Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: a key to advanced biotransformation.

The kinetics of immobilized enzymes can not be analyzed by means of the simple Michaelis-Menten concept, which generally fails to describe the immobilized state due to both its probable barriers, and because the active concentration of the enzyme approaches, or even exceeds this of its substrate(s). In such cases, the various experimental data are usually treated by complex rate equations comprising too many parameters acquiring different natures and meanings, depending on both the properties of the immobilization state and the experimental conditions; thus, more likely, only apparent values of the Michaelis-Menten kinetic parameters can be estimated experimentally. Likewise, immobilization is often a key method in optimizing the operational performance of enzymes, in both laboratory and industrial scale, and affects considerably the kinetics in non-aqueous and non-conventional media due to several issues as the structural changes of the enzyme molecule, the heterogeneity of the system, and the partial or total absence of water. In this work a theoretical approach is described on the formulation of simplified rate equations, reflecting also the actual mass balances of the reactants, in the case where esterification synthetic reactions are catalyzed by immobilized lipases, in either a non-aqueous organic solvent or in a non-solvent system.

Purification and characterization of a novel extracellular protease from a halo-alkaliphilic Bacillus sp. 17N-1, active in polar organic solvents.

A novel enzyme of molecular mass about 29 kDa was purified from the strain halo-alkaliphilic Bacillus sp. 17N-1 and designated protease-B-17N-1. This enzyme is likely to be a cysteine protease; it was found active in media containing EDTAK2 and dithiothreitol, it maintained considerable activity at temperatures 14 degrees C to 33 degrees C and pH 6.50 to 8.50 with optimum k(cat)/Km and/or k(cat) values at pH 7.00 and 25 degrees C. The activity of protease-B-17N-1 was strongly affected by the specific irreversible inhibitor of cysteine proteases E-64, while it remained unaffected by the 3,4-dichloro-isocoumarine, an irreversible inhibitor specific for serine proteases. Protease-B-17N-1 retained full activity at 25 degrees C after 30 min incubation at 8 degrees C or at 33 degrees C; moreover, it was found to be stable and active in the polar organic solvents DMSO and acetonitrile. The enzyme hydrolyzed the substrate Cbz-FR-pNA via Michaelis-Menten kinetics, while it showed insignificant activity for the substrate Suc-AAA-pNA. Valuable pK(a)s, rate constants, activation energies and other important features were estimated from the profiles of parameters k(cat)/Km, k(cat) and Km, versus pH, temperature, and [NaCl]. In addition, interesting results were obtained from the effect of different metallic ions and polar organic solvents on the Michaelis-Menten parameters of protease-B-17N-1, showing that it performs catalysis via a (Cys)-S(-)/(His)-Im(+)H ion-pair, as well as its industrial and biotechnological potential, respectively.

Investigation of the active center of rat pancreatic elastase.

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.

Insight into catalytic mechanism of papain-like cysteine proteinases: the case of D158.

We studied the role of D158 in papain-like cysteine proteinases by using subtilisin Carlsberg, and its chemically modified analog thiolsubtilisin, by applying the proton inventory (PI) method and also by taking into account the pH profiles of the kcat/Km parameter. In the case of thiolsubtilisin, we estimated large inverse solvent isotope effects for kcat/Km, as in papain, whereas for subtilisin we found "dome-shaped" PI, suggesting a completely different mechanism. Finally, the kinetic behavior of thiolsubtilisin presented similarities as well as differences, compared to papain, suggesting a possible role for D158 as part of a catalytic triad in papain-like cysteine proteinases.

Aristophanes' Wealth: ancient alternative medicine and its modern survival.

The miraculous cure of the blind god Plutos ("Wealth') in Aristophanes' play illuminates some of the reasons why people have sought help in alternative medicine over the ages. Apart from limitations of conventional medicine these factors can be social, political, religious, psychological, and scientific. Alternative medicine may function in a complementary way to the conventional. Nevertheless, an overestimation of its therapeutic potentials by the public can lead to the domination of irrationalism, all in the name of liberation from the shackles of a mechanistic rationalism.

Modeling growth, substrate consumption and product formation of Penicillium nalgiovense grown on meat simulation medium in submerged batch culture.

Penicillium nalgiovense is the most widely used starter mold for cured and fermented meat products. The development of a biomass film on the surface of these products prevents a large degree undesirable growth of various fungal contaminants and contributes to the ripening process with production of metabolites. This work presents an attempt to model the growth of P. nalgiovense and to relate it to substrate consumption and product release. Because of the extremely complex nature of the meat product fermentation, submerged culture was employed in a bioreactor system that enabled on-line monitoring, using a meat simulation medium, which contained peptones and lactate as carbon, nitrogen and energy sources. The unstructured model presented is based on a partial association of substrate assimilation and product formation with growth. Experimentally derived values for peptones and lactate were compared with model-derived values and their proportions corresponding to growth associated parts, used for biosynthesis, and non-growth associated parts, used for maintenance. The model was applied for the products ammonia, carbon dioxide and protons. Both peptones and lactate were used mainly for biosynthesis (85 and 80% of the total amounts provided, respectively). Assimilation of lactate and ammonia formation from amino acid metabolism resulted in a proton exchange, which was mainly growth associated. The contribution of the growth associated mechanism to the total proton exchange was estimated to be 75% while the contribution of the non-growth associated mechanism increased during the growth phase and reached a maximum of 25%. For carbon dioxide production, the contribution of a maintenance mechanism was evident at 40 h, while production was growth-associated and remained such even at the end of fermentation at 168 h when growth rate was very low. The partially growth associated model showed good agreement with the experimental data and allows accurate determination of the proportions of substrates or products related to biosynthesis and cell maintenance.

[The cost of treating schizophrenia in Greece].

The objective of this study was to estimate the direct annual cost of treating patients with schizophrenia in Greece in 2005. Due to the lack of quantitative data, information on the treatment pathway and medical resource utilization of patients were collected from a consensus panel of 9 psychiatrists and 5 health economists. For estimating the costs a bottom up approach from the National Health System perspective was used. The panel of experts defined three patient categories based on the severity of the disease and the medical setting where treatment is received: (a) outpatient setting, (b) ambulatory care, (c) inpatient setting and long-term care. The annual direct cost of treatment per patient was found to be 3,187 € (2,659-4,166 €) in the first category, 10,135 € (7,429-13,972 €) in the second category and 20,782 € (17,482-25,462 €) in the third category. The total cost of treatment increased with the severity of the disease and the use of hospitalization. Systematic data collection on medical resource utilization must be established at the national level to facilitate further research, guide the efficient use of resources and improve the healthcare provision.

Removal of foreign bodies from subsegmental bronchi with the fiberbronchoscope.

Though the fiberbronchoscope has been designed for diagnostic purposes, it can be used, as well, for the removal of small foreign bodies from segmental and subsegmental bronchi; In this paper we report two cases in which we successfully removed from subsegmental bronchi a pulp-canal reamer and a melon seed. This fact proves that in some of these cases we can avoid thoracotomies.

Insight into the catalysis of hydrolysis of four newly synthesized substrates by papain: a proton inventory study.

We synthesized the following four new peptide substrates, Suc-Phe-Leu-pNA, Suc-Phe-Leu-NMec, Suc-Phe-Leu-ONPh, and Pht-Phe-Leu-pNA, and we applied the proton inventory method to their hydrolysis by papain. Useful relationships between the rate constants of the catalytic reaction have been established and contributed to the elucidation of the hydrolytic mechanism of papain. For all amide substrates, the parameter K(S) and the rate constants k(1), k(-)(1), and k(2) were estimated. Moreover, it was found that k(cat)/K(m) = k(1) for all four substrates, while two exchangeable hydrogenic sites, one in the ground state and another in the transition state, generate an inverse isotope effect during the reaction governed by this parameter. The proton inventories of both k(2) and k(3) are essentially linear, whatever the acyl moiety and/or the leaving group of the substrate. The proton inventories of K(S) are also essentially linear for all amide substrates, while the observed large isotope effect of about 3 to 9 originates from a single hydrogenic site in the product state. This latter, in agreement to both the small transition state fractionation factors found for k(cat)/K(m) (or k(1)) and the unit ground-state fractionation factors found for k(2), argues for the formation of a tetrahedral adduct during the reaction governed by the k(1) parameter. Furthermore, papain acts as a one-proton catalyst during acylation or deacylation, both of which proceed through similar concerted reaction pathways, where a nucleophilic attack is accompanied by the movement of one proton.

Interaction of the peptide CF3-Leu-Ala-NH-C6H4-CF3 (TFLA) with porcine pancreatic elastase. X-ray studies at 1.8 A.

The peptide trifluoroacetyl-Leu-Ala-(p-trifluoromethylanilide), is a reversible inhibitor of pancreatic porcine elastase and is characterized by a Km of 2.5 x 10(-8) M. Co-crystals of the 1:1 complex were obtained in an acetate buffer + dimethylformamide solution at pH 5.7. Diffraction data were recorded on films at the LURE synchrotron facility. The inhibitor was localized on difference Fourier maps, and the refinement of the structure was performed by simulated annealing (XPLOR). The current agreement factor is R = 19% (for 13224 observed structure factors and 1.8 A effective resolution). The RMS deviations from ideality of bond distances and angles are 0.02 A and 2 degrees, respectively. The inhibitor molecule was found in the active site, bent around the side chain of Phe-215 in a geometry that resembles the previously reported structure of the CF3-Lys-Ala complex at 2.5 A, in a parallel beta-sheet association with the loop 214-216. The analysis of the close contacts (less than 3.5 A) indicates that the trifluoromethylamide bond interacts with the active site and not the Leu-Ala or Ala-anilide bonds. The two fluorinated groups of the inhibitor exhibit different specificities: the trifluoroacetyl group (N terminus) is tightly stacked between the two chain loops 191-195 and 213-215, while the trifluoromethylanilide (C terminus) shows less specificity and only a single contact.

A Zymomonas mobilis mutant with delayed growth on high glucose concentrations.

Exponentially growing cells of Zymomonas mobilis normally exhibit a lag period of up to 3 h when transferred from 0.11 M (2%) to 0.55 M (10%) glucose liquid medium. A mutant of Z. mobilis (CU1Rif2), fortuitously isolated, showed more than a 20-h lag period when grown under the same conditions, whereas on 0.55 M glucose solid medium, it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable (0.55 M sucrose or fructose) or nonfermentable (0.11 M glucose plus 0.44 M maltose or xylose) sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew without any delay on 0.55 M glucose on which wild-type cells had been incubated for 3 h and removed at the beginning of their exponential phase. This apparent preconditioning was not observed with medium obtained from wild-type cells grown on 0.11 M glucose and supplemented to 0.55 M after removal of the wild-type cells. Undelayed growth of CU1Rif2 on 0.55 M glucose previously conditioned by the wild type was impaired by heating or protease treatment. It is suggested that in Z. mobilis, a diffusible proteinaceous heat-labile factor, transitionally not present in 0.55 M glucose CU1Rif2 cultures, triggers growth on 0.55 M glucose. Biochemical analysis of glucose uptake and glycolytic enzymes implied that glucose assimilation was not directly involved in the phenomenon. By use of a wild-type Z. mobilis genomic library, a 4.5-kb DNA fragment which complemented in low copy number the glucose-defective phenotype as well as glucokinase and glucose uptake of CU1Rif2 was isolated. This fragment carries a gene cluster consisting of four putative coding regions, encoding 167, 167, 145, and 220 amino acids with typical Z. mobilis codon usage, -35 and -10 promoter elements, and individual Shine-Dalgarno consensus sites. However, strong homologies were not detected in a BLAST2 (EMBL-Heidelberg) computer search with known protein sequences.