 of ecamulin hydrolyze chromogenic substrates of plasma kallikrein S2302 and glandular kallikrein 2266. Ecamulin does not hydrolyze BAEE, TAME, LEE, thrombin substrates Chromozym TH and S2160, factor Xa-S2222, protein Ca-Chromozym PCa and Plasmin S2251. The amidase activity is nonreversibly inhibited by EDTA, o-phenanthroline (the activity is recovered by addition of Zn2+), Cys or DTT, EGTA, DFP, PMSF or pCMB do not inhibit the enzyme activity. Ecamulin converts prothrombin to alpha-thrombin passing by a shunt via the meizothrombin stage. The reaction of prothrombin activation does not require Ca2+, phospholipids of factor Va. Part of this work was presented at the International Conference "Fibrinogen and fibrinolysis", Yalta, September 23-28, 1995.

Amino Acids↗

PubMed1977

Cathepsin H: an endoaminopeptidase from rat liver lysosomes.

1. Cathepsin H is an endoaminopeptidase belonging to the group of thiol enzymes. It was purified from rat liver lysosomes by gel filtration on Sephadex G-75, chromatography on CM-Sephadex C-50, on DEAE-Cellulose DE-52 and subsequently on an organomercurial absorbent. 2. The molecular weight of cathepsin H was found to be 28,000 and the isoelectric point was estimated to be at pH 7.1 by analytical isoelectric focusing. 3. Cathepsin H has to be designated as endoaminopeptidase, because it catalyzes the hydrolysis of proteins, N-terminal substituted proteins and amino acid derivatives, respectively, as well as of peptides of various chain length and N-terminal free amino acid derivatives. Cathepsin H shows amidase and esterase activity, but it does not show carboxypeptidase activity. The finding of the amino- and endopeptidase nature of cathepsin H has been revealed mainly by the results obtained with inhibitors and by the rather high temperature stability of the enzyme. The chlormethyl ketone of leucine proves to be the strongest inhibitor of the aminopeptidase as well as of the endopeptidase activity, whereas leupeptin endopeptidase activity and endopeptidase substrates inhibit competitively the aminopeptidase activity. 5. Cathepsin H shows highest activity at pH 6.0 in the presence of 1--5 mM GSH and EDTA. 6. The enzyme is stable for several months at slightly acid pH values in a deep frozen state.

Animals↗

PubMed1997

Glycosylation properties of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator.

The oligosaccharide structures present on Asn5 of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator [(r)-[K2tPA]] have been determined by a combination of techniques, including HPLC, FPLC, gel filtration, endoglycosidase digestions and mass spectrometry. The major oligosaccharides identified after their liberation by either hydrazinolysis or by the enzyme peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase, were in the oligomer range of (mannose)8(N-acetylglucosamine)2 (Man8GN2) to Man18GN2. The preponderance of these glycans spanned Man9GN2 to Man12GN2, and the major overall product was Man10GN2. An additional (less than 5%) amount of the polypeptide was hyperglycosylated. In contrast with glycoproteins produced in Saccharomyces cerevisiae, our results with specific mannosidase digestions were consistent with previous studies showing that (alpha 1,3)-linked mannose residues were not present in extensions of the core Man8GN2 unit. The results show that the N-linked glycosylation pathways in P. pastoris are substantially different from those found in S. cerevisiae, with shorter Man(alpha 1,6) extensions to the core Man8GN2 and the apparent lack of significant Man(alpha 1,3) additions representing the major processing modality of N-linked glycans in P. pastoris.

Chromatography, Gel↗

PubMed1997

Characterization of the acidic oligosaccharides assembled on the Pichia pastoris-expressed recombinant kringle 2 domain of human tissue-type plasminogen activator.

The N-linked glycans assembled in Pichia pastoris on the recombinant kringle 2 domain of human tissue-type plasminogen activator (r-[K2tPA]) are composed of approx. 80% neutral and 20% charged species. After peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase-catalysed liberation of the oligosaccharides from the purified glycopeptide, the glycan mixture was resolved by HPLC on amino-silica-based resin. Oligosaccharide mapping of the resulting mixture by HPLC, gel filtration and time-of-flight matrix-assisted laser-desorption-ionization-with-delayed-extraction mass spectrometry (TOF-MALDI DE-MS) revealed that > 90% of the charged species consisted of a series of oligosaccharides possessing molecular masses that were consistent with a range of saccharides comprising phospho-Man10GlcNAc2-phospho-Man14GlcNAc2, with phospho-Man11GlcNAc2 representing the major species. The remaining material in the charged fraction contained identifiable phosphorylated glycans that were one or two mannose units shorter, and one to four mannose units longer, than those present in the above range of oligosaccharides. Treatment of the native charged glycan pool with alkaline phosphatase did not result in molecular-size alterations, showing that phosphomonoesters are not present. Mild acid hydrolysis of the glycans led to a decrease in the size of all charged glycans by one mannose residue, providing phospho-Man9GlcNAc2-phospho-Man13GlcNAc2. Following this procedure, treatment with alkaline phosphatase resulted in size decreases that were equivalent to the loss of one phosphate group from each glycan. This demonstrates that all charged glycans isolated contained phosphate in phosphodiester bonds to two mannose units. The present study shows that P. pastoris cells possess the capability of assembling phosphorylated glycans having the phosphate moiety present in phosphodiester linkages with two mannose units. These saccharides, like the neutral oligosaccharides, contain considerably smaller amounts of mannose than glycans present in other strains of yeast.

Carbohydrate Sequence↗

PubMed1998

Synthesis and characterization of diazomethylarachidonyl ketone: an irreversible inhibitor of N-arachidonylethanolamine amidohydrolase.

N-Arachidonylethanolamine (AEA), a putative endogenous agonist of neuronal (CB1) cannabinoid receptors, is a substrate for N-arachidonylethanolamine amidohydrolase (AEA amidohydrolase), a serine amidase present in cell membranes. Following a strategy that has been used to develop inhibitors that covalently bind to the active site of serine peptidases, diazomethyl arachidonyl ketone (DAK) was synthesized and its effects on AEA amidohydrolase were determined. DAK inhibits the hydrolysis of AEA by rat brain membranes with an IC50 value of 0.5 microM. At low concentrations, DAK reduces the Vmax and increases the K(m) of the enzyme for its substrate AEA, which suggests that it is both a competitive and noncompetitive inhibitor. At higher concentrations, DAK inhibition is completely noncompetitive. DAK inhibition of membrane-associated AEA amidohydrolase is irreversible because hydrolytic activity is not restored with extensive washing or dialysis of the membranes. Furthermore, DAK inhibition is not reversible by anion exchange chromatography of the subsequently solubilized enzyme. In contrast, DAK inhibition of detergent-solubilized enzyme exhibits competitive kinetics and is reversible upon ion exchange chromatography. Exposure of C6 glioma cells to DAK results in concentration-related inhibition of AEA amidohydrolase activity in cellular membranes with an IC50 value of 0.3 microM. In summary, these studies demonstrate that DAK is an irreversible inhibitor of AEA amidohydrolase in its native membrane and provides a useful tool with which to study the role of AEA amidohydrolase in the termination of action of AEA.

Amidohydrolases↗

PubMed1998

A hydrolase enzyme inactivating endogenous ligands for cannabinoid receptors.

Cannabinoids are psychoactive components of marijuana, and bind to specific G protein-coupled receptors in the brain and other mammalian tissues. Anandamide (arachidonoylethanolamide) was discovered as an endogenous agonist for the cannabinoid receptors. Hydrolysis of anandamide to arachidonic acid and ethanolamine results in the loss of its biological activities. The enzyme responsible for this hydrolysis was solubilized, partially purified from the microsomes of porcine brain, and referred to as anandamide amidohydrolase. In addition to the anandamide hydrolysis, the enzyme preparation catalyzed anandamide synthesis by the condensation of arachidonic acid with ethanolamine. Several lines of enzymological evidence suggested that a single enzyme catalyzes both the hydrolysis and synthesis of anandamide. This reversibility was confirmed by the use of a recombinant enzyme of rat liver overexpressed in COS-7 cells. However, in consideration of the high Km value for ethanolamine as a substrate for the anandamide synthesis, the enzyme was presumed to act as a hydrolase rather than a synthase under physiological conditions. The recombinant enzyme acted not only as an amidase hydrolyzing anandamide and other fatty acid amides but also as an esterase hydrolyzing methyl ester of arachidonic acid. 2-Arachidonoylglycerol, which was found recently to be another endogenous ligand, was also efficiently hydrolyzed by the esterase activity of the same enzyme. The anandamide hydrolase and synthase activities were detected in a variety of rat organs, and liver showed by far the highest activities. A high anandamide hydrolase activity was also detected in small intestine but only after the homogenate was precipitated with acetone to remove endogenous lipids inhibiting the enzyme activity. The distribution of mRNA of the enzyme was in agreement with that of the enzyme activity.

Amidohydrolases↗

PubMed1998

Production of D-phenylglycine from racemic (D,L)-phenylglycine via isoelectrically-trapped penicillin G acylase.

Penicillin G acylase (PGA) is exploited for producing pure D-phenylglycine from a racemate mixture, via an acylation reaction onto a cosubstrate, the ester methyl-4-hydroxyphenyl acetate. The reaction, when carried in a batch, is severely hampered by the reverse process, by which the product, 4-hydroxyphenylacetyl-(L)phenyl glycine, upon consumption of L-phenylglycine, is converted by the enzyme back into free substrate and 4-hydroxyphenyl acetic acid via lysis of the amido bond. To prevent this noxious reaction, a multicompartment electrolyzer with isoelectric membranes (MIER) is used as enzyme reactor, operating in an electric field. PGA is trapped between pI 5.5 and pI 10.5 membranes, together with an amphoteric, isoelectric buffer (lysine). As the 4-hydroxyphenylacetyl-(L)phenyl glycine product is formed, it vacates the reaction chamber by electrophoretic transport and is collected close to the anode, in a chamber delimited by pI 2.5 and 4.0 membranes. The same fate occurs to the free acid 4-hydroxyphenyl acetic acid, formed upon spontaneous (and enzyme-driven) hydrolysis of the methyl ester in the reaction chamber. These combined processes leave behind, in the enzyme reaction chamber, the desired product, pure D-phenylglycine. The advantages of the MIER reactor over batch operations: the consumption of the L-form in the racemate is driven to completion and the enzyme is kept in a highly stable form, maintaining 100% activity after one day of operation, during which time the PGA enzyme, in the batch reactor, has already lost >75% catalytic activity.

Electrophoresis, Capillary↗

PubMed1999

Electrically immobilized enzyme reactors: bioconversion of a charged substrate. Hydrolysis Of penicillin G by penicillin G acylase.

The possibility of using the multicompartment immobilized enzyme reactor (MIER) in presence of a charged substrate is here explored. Penicillin G acylase is used to convert penicillin G (a free acid, with a pK of 2.6) into two charged products: phenyl acetic acid (PAA, with a pK of 4.2) and 6-aminopenicillanic acid (6-APA, a zwitterion with a pI of 3.6). The enzyme is trapped by an isoelectric mechanism in a chamber of the electrolyzer delimited by a pI 5.0 and a pI 9.0 amphoteric, isoelectric membranes. Under normal operating conditions (continuous substrate feeding in the presence of an electric field), only a low substrate conversion can be achieved, due to rapid electrophoretic transport of unreacted penicillin G out of the reaction chamber towards the anode. Excellent conversion rates (>96%) are obtained under a "doubly-discontinuous" operation mode: a time-lapse substrate feeding, accompanied by short times (4-8 min) of electric field interruption. The product of interest (6-APA, a precursor of semisynthetic penicillins), by virtue of its amphoteric nature, is trapped in a chamber delimited by a pI 3.5 membrane and a pI 5.5 membrane, adjacent to the reaction chamber on its anodic side. The other contaminant product (PAA) first accumulates in the same chamber and then progressively vacates it to collect in the anodic reservoir, leaving behind a pure 6-APA solution. In this operation mode, vanishing amounts of unreacted substrate (penicillin G) leave the reaction chamber to contaminate the adjacent, anodic chambers. A novel class of zwitterionic buffers is additionally reported, able to cover very thoroughly any pH value along the pH 3-10 interval: polymeric, zwitterionic buffers, synthesized with the principle of the Immobiline (acrylamido weak acids and bases) chemicals. Enhanced enzyme reactivity is found in this macromolecular buffers as compared to conventional ones.

Bioreactors↗

PubMed2000

Effects of retinoic acid on N-glycosylation and mRNA stability of the liver/bone/kidney alkaline phosphatase in neuronal cells.

Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We investigated the effects of RA on N-linked glycosylation of the tissue nonspecific liver/bone/kidney- type of ALP (L/B/K ALP), on ALP transcripts, and on total protein glycosylation in two neuronal cell lines, P19 and NG108CC15, and in primary cultures of neonatal rat brain. ALP activity was determined in cell extracts and found to be induced by RA. Tunicamycin was used at various concentrations to inhibit protein N-glycosylation. After treatment of cells with low concentrations (0.1 and 0.125 microgram/ml) of tunicamycin for 48 h, uninduced and RA-induced ALP activity declined while incubation with a protease inhibitor restored activity, indicating that the L/B/K ALP bear N-linked oligosaccharide chains important for maintaining enzymatic activity. Interestingly, ALP activity in RA-treated cultures was less inhibited by tunicamycin compared to untreated controls suggesting that RA may have an impact on ALP N-glycosylation. To investigate effects of RA on ALP glycosylation further, incorporation of [(14)C]mannose and [(35)S]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mannosylation and biosynthesis demonstrate that incubation of cells with RA increased [(14)C]mannose incorporation into ALP molecules. Also, the release of free [(14)C]mannose from ALP molecules relative to the amount of protein by N-Glycanase was increased in RA-treated cultures. In addition, mannosylation of total protein was found to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosylation of the polypeptides. Furthermore, tunicamycin decreased the stability of ALP mRNA, an effect that was reduced by cotreatment with RA. Thus, the degree of N-glycosylation of the L/B/K ALP as well as mRNA and protein levels of this enzyme are affected by RA. The P19 cell line provides a useful model system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further.

Alkaline Phosphatase↗

PubMed1996

Identification of the rat epididymis-secreted 4E9 antigen as protein E: further biochemical characterization of the highly homologous epididymal secretory proteins D and E.

Epididymis-secreted proteins D and E have been purified to homogeneity and partially characterized, and it is shown that monoclonal antibody (MAb) 4E9 (raised against a detergent extract of rat caudal epididymal sperm [Moore et al., 1994: Mol Reprod Dev 37(2):181-194]) recognizes protein E, but not protein D. The molecular weight of protein D (approximately 30 kD) is approximately 2 kD lower than protein E (approximately 32 kD). The NH2-terminus of each protein is blocked; however, microsequencing of internal peptides confirms earlier reports of significant sequence identity between the two proteins. High performance liquid chromatography tryptic peptide mapping showed peak differences between the two proteins, but it was not possible to obtain amino acid sequence in the peaks that were different. The epitope for MAb 4E9 was localized in the blocked NH2-terminus-CNBr peptide derived from protein E. The epitope was destroyed by protease treatment of protein E. Removal of N-linked oligosaccharides did not destroy the epitope for MAb 4E9 and did not affect the molecular weight difference between the proteins.

Amidohydrolases↗

PubMed1998

Preliminary studies on continuous recovery and purification of the penicillin acylase under pseudo-affinity conditions using phenyl-Sepharose gel.

A continuous system for the recovery and purification of the penicillin acylase from crude extracts by recycling phenyl-Sepharose gel through three agitated vessels with disc filters of stainless steel is presented. The penicillin acylase present in the crude extract was absorbed into the phenyl-Sepharose gel under pseudo-affinity conditions (16% w/v of ammonium sulphate). After gel washing under the same conditions in the second vessel, enzyme desorption was performed using the same salt but at a lower concentration (6% w/v) in the third vessel. The preliminary studies reported here occurred without experimental difficulties, even at a gel concentration as high as 40% (v/v). The recovery of the penicillin acylase was achieved with high yield (74%), but a low purification factor of 2.4 was obtained owing to the use of a crude extract with low specific activity.

Affinity Labels↗

PubMed1999

Salivary proteins in rheumatoid arthritis and Sjögren's syndrome: one-dimensional and two-dimensional electrophoretic studies.

Parotid saliva from patients with rheumatoid arthritis and Sjögren's syndrome contains elevated levels of multiple anionic proteins of pI approximately 3.75-4.75 and Mr approximately 32,000. Further studies on these components involving narrow range pH 3.5-5.0 immobilized pH gradients (IPGs) and two-dimensional (2D) electrophoresis with narrow- or broad-range IPGs in the first dimension have confirmed their association with these disorders. Immunoblotting showed that these proteins include multiple forms of tissue kallikrein. Treatment with neuraminidase results in removal of these anionic substances from the pH 3.75-4.75 region of gels, thereby indicating that heterogeneity arises from differences in sialation of the carbohydrate residues. The results of treatment with endo-beta-N-acetylglucosaminidase (Endo F) or peptide N-glycosidase (PNGase F) and comparison of the results with studies on human urinary kallikrein suggest that proteins other than kallikrein may also comigrate in the anionic region of gels and that deglycosylation of kallikrien is incomplete in the experimental conditions used, probably because of inadequate denaturation. The paper also reviews the limitations of current criteria used in the investigation of salivary gland function associated with connective tissue disorders and the diagnosis of these. It assesses the potential of electrophoresis in forming the basis of new diagnostic techniques and furthering the understanding of the nature of these diseases. The findings presented in this paper could make a key contribution to this.

Amidohydrolases↗

PubMed2001

Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast).

BACKGROUND: Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. METHODS: Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. RESULTS: We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. CONCLUSIONS: Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.

Amidohydrolases↗

PubMed2000

Functional reconstitution of an active recombinant human chymase from Pichia pastoris cell lysate.

We have previously reported efficient production of mature human chymase (h-chymase) using an original system of expression in Pichia pastoris (Nakakubo et al., 2000), whereby recombinant h-chymase (rh-chymase) was secreted as a mature form with the correct N-terminal amino acid sequence and was easily purified. In the course of investigation of secretory rh-chymase, we also found large amounts of chymase to be present in insoluble form in the transformant cell. Although the cellular rh-chymase had no proteolytic activity, its chymotryptic activity was restored in a reconstitution process utilizing guanidine and glutathione. As with secretory rh-chymase, efficient purification was possible by heparin affinity chromatography. The purified cellular rh-chymase showed the same mobility as secretory rh-chymase in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after deglycosylation. N-terminal amino acid sequence analysis revealed that the signal peptide had been correctly removed. K(m) value (5.93 mM), as well as pH profile and inhibition profile toward protease inhibitors of reconstituted cellular rh-chymase, indicated that the rh-chymase enzymatically closely resembles native h-chymase. Furthermore, it showed a greatly restricted proteolytic activity towards Ang I, and formed Ang II without the further cleavage which is a feature of h-chymase. It was thus found that the insoluble rh-chymase stored in the cells could be solubilized and reconstituted to give the same structure as h-chymase, not only in terms of enzyme active site but also of substrate recognition site.

Amidohydrolases↗

PubMed2000

A systematic approach to the validation of process control parameters for monoclonal antibody production in fed-batch culture of a murine myeloma.

A systematic approach to the validation of control ranges of control parameters for a cell culture process producing a monoclonal antibody is described. Specifically, the structure and functional activity of a monoclonal IgG1 antibody produced at the outer limits of numerical ranges of fed-batch culture control parameters such as pH and temperature were examined, with the aim of providing assurance that antibody produced under varying culture conditions was of consistent quality based on a carefully defined set of specifications. An experimental design was created using a half-fractional factorial design for fed-batch culture incorporating half of the thirty two possible combinations of five selected control parameters at high and low levels. Statistical analysis of all data gathered from the study allowed an assessment of the effects of the process control parameters at either high or low outer limits on fed-batch culture response variables such as growth rate and specific antibody productivity. Measured values for the responses of growth rate and specific antibody productivity throughout this study ranged from 0.22-0.44 d(-1) and 6.4-32 microg monoclonal antibody/10(6) cells/d respectively. Analytical characterisation of monoclonal antibody purified from each fed-batch culture considered the purity, structure and biological activity of the glycoprotein. All antibody preparations were identical to each other and to the current antibody reference standard or control. Glycosylation analysis of certain samples from the study demonstrated that the distribution of glycoforms of the antibody was not affected by the varying process control conditions of the fed-batch cultures.

Amidohydrolases↗

PubMed2000

Characterization of an industrial biocatalyst: immobilized glutaryl-7-ACA acylase.

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.

Amidohydrolases↗

PubMed2000

Equilibrium position, kinetics, and reactor concepts for the adipyl-7-ADCA-hydrolysis process.

One of the building blocks of cephalosporin antibiotics is 7-amino-deacetoxycephalosporanic acid (7-ADCA). It is currently produced from penicillin G using an elaborate chemical ring-expansion step followed by an enzyme-catalyzed hydrolysis. However, 7-ADCA-like components can also be produced by direct fermentation. This is of scientific and economic interest because the elaborate ring-expansion step is performed within the microorganism. In this article, the hydrolysis of the fermentation product adipyl-7-ADCA is studied. Adipyl-7-ADCA can be hydrolyzed in an equilibrium reaction to adipic acid and 7-ADCA using glutaryl-acylase. The equilibrium reaction yield is described as a function of pH, temperature, and initial adipyl-7-ADCA concentration. Reaction rate equations were derived for adipyl-7-ADCA-hydrolysis using three (pH-independent) reaction rate constants and the apparent equilibrium constant. The reaction rate constants were calculated from experimental data. Based on the equilibrium position and reaction rate equations the hydrolysis reaction was optimized and standard reactor configurations were evaluated. It was found that equilibrium yields are high at high pH, high temperature and low-initial adipyl-7-ADCA concentration. The course of the reaction could be described well as a function of pH (7-9), temperature (20-40 degrees C) and concentration using the reaction rate equations. It was shown that a series of CSTR's is the best alternative for the process.

Adipates↗