Generation of molecular chiral asymmetry through stirred crystallization

In our earlier work we established that stirred crystallization of achiral compounds that crystallize in enantiomeric forms result in spontaneous chiral symmetry breaking. The asymmetry thus spontaneously generated is confined to the solid state. In this article, we present a case in which the crystal enantiomeric excess (CEE) can be converted to molecular enantiomeric excess (EE) through a solid state reaction which relates the enantiomeric form of the crystal to the enantiomeric form of the product. Such a process not only provides a means of detecting the CEE generated in stirred crystallization but it is also a means through which chiral asymmetry generated spontaneously is "propagated" to generate chiral compounds with enantiomeric excess.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Application of protease from Nocardiopsis sp. as a laundry detergent additive

The application of protease as a laundry detergent additive from a newly isolated Nocardiopsis sp., isolated from a soil sample collected in Northeast Brazil is reported. The optimal pH and temperature for protease activity were pH 10.5 and 50 °C, respectively. The enzyme was stable in a long-term incubation, showed 73.5% of initial activity at pH 10.5 and 61.7% at pH 12.0 for 120 min. Approximately 60% of initial activity remained after 120 min at 50 °C or after 30 min at 80 °C. Almost 87% of enzyme activity was retained in the presence of 10% (v/v) of peroxide at 40 °C, after 1 h. The protease also was stable in the presence of oxidants and surfactants such as SDS, saponin, Tween 20 and Tween 80 after 30 min. In the presence of Omo^®, the enzyme retained 64% of its activity at 40 °C for 1 h. An increase in the proteolytic activity (6–17%) was observed with K⁺, Na⁺, and Mg⁺⁺ ions. At pH 8.0, the protease hydrolysed casein maximally (50 U/mg).     

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes,enzymes and exopolysaccharide production engineering

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 170–176 doi:10.1038/sj.jim.7000266 Received 11 February 2002/ Accepted in revised form 09 April 2002     

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.     

Decolourisation of Remazol Brilliant Blue R via a novel Bjerkandera sp. strain

A novel strain of Bjerkandera sp. (B33/3), with particularly high decolourisation activities upon Poly R-478 and Remazol Brilliant Blue R (RBBR) dyes, was isolated. The role of the ligninolytic extracellular enzymes produced by this strain on decolourisation of RBBR was studied in some depth. The basis of decolourisation is an enzyme-mediated process, in which the main enzyme responsible is a recently described peroxidase with capacity for oxidation of manganese, as well as veratryl alcohol and 2,6-dimethoxyphenol in a manganese-independent reaction.     

Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.     

Acyloxymethyl as a drug protecting group. Part 6: N-acyloxymethyl- and N-[(aminocarbonyloxy)methyl]sulfonamides as prodrugs of agents containing a secondary sulfonamide group

Tertiary N-acyloxymethyl- and N-[(aminocarbonyloxy)methyl]sulfonamides were synthesised and evaluated as novel classes of potential prodrugs of agents containing a secondary sulfonamide group. The chemical and plasma hydrolyses of the title compounds were studied by HPLC. Tertiary N-acyloxymethylsulfonamides are slowly and quantitatively hydrolysed to the parent sulfonamide in pH 7.4 phosphate buffer, with half-lives ranging from 20 h, for 7d, to 30 days, for 7g. Quantitative formation of the parent sulfonamide also occurs in human plasma, the half-lives being within 0.2-2.0 min for some substrates. The rapid rate of hydrolysis can be ascribed to plasma cholinesterase, as indicated by the complete inhibition observed at [eserine] = 0.10 mM. These results suggest that tertiary N-acyloxymethylsulfonamides are potentially useful prodrugs for agents containing a secondary sulfonamide group, especially with pKa < 8, combining a high stability in aqueous media with a high rate of plasma activation. In contrast, N-[(aminocarbonyloxy)methyl]sulfonamides 7h-j do not liberate the parent sulfonamide either in aqueous buffers or in human plasma and thus appear to be unsuitable for development as sulfonamide prodrugs.     

Morphological Differentiation and Possible Origin of B Chromosomes in Natural Brazilian Population of Astyanax Scabripinnis (PISCES, CHARACIDAE)

Specimens of Astyanax scabripinnisfrom three different altitudes (1920, 1800 and 700?m) along the Ribeirão Grande stream in the Campos do Jordão region (São Paulo State, Brazil) were investigated. The same diploid number, 2n?=?50, was detected in the three populations, with the following karyotypic constitution: 6M, 22SM, 10ST and 12A. The populations located at 1920 and 1800?m altitude presented a high incidence of B chromosomes varying in number (0–2), shape (meta- and submetacentrics), size (large and small) and sex-related frequency (they were more frequent among females). The two morphologically variant B chromosomes probably evolved from a metacentric macrochromosome, which is the most commonly observed B chromosome in several A. scabripinnispopulations.