130,668 research outputs found
Studies on the structural and functional aspects of Rhodotorula gracilis D-amino acid oxidase by limited trypsinolysis
No full textThe structure-function relationships of purified Rhodotorula gracilis D-amino acid oxidase (in its holo-, apo- and holo-enzyme-benzoate complex forms) was analysed by digestion with trypsin. In all cases trypsin cleaves this 80 kDa dimeric enzyme at the C-terminal region, since the peptide bonds sensitive to proteinase attack are clustered in this region. Digestion of native enzyme with trypsin produced a nicked and truncated form of 38.3 kDa containing two polypeptides of 34 and 5 kDa starting from Met1 and Ala319 respectively, and with detachment of the Thr306-Arg318 and Glu365-Leu368 peptides. Our results show that this 'core', folded into a compact structure, is catalytically competent. The acquisition of this nicked form was marked by a shift from a dimeric to a monomeric active enzyme, a result never previously obtained. The deleted sequences, Thr306-Arg318 and Glu365-Leu368, are essential for the monomer-monomer interaction, and, in particular, the region encompassing Thr306-Arg318 should play an essential role in the dimerization process. interestingly, the Ser308-Lys321 sequence present in the lost peptide corresponds to a sequence not present in other known D-amino acid oxidases [Faotto, Pollegioni, Ceciliani, Ronchi and Pilone (1995) Biotechnol. Lett. 17, 193-198]. A role of the cleaved-off region for the thermostabilization of the enzyme is also discussed
Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase
No full textWhen analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilis D-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of ≃ 34 kDa (Met1-Leu312) and one of ≃ 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilis D-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers
Additively manufactured CuCrZr alloy: improvement of mechanical properties by heat treatment
Get PDFCuCrZr alloy plays a fundamental role for the production of critical components because it is characterized by good thermal and electrical conductivity and by high mechanical strength after precipitation hardening treatment. In the framework of a wider research on the mechanical behaviour of additively manufactured CuCrZr alloy, this study focuses on the effects of heat treatment parameters on the alloy strength. The additive manufacturing process, characterized by very high cooling rates, determines the formation, in the as-built condition, of a supersaturated solid solution. The results obtained reveal that aging temperature and time are critical parameters for improving the mechanical behaviour of CuCrZr alloy which behaves differently than the alloy produced through the use of traditional techniques
Investigation on some factors affecting crack formation in high resistance aluminum alloys
Get PDFAluminum alloys having good mechanical properties are Al-ZnMg alloys (7xxx) and Al-Cu-Li alloys (Weldalite). These alloys may be subjected to stress corrosion cracking. In order to overcome this problem the Al 7050 alloy has been developed and it is widely used for aerospace applications. Despite that, some components made of this alloy cracked during the manufacturing process including machining and chemical anodization. In a previous work cracked Al 7050 components have been analyzed in order to identify possible causes of crack formation. In this work the susceptibility of this alloy to intergranular corrosion has been analysed and compared with that of other high resistance aluminum alloy
Enzymatic detection of D-amino acids
No full textD: -Amino acids play several key roles and are widely diffused in living organisms, from bacteria (in which D-alanine is a component of the cell wall) to mammals (where D-serine is involved in glutamatergic neurotransmission in the central nervous system). The study of the biological processes involving D-amino acids and their use as clinical or biotechnological biomarkers requires reliable methods of quantifying them. Although "traditional" analytical techniques have been (and still are) employed for such tasks, enzymatic assays based on enzymes which possess a strict stereospecificity (i.e., that are only active on the D-enantiomers of amino acids) allowed the set-up of low-cost protocols with a high sensitivity and selectivity and suitable for determining the D-amino acid content of complex biological samples. The most exploited enzyme in these assays is D-amino acid oxidase, a flavoenzyme that exclusively oxidizes D-amino acids and possesses with a broad substrate specificity and a high kinetic efficiency
Limited proteolysis and site directed mutagenesis revealed the origin of microheterogeneity of Rhodotorula gracilis D-amino acid oxidase
No full textHigh-temperature oxidation behaviour of a TiAl-based alloy subjected to aluminium hot-dipping
Get PDFIn this research the oxidation resistance at high temperature of a TiAl-based alloy has been improved by hot-dipping the alloy in molten aluminium and by performing an interdiffusion process. After selecting the best process parameters, a compact TiAl3 coating characterized by an almost constant thickness was formed on the surface. Isothermal oxidation tests, carried out at 900, 950 and 1000 °C, showed that the coated alloy is able to form a continuous and thin alumina layer that is very protective. Microstructural investigations highlighted that, above 900 °C, long residence times at high temperature determine the diffusion through the TiAl3 layer of Cr that favours migration toward the outer surface of Al and thus the formation of a self-healing alumina layer
Analyzing the d-amino acid content in biological samples by engineered enzymes
No full textThe aim of our present research is to produce mutant forms of d-amino acid oxidase from Rhodotorula gracilis in order to determine d-amino acid content in different biological samples. During the past few years, our group has produced yeast d-amino acid oxidase variants with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all d-amino acids) both by rational design and directed evolution methods. Now, the kinetic constants for a number of amino acids (even for unnatural ones) of the most relevant d-amino acid oxidase variants have been investigated. This information constitutes the basis for considering potential analytical applications in this important field
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