Penicillin G acylase-fatty lipid biocomposite films show excellent catalytic activity and long term stability/reusability

The formation of biocomposite films of the pharmaceutically important enzyme penicillin G acylase (PGA) and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein the enzyme diffuses into the lipid film during immersion in the enzyme solution, thereby leading to the formation of a biocomposite film. The incorporation of the enzyme in both cationic as well as anionic lipids suggests the important role of secondary interactions such as hydrophobic and hydrogen bonding in the enzyme immobilization process. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimentry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by Fourier transform infrared spectroscopy (FTIR) and biocatalytic activity measurements. Whereas the biological activity of the lipid-immobilized enzyme was marginally higher than that of the free enzyme, the biocomposite film exhibited increased thermal/temporal stability. Particularly exciting was the observation that the biocomposite films could be reused in biocatalysis reactions without significant loss in activity, which indicates potentially exciting biomedical/industrial application of these films.     

Invertase-lipid biocomposite films: preparation, characterization, and enzymatic activity

The formation of biocomposite films of the industrially important enzyme invertase and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein invertase diffuses into the cationic lipid octadecylamine during immersion of the lipid film in the enzyme solution. Entrapment of invertase in the octadecylamine film is highly pH-dependent, underlining the role of attractive electrostatic interactions between the enzyme and the lipid in the biocomposite film formation. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimetry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by fluorescence spectroscopy and biocatalytic activity measurements. The biocatalytic activity of the invertase-lipid biocomposite films was comparable to that of the free enzyme in solution and showed marginally higher temperature stability. Particularly exciting was the excellent reuse characteristics of the biocomposite films, indicating potential industrial application of these films.     

Enhancing the reusability of endoglucanase-gold nanoparticle bioconjugates by tethering to polyurethane microspheres

The synthesis of polyurethane microsphere-gold nanoparticle "core-shell" structures and their use in the immobilization of the enzyme endoglucanase are described. Assembly of gold nanoparticles on the surface of polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles, thereby precluding the need for modifying the polymer microspheres to enable such nanoparticle binding. Endoglucanse could thereafter be bound to the gold nanoparticles decorating the polyurethane microspheres, leading to a highly stable biocatalyst with excellent reuse characteristics. The immobilized enzyme retains its biocatalytic activity and exhibits improved thermal stability relative to free enzyme in solution. The high surface area of the host gold nanoparticles renders the immobilized enzyme "quasi free", while at the same time retaining advantages of immobilization such as ease of reuse, enhanced temporal and thermal stability, etc.     

Transgenic tomato plants with decreased sucrose synthase are unaltered in starch and sugar accumulation in the fruit

Sucrose is the photoassimilate transported from the leaves to the fruit of tomato yet the fruit accumulates predominantly glucose and fructose. Hydrolysis of sucrose entering the fruit can be accomplished by invertase or sucrose synthase. Early in tomato fruit development there is a transient increase in sucrose synthase activity and starch which is correlated with fruit growth and sink strength suggesting a regulatory role for sucrose synthase in sugar import. Using an antisense sucrose synthase cDNA under the control of a fruit-specific promoter we show that sucrose synthase activity can be reduced by up to 99% in young fruit without affecting starch or sugar accumulation. This result calls into question the importance of sucrose synthase in regulating sink strength in tomato fruit.     

A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone

A -D-glucosidase has been purified to apparent homogeneity from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seedlings during the mobilization of the xyloglucan stored in the cotyledonary cell walls. The purified protein (M_r 76 000; a glycoprotein; pI > 9.5; apparent pH optimum 4.5; temperature optimum 30°C) catalysed the hydrolysis of p-nitrophenyl--D-glucopyranoside, cello-oligosaccharides, -linked glucose disaccharides, and certain xyloglucan oligosaccharides. Glucose disaccharides with different linkages were hydrolysed at different rates [(1ν3) > (1ν4) > (1ν2) > (1ν6)] with significant transglycosylation occurring in the early stages of the reaction. Cello-oligosaccharide hydrolysis was also accompanied by extensive transglycosylation to give transitory accumulations of higher oligosaccharides. At least some of the glycosyl linkages formed during transglycosylation were (1ν6)-. Xyloglucan oligosaccharides xylose-substituted at the non-reducing terminal glucose residue (XXXG, XXLG, XLXG and XLLG, where G is an unsubstituted glucose residue, X is a xylose-substituted glucose residue, and L is a galactosylxylose-substituted glucose residue) were not hydrolysed. Some xyloglucan oligosaccharides with an unsubstituted non-reducing terminal glucose residue (GXXG, GXLG and GXG) were hydrolysed, but others (GLXG and GLLG) were not. This indicated steric hindrance by L but not X substitution at the glucose residue next to the one at the non-reducing end of the oligosaccharide. Hydrolysis of xyloglucan oligosaccharides was not accompanied by transglycosylation. Natural xyloglucan subunit oligosaccharides (XXXG, XXLG, XLXG, XLLG) were totally degraded to their monosaccharide components when treated with nasturtium -D-galactosidase ( Edwards et al. (1988 ) J. Biol. Chem. 263, 4333–4337), followed by alternations of nasturtium xyloglucan-specific α-xylosidase ( Fanutti et al. (1991 ) Planta 184, 137–147) and this enzyme. Several extensively overlapping cDNA clones were obtained by RT–PCR and by screening cDNA libraries. A composite, full-length DNA had an open reading frame of 1962 bp, encoding a polypeptide of 654 amino acids, including all N-terminal and internal sequences obtained from the purified -glucosidase protein, and a motif resembling plant signal sequences thought to direct proteins to the cell wall. Database searches revealed homology with -glucosidases from several sources (plant, bacteria, yeast), notably with glycosylhydrolases of ‘Family 3’, according to the classification of Henrissat ( Henrissat (1991) Biochem. J. 280, 309–316). There was strong sequence homology with a -glucan exo-hydrolase from barley ( Hrmova et al. (1996 ) J. Biol. Chem. 271, 5277–5286). The nasturtium -glucosidase is ascribed a role in xyloglucan mobilization, and its interaction with the α-xylosidase and the -galactosidase is modelled.     

Histatin 5 Resistance of Candida glabrata Can Be Reversed by Insertion of Candida albicans Polyamine Transporter-Encoding Genes DUR3 and DUR31

Candida albicans and Candida glabrata are predominant fungi associated with oral candidiasis. Histatin 5 (Hst 5) is a small cationic human salivary peptide with high fungicidal activity against C. albicans, however many strains of C. glabrata are resistant. Since Hst 5 requires fungal binding to cell wall components prior to intracellular translocation, reduced Hst 5 binding to C. glabrata may be the reason for its insensitivity. C. glabrata has higher surface levels of β-1,3-glucans as compared with C. albicans; however these differences did not account for reduced Hst 5 uptake and killing in C. glabrata. Similarly, the biofilm matrix of C. glabrata contained significantly higher levels of β-1,3-glucans compared with C. albicans, but it did not reduce the percentage of Hst 5 positive fungal cells in the biofilm. Hst 5 enters C. albicans cell through polyamine transporters Dur3p and Dur31p that are uncharacterized in C. glabrata. C. glabrata strains expressing CaDur3 and CaDur31 had two-fold higher killing and uptake of Hst 5. Thus, neither C. glabrata cell surface or biofilm matrix β-1,3-glucan levels affected Hst 5 toxicity; rather the crucial rate limiting step is reduced uptake that can be overcome by expression of C. albicans Dur proteins in C. glabrata.     

Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design

Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.     

Diabetic nephropathy: mechanisms of renal disease progression

Diabetic nephropathy is characterized by excessive amassing of extracellular matrix (ECM) with thickening of glomerular and tubular basement membranes and increased amount of mesangial matrix, which ultimately progress to glomerulosclerosis and tubulo-interstitial fibrosis. In view of this outcome, it would mean that all the kidney cellular elements, i.e., glomerular endothelia, mesangial cells, podocytes, and tubular epithelia, are targets of hyperglycemic injury. Conceivably, high glucose activates various pathways via similar mechanisms in different cell types of the kidney except for minor exceptions that are related to the selective expression of a given molecule in a particular renal compartment. To begin with, there is an obligatory excessive channeling of glucose intermediaries into various metabolic pathways with generation of advanced glycation products (AGEs), activation of protein kinase C (PKC), increased expression of transforming growth factor-beta (TGF-beta), GTP-binding proteins, and generation of reactive oxygen species (ROS). The ROS seem to be the common denominator in various pathways and are central to the pathogenesis of hyperglycemic injury. In addition, there are marked alterations in intraglomerular hemodynamics, i.e., hyperfiltration, and this along with metabolic derangements adversely compounds the hyperglycemia-induced injury. Here, the information compiled under various subtitles of this article is derived from an enormous amount of data summarized in several excellent literature reviews, and thus their further reading is suggested to gain in-depth knowledge of each of the subject matter.     

Optimization of Alkaline Protease Production from Bacillus sp. by Response Surface Methodology

High yields (1939 U/ml) of an alkaline protease were obtained in batch fermentation of a Bacillus sp. using a response surface methodology. The interaction of four variables, viz., starch, peptone, incubation time, and inoculum density, suggested inoculum density to be an insignificant variable. However, incubation time had a profound effect on protease yields at all the concentrations of carbon and nitrogen used. The response surface raised and flattened with increase in time of incubation, and maximum protease production up to 1939 U/ml was obtained after 96 h of incubation. The model equation obtained was validated experimentally at maximum starch (15 mg/ml) and peptone (7.5 mg/ml) concentration with increased incubation time up to 144 h in the presence of minimum inoculum density (1%). An overall 2.6-fold increase in protease production was obtained as compared with mean observed response (750 U/ml) at zero level of all variables. Received: 19 July 2001 / Accepted: 15 August 2001