Long-term operation of slurry bioreactor for decomposition of food wastes

A pilot scale slurry bioreactor was used for the treatment of food wastes. Food wastes were continuously added (750 g wet weight per day) into the reactor and successfully decomposed to inorganic carbon without intermittent removal of suspended solids. During operation for 90 days, 91% reduction of food wastes was achieved. Microorganisms actively grew during the initial 20 days of operation, and reached a stationary phase with a cell concentration of around 5 x 10(10) cells ml(-1), which indicated that food waste was utilized as a respiratory substrate during this phase. Using data for time variation of dissolved oxygen, the oxygen requirement for decomposition of food wastes was estimated to be 5.0 g O2 g(-1) dry weight of food wastes.     

Separation and selective detection of lipoprotein particles of patients with coronary artery disease by frit-inlet asymmetrical flow field-flow fractionation

An analytical method to improve the characterization of lipoprotein fractions is presented. Human plasma samples were treated with Sudan Black B to stain the lipid component in lipoproteins, then the stained lipoproteins were separated by frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF), according to the lipoprotein particle sizes, with the selective detection of eluting lipoprotein fractions, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), at 610 nm. The capability of this technique has been evaluated with plasma samples obtained from patients with coronary artery disease (CAD), and it showed that the retention profile of patients' lipoprotein samples was clearly distinct from those of healthy persons. The potential of this technique comes with the direct injection of a stained lipoprotein sample without a prior procedure such as ultracentrifugation for sample preparation, and the size calculation of lipoprotein particles from the experimental retention time by theory. Since sample relaxation was achieved hydrodynamically in an FI-AFlFFF channel, sample injection and separation processes were continuously made without stopping the separation flow. This study demonstrated the potential of the FI-AFlFFF technique to be utilized as a powerful tool for the determination of the LDL profiles of patients with CAD.     

Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His(6) was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca(++)-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta mutant-derived GOD-His(6) on a protein basis was very similar to that of the hyperglycosylated GOD-His(6), although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His(6).     

GSH-dependent peroxidase activity of the rice (Oryza sativa) glutaredoxin,a thioltransferase

Glutaredoxin (Grx) is a 12-kDa thioltransferase that reduces disulfide bonds of other proteins and maintains the redox potential of cells. In addition to its oxidoreductase activity, we report here that a rice Grx (OsGrx) can also function as a GSH-dependent peroxidase. Because of this antioxidant activity, OsGrx protects glutamine synthetase from oxidative damage. Individually replacing the conserved Cys residues in OsGrx with Ser shows that Cys(23), but not Cys(26), is essential for the thioltransferase and GSH-dependent peroxidase activities. Kinetic characterization of OsGrx reveals that the maximal catalytic efficiency (V(max)/K(m)) is obtained with cumene hydroperoxide rather than H(2)O(2) or t-butyl hydroperoxide.     

A novel amylolytic enzyme from Thermotoga maritima,resembling cyclodextrinase and alpha-glucosidase,that liberates glucose from the reducing end of the substrates

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.     

Efficient generation of feasible pathways for protein conformational transitions

We develop a computationally efficient method to simulate the transition of a protein between two conformations. Our method is based on a coarse-grained elastic network model in which distances between spatially proximal amino acids are interpolated between the values specified by the two end conformations. The computational speed of this method depends strongly on the choice of cutoff distance used to define interactions as measured by the density of entries of the constant linking/contact matrix. To circumvent this problem we introduce the concept of using a cutoff based on a maximum number of nearest neighbors. This generates linking matrices that are both sparse and uniform, hence allowing for efficient computations that are independent of the arbitrariness of cutoff distance choices. Simulation results demonstrate that the method developed here reliably generates feasible intermediate conformations, because our method observes steric constraints and produces monotonic changes in virtual bond and torsion angles. Applications are readily made to large proteins, and we demonstrate our method on lactate dehydrogenase, citrate synthase, and lactoferrin. We also illustrate how this framework can be used to complement experimental techniques that partially observe protein motions.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.