Electrochemical determination of uric acid in urine and serum with uricase/carbon nanotube /carboxymethylcellulose electrode.

The detection of uric acid in blood and urine is clinically important in terms of suitable diagnosis and self-healthcare. An amperometric thin film biosensor composed of carbon nanotube and uricase enzyme is presented. The CNT is successfully dispersed in aqueous solution with carboxymethylcellulose surfactant. This enables thin film formation by a simple drop-casting layer-by-layer process. The uricase/carboxymethylcellulose dispersed carbon nanotube/gold thin film biosensor shows the best sensing performance compared to that with sodium cholate surfactant in terms of higher current and lower detection potential. The presented procedure shows good performance with neither electron transfer mediator nor complicated process. Cyclic voltammetry exhibited a sensitivity of 233 µA mM-1 cm-2 at +0.35 V, a linear range of 0.02-2.7 mM, and a detection limit of 2.8 µM. We quantify and graph uric acid data in actual physiological samples (serum and urine) for the first time and detection values showed good agreement with those obtained by a conventional analytical method (enzymatic colorimetry kit).

Identification of the gene PtMAT1 encoding acetyltransferase from the diastereomer type of mannosylerythritol lipid-B producer Pseudozyma tsukubaensis.

Mannosylerythritol lipids (MELs) are biosurfactants produced from feedstocks by basidiomycetous yeasts. MELs exhibit different properties depending on their structures, such as the degree of acetylation or acylation and the chirality of the mannosylerythritol moiety. Pseudozyma tsukubaensis produces a diastereomer type of MEL-B (mono-acetylated MEL); therefore, deletion of an acetyltransferase could yield a diastereomer type of MEL-D (deacetylated MEL), which has only been produced in in vitro reactions of lipase using MEL-B as a substrate. Here, we deleted the gene PtMAT1 in P. tsukubaensis 1E5 encoding an acetyltransferase related to MEL biosynthesis via targeted gene deletion and generated a producer of the diastereomer type of MEL-D. The uracil auxotrophic mutant of P. tsukubaensis 1E5 (PtURA5-mutant) was used as a host strain for gene deletion. The gene PtMAT1 was replaced with a PtURA5 cassette by homologous recombination using uracil auxotrophy as a selectable marker. According to thin-layer chromatography and nuclear magnetic resonation spectroscopy, we identified the strain ΔPtMAT1 as a producer of the diastereomer type of MEL-D instead of MEL-B.

Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase.

Direct and mediated electron transfer (DET and MET) in enzyme electrodes with a novel flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from fungi are compared for the first time. DET is achieved by placing a single-walled carbon nanotube (CNT) between GDH and a flat gold electrode where the CNT is close to FAD within the distance for DET. MET is induced by using a free electron transfer mediator, potassium hexacyanoferrate, and shuttles electrons from FAD to the gold electrode. Cyclic voltammetry shows that the onset potential for glucose response current in DET is smaller than in MET, and that the distinct redox current peak pairs in MET are observed whereas no peaks are found in DET. The chronoamperometry with respect to a glucose biosensor shows that (i) the response in DET is more rapid than in MET; (ii) the current at more than +0.45V in DET is larger than the current at the current-peak potential in MET; (iii) a DET electrode covers the glucose concentration range for clinical requirements and is not susceptible to interfering agents at +0.45 V; and (iv) a DET electrode with the novel fungal FAD-GDH does not affect sensing accuracy in the presence of up to 5 mM xylose, while it often shows a similar response level to glucose with other conventionally used fungus-derived FAD-GDHs. It is concluded that our DET system overcomes the disadvantage of MET.

Biosynthesis of mono-acylated mannosylerythritol lipid in an acyltransferase gene-disrupted mutant of Pseudozyma tsukubaensis.

The basidiomycetous yeast genus Pseudozyma produce large amounts of mannosylerythritol lipids (MELs), which are biosurfactants. A few Pseudozyma strains produce mono-acylated MEL as a minor compound using excess glucose as the sole carbon source. Mono-acylated MEL shows higher hydrophilicity than di-acylated MEL and has great potential for aqueous applications. Recently, the gene cluster involved in the MEL biosynthesis pathway was identified in yeast. Here, we generated an acyltransferase (PtMAC2) deletion strain of P. tsukubaensis 1E5 with uracil auxotrophy as a selectable marker. A PtURA5-mutant with a frameshift mutation in PtURA5 was generated as a uracil auxotroph of strain 1E5 by ultraviolet irradiation on plate medium containing 5-fluoro-orotic acid (5-FOA). In the mutant, PtMAC2 was replaced with a PtURA5 cassette containing the 5' untranslated region (UTR) (2000 bp) and 3' UTR (2000 bp) of PtMAC2 by homologous recombination, yielding strain ΔPtMAC2. Based on TLC and NMR analysis, we found that ΔPtMAC2 accumulates MEL acylated at the C-2' position of the mannose moiety. These results indicate that PtMAC2p catalyzes acylation at the C-3' position of the mannose of MEL.

Enhanced production of a diastereomer type of mannosylerythritol lipid-B by the basidiomycetous yeast Pseudozyma tsukubaensis expressing lipase genes from Pseudozyma antarctica.

Basidiomycetous yeasts in the genus Pseudozyma are known to produce extracellular glycolipids called mannosylerythritol lipids (MELs). Pseudozyma tsukubaensis produces a large amount of MEL-B using olive oil as the sole carbon source (> 70 g/L production). The MEL-B produced by P. tsukubaensis is a diastereomer type of MEL-B, which consists of 4-O-ß-D-mannopyranosyl-(2R,3S)-erythritol as a sugar moiety, in contrast to the conventional type of MELs produced by P. antarctica, which contain 4-O-ß-D mannopyranosyl-(2S,3R)-erythritol. In this study, we attempted to increase the production of the diastereomer type of MEL-B in P. tsukubaensis 1E5 by introducing the genes encoding two lipases, PaLIPAp (PaLIPA) and PaLIPBp (PaLIPB) from P. antarctica T-34. Strain 1E5 expressing PaLIPA exhibited higher lipase activity than the strain possessing an empty vector, which was used as a negative control. Strains of 1E5 expressing PaLIPA or PaLIPB showed 1.9- and 1.6-fold higher MEL-B production than the negative control strain, respectively, and oil consumption was also accelerated by the introduction of these lipase genes. MEL-B production was estimated using time course analysis in the recombinant strains. Strain 1E5 expressing PaLIPA produced 37.0 ± 1.2 g/L of MEL-B within 4 days of cultivation, whereas the strain expressing an empty vector produced 22.1 ± 7.5 g/L in this time. Overexpression of PaLIPA increased MEL-B production by P. tsukubaensis strain 1E5 from olive oil as carbon source by more than 1.7-fold.

The reaction mechanism of sarcosine oxidase elucidated using FMO and QM/MM methods.

Monomeric sarcosine oxidase (MSOX) is a flavoprotein that oxidizes sarcosine to the corresponding imine product and is widely used in clinical diagnostics to test renal function. In the past decade, several experimental studies have been performed to elucidate the underlying mechanism of this oxidation reaction. However, the details of the molecular mechanism remain unknown. In this study, we theoretically examined three possible reaction mechanisms, namely, the single-electron transfer, hydride-transfer, and polar mechanisms, using the fragment molecular orbital (FMO) and mixed quantum mechanics/molecular mechanics (QM/MM) methods. We found that, of the three possible reaction pathways, hydride-transfer is the most energetically favorable mechanism. Significantly, hydrogen is not transferred in the hydride state (H-) but in a hydrogen atom state (H˙). Furthermore, a single electron is simultaneously transferred from sarcosine to flavin through their overlapping orbitals. Therefore, based on a detailed theoretical analysis of the calculated reaction pathway, the reaction mechanism of MSOX can be labeled the "hydrogen-atom-coupled electron-transfer" (HACET) mechanism instead of being categorized as the classical hydride-transfer mechanism. QM/MM and FMO calculations revealed that sarcosine is moved close to the flavin ring because of a small charge transfer (about 0.2 electrons in state 1 (MSOX-sarcosine complex)) and that the positively charged residues (Arg49, Arg52, and Lys348) near the active site play a prominent role in stabilizing the sarcosine-flavin complex. These results indicate that strong Coulombic interactions primarily control amine oxidation in the case of MSOX. The new reaction mechanism, HACET, will be important for all the flavoprotein-catalyzed oxidation reactions.

A Gene Cluster for Biosynthesis of Mannosylerythritol Lipids Consisted of 4-O-ß-D-Mannopyranosyl-(2R,3S)-Erythritol as the Sugar Moiety in a Basidiomycetous Yeast Pseudozyma tsukubaensis.

Mannosylerythritol lipids (MELs) belong to the glycolipid biosurfactants and are produced by various fungi. The basidiomycetous yeast Pseudozyma tsukubaensis produces diastereomer type of MEL-B, which contains 4-O-ß-D-mannopyranosyl-(2R,3S)-erythritol (R-form) as the sugar moiety. In this respect it differs from conventional type of MELs, which contain 4-O-ß-D-mannopyranosyl-(2S,3R)-erythritol (S-form) as the sugar moiety. While the biosynthetic gene cluster for conventional type of MELs has been previously identified in Ustilago maydis and Pseudozyma antarctica, the genetic basis for MEL biosynthesis in P. tsukubaensis is unknown. Here, we identified a gene cluster involved in MEL biosynthesis in P. tsukubaensis. Among these genes, PtEMT1, which encodes erythritol/mannose transferase, had greater than 69% identity with homologs from strains in the genera Ustilago, Melanopsichium, Sporisorium and Pseudozyma. However, phylogenetic analysis placed PtEMT1p in a separate clade from the other proteins. To investigate the function of PtEMT1, we introduced the gene into a P. antarctica mutant strain, ΔPaEMT1, which lacks MEL biosynthesis ability owing to the deletion of PaEMT1. Using NMR spectroscopy, we identified the biosynthetic product as MEL-A with altered sugar conformation. These results indicate that PtEMT1p catalyzes the sugar conformation of MELs. This is the first report of a gene cluster for the biosynthesis of diastereomer type of MEL.

Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060.

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations.