Enzymatic characterization of a novel Xaa-Pro aminopeptidase XpmA from Aspergillus oryzae expressed in Escherichia coli.

Xaa-Pro aminopeptidases are peptidases responsible for the cleavage of any amino acid N-terminally adjacent to a proline residue. We identified a gene encoding a putative Xaa-Pro aminopeptidase in the genome of the filamentous fungus Aspergillus oryzae (genome database number: AO090701000720) and named this gene xpmA. We produced its enzyme in a C-terminally His6-tag-fused form in an Escherichia coli expression system and purified it. The purified recombinant XpmA (rXpmA) showed hydrolysis activity toward Xaa-Pro-oligopeptides, especially the two dipeptides Ala-Pro and Phe-Pro. The molecular weight of rXpmA was estimated to be 69 kDa by SDS-PAGE and 126 kDa by gel filtration, suggesting that it is a homodimer. The enzyme was activated by various divalent metal ions such as Mn2+, Co2+, and Mg2+; in particular, the enzyme activity was increased 27.6-times relative to the no-addition control by 1 mM Mn2+. Additionally, 10 mM EDTA suppressed its activity to 0.26-times of the control level. Therefore, rXpmA was a metalloprotease. Optimal hydrolytic activity of rXpmA was observed at 50°C and pH 8.5-9.0. The enzyme was stable up to 50°C and from pH 4.0 to 11.0. rXpmA showed substrate inhibition by Leu-Pro, Ser-Pro and Arg-Pro at concentrations over 4 mM, 10 mM, and 3 mM, respectively. NaCl increased the enzyme activity in the concentration range 0.5-3.0 M, suggesting that the enzyme is halophilic.

Telomere-mediated chromosomal truncation in Aspergillus oryzae.

We truncated the short arm of chromosome 3 to delete the aflatoxin biosynthesis gene homolog cluster using telomeric repeats in Aspergillus oryzae. The predicted deletion was confirmed by Southern blot analyses. This telomere-mediated chromosomal truncation method enables the development of an artificial chromosome in A. oryzae.

Evaluation of commercially available reagents as a reference material for intramolecular carbon isotopic measurements of acetic acid.

RATIONALE: Recent advances in analytical techniques for the intramolecular carbon isotopic ratio measurement of some organic compounds have provided important information on carbon cycles in biochemistry, organic geochemistry and food chemistry. These advances have made it necessary to prepare intramolecular isotopic reference materials (RMs) to use for inter-laboratory calibration and/or inter-calibration among different analytical methods. METHODS: We evaluated the feasibility of preparing RMs using commercially available reagents for intramolecular carbon isotopic ratio measurement of acetic acid. The intramolecular carbon isotopic distribution of nine acetic acid and four sodium acetate reagents was determined with high precision using off-line pyrolysis combined with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We also evaluated the potential alteration in the isotopic signature of acetic acid reagents by evaporation. RESULTS: The intramolecular carbon isotopic distributions for the acetic acid and sodium acetate reagents were determined with a precision of better than 0.45‰. We found that the isotopic values of these reagents spanned the carbon isotopic range of acetic acid in biological and environmental samples. We also found that the isotope fractionation associated with the evaporation of acetic acid occurs solely on the methyl position, the carboxyl position being unaffected. CONCLUSIONS: These commercially available reagents will be used as RMs in the future for inter-laboratory calibration and/or inter-calibration with another intramolecular isotopic measurement technique, namely quantitative (13) C NMR. In cases where acetic acid is being used as a RM, its storage must be carefully controlled to prevent evaporation.

Reduction of the degradation activity of umami-enhancing purinic ribonucleotide supplement in miso by the targeted suppression of acid phosphatases in the Aspergillus oryzae starter culture.

Miso (fermented soybean paste) is a traditional Japanese fermented food, and is now used worldwide. The solid-state culture of filamentous fungus, Aspergillus oryzae, grown on rice is known as rice-koji, and is important as a starter for miso fermentation because of its prominent hydrolytic enzyme activities. Recently, commercial miso products have been supplemented with purinic ribonucleotides, such as inosine monophosphate (IMP) and guanine monophosphate, to enhance the characteristic umami taste of glutamate in miso. Because the purinic ribonucleotides are degraded by enzymes such as acid phosphatases in miso, heat inactivation is required prior to the addition of these flavorings. However, heat treatment is a costly process and reduces the quality of miso. Therefore, an approach to lower acid phosphatase activities in koji culture is necessary. Transcriptional analysis using an A. oryzae KBN8048 rice-koji culture showed that eight of the 13 acid phosphatase (aph) genes were significantly down-regulated by the addition of phosphoric acid in the preparation of the culture in a concentration-dependent manner, while aphC expression was markedly up-regulated under the same conditions. The eight down-regulated genes might be under the control of the functional counterpart of the Saccharomyces cerevisiae transcriptional activator Pho4, which specifically regulates phosphatase genes in response to the ambient phosphate availability. However, the regulatory mechanism of aphC was not clear. The IMP dephosphorylation activities in rice-koji cultures of KBN8048 and the aphC deletion mutant (ΔaphC) were reduced by up to 30% and 70%, respectively, in cultures with phosphoric acid, while protease and amylase activity, which is important for miso fermentation, was minimally affected. The miso products fermented using the rice-koji cultures of KBN8048 and ΔaphC prepared with phosphoric acid had reductions in IMP dephosphorylation activity of 80% and 90%, respectively, without any adverse effects on amylase and protease activities. Thus, preparing the A. oryzae rice-koji culture under phosphate-sufficient conditions preferentially produces a fermentation starter of miso exhibiting low purinic ribonucleotide dephosphorylation activity. Moreover, aphC is a potential breeding target to reduce purinic ribonucleotide degradation activity further in commercial miso products.

Characterization of a (D)-stereoselective aminopeptidase (DamA) exhibiting aminolytic activity and halophilicity from Aspergillus oryzae.

ß-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the ß-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [ß-Ala-para-nitroanilide (pNA)], the enzyme preferred D-Leu-pNA and D-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward D-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0-11.0. DamA also exhibited aminolytic activity, producing D-Leu-D-Leu-NH2 from D-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from D-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a D-amino acid at the N-terminus as well as physiologically active peptides.

Comparison of IRMS and NMR spectrometry for the determination of intramolecular 13C isotope composition: application to ethanol.

Isotopic (13)C NMR is a relatively recent technique which allows the determination of intramolecular (13)C isotope composition at natural abundance. It has been used in various scientific fields such as authentication, counterfeiting or plant metabolism. Although its precision has already been evaluated, the determination of its trueness remains still challenging. To deal with that issue, a comparison with another normalized technique must be achieved. In this work, we compare the intramolecular (13)C isotope distribution of ethanol from different origins obtained using both Isotope Ratio Mass Spectrometry (IRMS) and Nuclear Magnetic Resonance (NMR) spectrometry techniques. The IRMS approach consists of the oxidation of ethanol to acetic acid followed by the degradation of the latter for the analysis of each fragments formed. We show here that the oxidation of ethanol to acetic acid does not bring any significant error on the determination of the site-specific δ(13)C (δ(13)C(i)) of ethanol using the IRMS approach. The difference between the data obtained for 16 samples from different origins using IRMS and NMR approaches is not statistically significant and remains below 0.3‰. These results are encouraging for the future studies using isotopic NMR, especially in combination with the IRMS approach.

Comparison of expression and enzymatic properties of Aspergillus oryzae lysine aminopeptidases ApsA and ApsB.

The apsA and apsB genes encoding family M1 aminopeptidases were identified in the industrial fungus Aspergillus oryzae. The apsB was transcriptionally up-regulated up to 2.5-fold in response to the deprivation of nitrogen or carbon sources in growth media, while up-regulation of apsA was less significant. The encoded proteins were bacterially expressed and purified to characterize their enzymatic properties. ApsA and ApsB were optimally active at pH 7.0 and 35 °C and stable at pH ranges of 6-10 and 4-10, respectively, up to 40 °C. The enzymes were inhibited by bestatin and EDTA, as has been reported for family M1 aminopeptidases that characteristically contain a zinc-binding catalytic motif. Both enzymes preferentially liberated N-terminal lysine, which is an essential amino acid and an important additive to animal feed. Enzymes that efficiently release N-terminal lysine from peptides could be useful for food and forage industries. Examination of the reactivity toward peptide substrate of varying length revealed that ApsB exhibited broader substrate specificity than ApsA although the reactivity of ApsB decreased as the length of peptide substrate decreased.

Enzymatic properties of the glycine D-alanine [corrected] aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji).

The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9.

Intramolecular carbon isotope distribution of acetic acid in vinegar.

Compound-specific carbon isotope analysis of acetic acid is useful for origin discrimination and quality control of vinegar. Intramolecular carbon isotope distributions, which are each carbon isotope ratios of the methyl and carboxyl carbons in the acetic acid molecule, may be required to obtain more detailed information to discriminate such origin. In this study, improved gas chromatography-pyrolysis-gas chromatography-combustion-isotope ratio mass spectrometry (GC-Py-GC-C-IRMS) combined with headspace solid-phase microextraction (HS-SPME) was used to measure the intramolecular carbon isotope distributions of acetic acid in 14 Japanese vinegars. The results demonstrated that the methyl carbons of acetic acid molecules in vinegars produced from plants were mostly isotopically depleted in (13)C relative to the carboxyl carbon. Moreover, isotopic differences (δ(13)C(carboxyl) - δ(13)C(methyl)) had a wide range from -0.3 to 18.2‰, and these values differed among botanical origins, C3, C4, and CAM plants.

Characterization of an Aspergillus oryzae cysteinyl dipeptidase expressed in Escherichia coli.

Cysteinyl dipeptidase from Aspergillus oryzae (CdpA) was produced in Escherichia coli and purified. The enzyme showed activity specific toward cysteine-containing dipeptides, but its substrate specificity was distinct from those of other cysteinyl dipeptidases of the M20 family. It was optimally active at pH 7-8 and stable at pH 6-9 and at up to 40 °C.

Overexpression and characterization of an extracellular leucine aminopeptidase from Aspergillus oryzae.

Leucine aminopeptidase (LAP), an enzyme used in the food industry, is an exopeptidase that removes an amino acid residue, primarily leucine (Leu), from the N-terminus of peptides and protein substrates. In this study, we focused on the leucine aminopeptidase A (lapA) gene from Aspergillus oryzae RIB40. To purify and characterize the LapA, lapA was overexpressed in A. oryzae RIB40 using the amyB promoter. LAP activity in the culture supernatant of one transformant harboring the lapA expression plasmid was 33 times that of the host strain. LapA was purified from the culture supernatant of this lapA-overexpressing strain by column chromatography. The purified recombinant LapA had a molecular mass of 33 kDa, and its N-terminal amino acid was the tyrosine at position 80 of the deduced amino acid sequence. Optimal enzyme activity was observed at 60°C and pH 8.5, and the enzyme was stable at temperatures up to 60°C and in the pH range 7.5-11. In transcriptional analysis, lapA was induced under alkaline conditions and expressed at a relatively low level under normal conditions. LapA showed maximum hydrolyzing activity for the substrate leucine para-nitroanilide (Leu-pNA), followed by substrates Phe-pNA (39% activity compared with Leu-pNA), Met-pNA, Lys-pNA, and Arg-pNA. In addition, LapA preferentially hydrolyzed peptides longer than tripeptides.

Determination of true absorption and fecal endogenous loss of zinc in goats.

We determined the true absorption and endogenous fecal loss of zinc (Zn) in goats using its stable isotope. Three goats were fed with the diet containing 50 mg/kg Zn twice a day for 17 days. In the morning of day 11, the goats were given a meal labeled by (67) Zn as the tracer with dysprosium as the unabsorbed marker. Then the goats were given unlabeled diet as the rest of the morning feed. We measured dietary and fecal Zn concentration, (67) Zn abundance and dysprosium concentration in feces. The excretion pattern of the tracer Zn into feces differed from that of dysprosium. Therefore, we directly calculated the true absorption of Zn from Zn concentration and (67) Zn abundance in fecal samples collected after the labeled diet was given. The apparent absorption of Zn was -0.009 ± 0.016 mg/kg bodyweight (fractional absorption, -1.07 ± 1.85%). The true absorption of Zn was 0.162 ± 0.018 mg/kg bodyweight (fractional absorption, 18.25 ± 2.01%). The endogenous fecal loss of Zn was 0.172 ± 0.004 mg/kg bodyweight and the intestinal secretion of Zn was 0.210 ± 0.009 mg/kg bodyweight. The present experiment indicates that stable isotopic Zn is a powerful tool for examining Zn metabolism in ruminants.

Determination of carbon isotope ratios of methanol and acetaldehyde in air samples by gas chromatography-isotope ratio mass spectrometry combined with headspace solid-phase microextraction.

Isotopic signatures of atmospheric methanol and acetaldehyde have the potential to improve our ability to quantitatively assess their importance in atmospheric chemistry. However, isotopic measurements of atmospheric methanol and acetaldehyde and their individual source and sink processes have been limited. In this study, we examined gas chromatography-isotope ratio mass spectrometry combined with headspace solid-phase microextraction to measure the carbon isotope ratios of methanol and acetaldehyde in air samples. The method enabled us to determine carbon isotope ratios with a precision (1 standard deviation) of ± 0.6 ‰ for 20 ml of air sample containing more than 3 ppm of methanol and ± 0.7 ‰ for 20 ml of air sample containing more than 2 ppm of acetaldehyde. Moreover, the applicability of this method to determine isotope ratios of methanol and acetaldehyde emitted from detached plant leaves was demonstrated.

Measurement of the isotope ratio of acetic acid in vinegar by HS-SPME-GC-TC/C-IRMS.

Acetic acid is the main ingredient of vinegar, and the worth of vinegar often depends on the fermentation of raw materials. In this study, we have developed a simple and rapid method for discriminating the fermentation of the raw materials of vinegar by measuring the hydrogen and carbon isotope ratio of acetic acid using head space solid-phase microextraction (HS-SPME) combined with gas chromatography-high temperature conversion or combustion-isotope ratio mass spectrometry (GC-TC/C-IRMS). The measurement of acetic acid in vinegar by this method was possible with repeatabilities (1sigma) of +/-5.0 per thousand for hydrogen and +/-0.4 per thousand for carbon, which are sufficient to discriminate the origin of acetic acid. The fermentation of raw materials of several vinegars was evaluated by this method.

Dietary phytase increases the true absorption and endogenous fecal excretion of zinc in growing pigs given a corn-soybean meal based diet.

We investigated the effect of dietary phytase on the true absorption and endogenous fecal excretion of zinc (Zn) using (67)Zn in growing pigs given a corn-soybean meal based diet. Ten crossbred barrows were fed the control diet containing 90-mg/kg Zn, 2.3-g/kg phytate-phosphorus and 3.7-g/kg non-phytate-phosphorus or the phytase diet containing similar amounts of Zn and phytate-phosphorus, and 1.4-g/kg non-phytate-phosphorus with 750-PU/kg phytase for 12 h/day. On day 6, the pigs were given 200 g of the corresponding diet labeled by (67)Zn for 2 h. We measured feed intake, fecal Zn concentration and (67)Zn abundance for the determination of apparent absorption, true absorption and endogenous fecal excretion of Zn. Although the apparent absorption of Zn did not significantly differ between the dietary groups, the phytase group had significantly more (P < 0.05) true absorption of Zn than the control group. The endogenous fecal excretion of Zn tended to be more (P = 0.07) in the phytase group than in the control group. These results suggest that dietary phytase improves Zn bioavailability through increasing the true absorption of Zn in growing pigs, which results in stimulating the endogenous fecal excretion of Zn when dietary Zn satisfies its requirement.

An improved method for the measurement of the isotope ratio of ethanol in various samples, including alcoholic and non-alcoholic beverages.

The isotope ratios of ethanol, an important constituent or ingredient of some foods and various beverages and fuels, provide information about biological and geographical origin and quality. We have developed an improved method for measuring the isotope ratio of ethanol in various samples by gas chromatography-high temperature conversion or combustion-isotope ratio mass spectrometry (GC-TC/C-IRMS) with headspace solid-phase microextraction (HS-SPME). A HS-SPME method was developed by optimizing several different parameters, including salt addition, incubation temperature and time, and extraction time. The HS-SPME method enabled us to determine the isotope ratio at low ethanol concentrations (0.08 mM) in 50 min with good precision (+/-0.3 per thousand for delta(13)C and +/-5 per thousand for deltaD). An advantage of this technique is that it can be adapted for use with samples which have high viscosity and contain many matrix compounds, such as alcoholic and non-alcoholic beverages.

A preliminary study of anti-1-amino-3-18F-fluorocyclobutyl-1-carboxylic acid for the detection of prostate cancer.

UNLABELLED: We evaluated the feasibility of anti-1-amino-3-(18)F-fluorocyclobutyl-1-carboxylic acid (anti-(18)F-FACBC) in diagnosing prostate cancer (PCa), using a rat orthotopic prostate cancer transplantation (OPCT) model. Furthermore, using in vivo experiments, we examined the potential of anti-(18)F-FACBC for differentiating between PCa and inflammation and between PCa and benign prostatic hyperplasia (BPH). METHODS: The OPCT model was developed by transplanting DU145, a human PCa cell line, into the ventral prostate of athymic F344 rats. To develop a dual PCa and inflammation (DPCI) model, MAT-Ly-Lu-B2--a rat PCa cell line--was transplanted subcutaneously into male Copenhagen rats. Streptozotocin was injected into the hind footpad of these rats for inducing popliteal lymphadenitis. For inducing the BPH, normal F344 rats were castrated and injected subcutaneously with testosterone propionate. In biodistribution studies, the rats were injected with anti-(18)F-FACBC or (18)F-FDG and sacrificed at 15 or 60 min after injection. We performed dynamic small-animal PET of the abdominal portion of the OPCT rats for 60 min after the injection of anti-(18)F-FACBC or (18)F-FDG. RESULTS: The biodistribution in the OPCT rats at 60 min after injection showed that the uptake of anti-(18)F-FACBC and (18)F-FDG into the PCa tissue was 1.58 +/- 0.40 %ID/cm(3) (percentage injected dose per cm(3)) and 1.48 +/- 0.90 %ID/cm(3), respectively (P > 0.05). The accumulation of anti-(18)F-FACBC in the urinary bladder at 60 min after injection was 3.09 +/- 1.43 %ID/cm(3), whereas that of (18)F-FDG was 69.31 +/- 16.55 %ID/cm(3) (P < 0.05). Consequently, small-animal imaging with anti-(18)F-FACBC facilitated the visualization of the PCa tissue of the OPCT rats with higher contrast than (18)F-FDG. Furthermore, in comparison with (18)F-FDG, apparently higher ratios of PCa to inflammation and PCa to BPH accumulation of anti-(18)F-FACBC were demonstrated in the animal models. CONCLUSION: FACBC PET is believed to be useful not only for the visualization of human PCa but also for differentiating between PCa and inflammation and between PCa and BHP.

Multielement determination of trace metals in river water (certified reference material, JSAC 0301-1) by high efficiency nebulization ICP-MS after 100-fold preconcentration with a chelating resin-packed minicolumn.

The determination of 34 trace metals in a river water certified reference material (CRM), i.e. JSAC 0301-1, which was issued by the Japan Society for Analytical Chemistry in January 2004, was performed by ICP-MS with a high efficiency nebulizer after preconcentration with a laboratory-made chelating resin-packed minicolumn, with which trace metals were concentrated 100-fold from 50 mL of a river water sample to 0.5 mL of the final analysis solution. Trace metals in JSAC 0301-1 were observed in the concentration range from 19 microg L(-1) of Al to 0.000053 microg L(-1) of Bi. It was found that most of the concentrations of trace metals, including rare earth elements (REEs), in JSAC 0301-1 were lower than those in JAC 0031, which was also a previously issued CRM prepared with water from the same river as that of JSAC 0301-1. The low concentrations of trace metals in JSAC 0301-1 might be attributed to the fact that there was a heavy rain before collecting the original water sample to prepare the present CRM. Furthermore, the REE distribution patterns of JSAC 0301-1, JAC 0031 and the average values of river water samples in Japan were parallel to each other. These results indicate that the distributions of REEs in JSAC 0301-1 and JAC 0031 were the typical ones of river water samples in Japan.