Marine bacteria have multiple polyamide 4-degrading enzymes.

Polyamide 4 (PA4) is expected to solve the issue of marine plastic pollution due to its excellent mechanical properties and biodegradability. In this study, to reveal the mechanism of PA4 biodegradation in the marine environment, we isolated 5 strains of PA4-degrading bacteria belonging to Aliiglaciecola, Dasania, and Pseudophaeobacter from a marine environment. The isolated 5 strains are novel PA4-degrading bacteria that are phylogenetically distinct from those isolated in previous studies. In addition, we compared the PA4-degrading activities and structures of the PA4-degrading enzymes secreted by the 5 strains and PA4-degrading strains isolated in our previous study. The PA4-degrading activity in the supernatant of the cultivation solutions differed among the strains. Native-PAGE and zymography using a polyacrylamide gel containing a PA4 emulsion demonstrated that PA4-degrading enzymes are classified into no less than three types of structures. These results suggested that marine PA4-degrading bacteria have multiple PA4-degrading enzymes. Our findings will contribute to a better understanding of the microbial degradation of PA4 in the marine environment.

Novel dicarbonyl metabolic pathway via mitochondrial ES1 possessing glyoxalase III activity.

Glycation, caused by reactive dicarbonyls, plays a role in various diseases by forming advanced glycation end products. In live cells, reactive dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) are produced during cell metabolism, and these should be removed consistently. However, the dicarbonyl metabolic system in the mitochondria remains unclear. It has been speculated that the mammalian mitochondrial protein ES1 is a homolog of bacterial elbB possessing glyoxalase III (GLO3) activity. Therefore, in this study, to investigate ES1 functions and GLO3 activity, we generated ES1-knockout (KO) mice and recombinant mouse ES1 protein and investigated the biochemical and histological analyses. In the mitochondrial fraction obtained from ES1-KO mouse brains, the GO metabolism and cytochrome c oxidase activity were significantly lower than those in the mitochondrial fraction obtained from wildtype (WT) mouse brains. However, the morphological features of the mitochondria did not change noticeably in the ES1-KO mouse brains compared with those in the WT mouse brains. The mitochondrial proteome analysis showed that the MGO degradation III pathway and oxidative phosphorylation-related proteins were increased. These should be the response to the reduced GO metabolism caused by ES1 deletion to compensate for the dicarbonyl metabolism and damaged cytochrome c oxidase by elevated GO. Recombinant mouse ES1 protein exhibited catalytic activity of converting GO to glycolic acid. These results indicate that ES1 possesses GLO3 activity and modulates the metabolism of GO in the mitochondria. To our knowledge, this is the first study to show a novel metabolic pathway for reactive dicarbonyls in mitochondria.

Improvement of solubility of phospholipase D from Streptomyces antibioticus in recombinant Escherichia coli and its application for the enzymatic synthesis of a non-natural plasmalogen.

Plasmalogens are a subclass of glycerophospholipids that have a vinyl-ether bond at the sn-1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in the cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with ∼730 mU mg-1 protein of specific activity, and the yield was ∼420 mU l-1 culture, corresponding to 76 mU per gram of wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn-3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens.

Construction of a conjugation method for the transformation of Cobetia sp. IU180733JP01 (5-11-6-3) that accumulates poly(3-hydroxybutyrate) from seaweeds.

Cobetia bacteria are considered useful hosts for industrial applications owing to their fast growth, high cell density, and halophilicity. Here, we constructed an efficient conjugation method to obtain Cobetia sp. IU180733JP01 (5-11-6-3) transformants, which can produce bioplastics from alginate or seaweed waste. Lysogeny Broth medium containing 2% NaCl was used to co-cultivate the 5-11-6-3 strain (plasmid recipient) with Escherichia coli S17-1 (plasmid donor). Transformants with the highest conjugation efficiency [(2.92 ± 1.37) × 10-3] were obtained at a donor:recipient cell number ratio of 5:1. This is the first study reporting the creation of recombinant strains in the genus Cobetia. This method will contribute to creating a platform strain for the production of bioplastics and other useful materials from marine biomass.

Utilization of various carbon sources for poly(3-hydroxybutyrate) [P(3HB)] production by Cobetia sp. IU180733JP01 (5-11-6-3) which is capable of producing P(3HB) from alginate and waste seaweed.

The marine bacterium Cobetia sp. IU180733JP01 (5-11-6-3) can accumulate poly(3- hydroxybutyrate) [P(3HB)] during cultivation on alginate or waste Laminaria sp. Here, we examined this strain's ability to utilize various carbon sources for P(3HB) production. When cultured in mineral salt medium containing 1% (w/v) glucose, fructose, glycerol, or gluconic acid, the strain showed better growth and higher P(3HB) production than on alginate, with fructose enabling the highest P(3HB) yield (0.8 ± 0.06 g/L). We also predicted metabolic pathways for P(3HB) synthesis based on draft genome sequence analysis, in which carbon sources are assimilated through Entner-Doudoroff and Embden-Meyerhof pathways, and the resultant acetyl-CoA is converted into P(3HB). Our findings reveal the potential of the 5-11-6-3 strain for application in bioplastic production from not only marine biomass but also other biomass and industrial wastes.

Lymphatic Absorption of Microbial Plasmalogens in Rats.

Plasmalogens, functional glycerophospholipids with biological roles in the human body, are associated with various diseases. Although a variety of saturated and/or unsaturated fatty acids in plasmalogens are presumed to have different functions in the human body, there are limited reports validating such functions of plasmalogens. In this study, we focused on the bacterial plasmalogen derived from Selenomonas ruminantium subsp. lactilytica (NBRC No. 103574) with different main species of hydrocarbon chains at the sn-1 position and shorter fatty acids at the sn-2 position than animal plasmalogens. Optimum culture conditions of S. ruminantium for high-yield production of plasmalogens, such as pH and the concentration of caproic acid, were investigated under anaerobic conditions using a 2-L scale jar fermenter. The obtained plasmalogen mainly consisted of the ethanolamine plasmalogen (PlsEtn). The molar ratios of PlsEtn species obtained from S. ruminantium, at sn-1/sn-2 positions, were p16:1/14:0 (68.4%), p16:1/16:1 (29.2%), p16:1/16:0 (0.7%), p16:1/15:0 (0.3%), and p17:1/14:0 (0.3%). Subsequently, duodenal infusion of the emulsion carrying the lipid extracted from S. ruminantium was carried out in lymph duct-cannulated rats. In the lymphatic plasmalogen of rats, the level of PlsEtns with molar ratios p16:1/14:0 and p16:1/16:1, the main species of plasmalogens from S. ruminantium, increased gradually until 3-4 h after lipid injection and then gradually decreased. In addition, the level of PlsEtns with p16:1/20:4 and p16:1/22:6 rapidly increased, peaking at 1-1.5 h and 1.5-2 h after lipid injection, respectively. The increase in the number of PlsEtns with p16:1/20:4 and p16:1/22:6 suggested that 20:4 and 22:6, the main fatty acids at the sn-2 position in the rat lymphatic plasmalogen, were preferentially re-esterified at the sn-2 position, regardless of the types of hydrocarbon chains at the sn-1 position. Thus, we showed that bacterial PlsEtns with "unnatural" structures against rats could be absorbed into the lymph. Our findings provide insights into the association between the chemical structure of plasmalogens and their biological functions in humans.

Cobetia sp. Bacteria, Which Are Capable of Utilizing Alginate or Waste Laminaria sp. for Poly(3-Hydroxybutyrate) Synthesis, Isolated From a Marine Environment.

We isolated the Cobetia sp. strains IU 180733JP01 (5-11-6-3) and 190790JP01 (5-25-4-2) from seaweeds and showed that both strains accumulate poly(3-hydroxybutyrate) [P(3HB)] homopolymer in a nitrogen-limiting mineral salt medium containing alginate as a sole carbon source. Genome sequence analysis of the isolated strains showed that they have putative genes which encode enzymes relevant to alginate assimilation and P(3HB) synthesis, and the putative alginate-assimilating genes formed a cluster. Investigation of the optimum culture conditions for high accumulation of P(3HB) showed that when the 5-11-6-3 strain was cultured in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and 3% (w/v) alginate as a sole carbon source for 2 days, the P(3HB) content and P(3HB) production reached 62.1 ± 3.4 wt% and 3.11 ± 0.16 g/L, respectively. When the 5-25-4-2 strain was cultured in a nitrogen-limiting mineral salt medium (pH 4.0) containing 5% NaCl and 3% (w/v) alginate for 2 days, the P(3HB) content and P(3HB) production reached 56.9 ± 2.1 wt% and 2.67 ± 0.11 g/L, respectively. Moreover, the 5-11-6-3 strain also produced P(3HB) in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and freeze-dried and crushed waste Laminaria sp., which is classified into brown algae and contains alginate abundantly. The resulting P(3HB) content and P(3HB) productivity were 13.5 ± 0.13 wt% and 3.99 ± 0.15 mg/L/h, respectively. Thus, we demonstrated the potential application of the isolated strains to a simple P(3HB) production process from seaweeds without chemical hydrolysis and enzymatic saccharification.

Cloning of the SPO11 gene that complements a meiotic recombination defect in sake yeast.

Cross hybridization breeding of sake yeasts is hampered by difficulty in acquisition of haploid cells through sporulation. We previously demonstrated that typical sake yeast strains were defective in meiotic chromosome recombination, which caused poor sporulation and loss of spore viability. In this study, we screened a single copy plasmid genomic DNA library of the laboratory Saccharomyces cerevisiae GRF88 for genes that might complement the meiotic recombination defect of UTCAH-3, a strain derived from the sake yeast Kyokai no. 7 (K7). We identified the SPO11 gene of the laboratory strain (ScSPO11), encoding a meiosis-specific endonuclease that catalyzes DNA double-strand breaks required for meiotic recombination, as a gene that restored meiotic recombination and spore viability of UTCAH-3. K7SPO11 could not restore sporulation efficiency and spore viability of UTCAH-3 and a laboratory strain BY4743 spo11Δ/spo11Δ, indicating that K7SPO11 is not functional. Sequence analysis of the SPO11 genes of various Kyokai sake yeasts (K1, and K3-K10) revealed that the K7 group of sake yeasts (K6, K7, K9, and K10) had a mutual missense mutation (C73T) in addition to other three common mutations present in all Kyokai yeasts tested. ScSPO11C73T created through in vitro mutagenesis could not restore spore viability of BY4743 spo11Δ/spo11Δ. On the other hand, K8SPO11, which have the three common mutations except for C73T could restore spore viability of BY4743 spo11Δ/spo11Δ. These results suggest that C73T might be a causative mutation of recombination defect in K7SPO11. Moreover, we found that the introduction of ScRIM15 restored sporulation efficiency but not spore viability.

Expression of alcohol oxidase gene from Ochrobactrum sp. AIU 033 in recombinant Escherichia coli through the twin-arginine translocation pathway.

We cloned a set of genes encoding alcohol oxidase from Ochrobactrum sp. AIU 033 (OcAOD), which exhibits the appropriate substrate specificity for glyoxylic acid production from glycolic acid. The set of genes for OcAOD contained two open reading frames consisting of 555-bp (aodB) and 1572-bp (aodA) nucleotides, which encode the precursor for the ß-subunit and α-subunit of OcAOD, respectively. We expressed the cloned genes as an active product in Escherichia coli BL21(DE3). The recombinant OcAOD oxidized glycolic acid and primary alcohols with C2-C8 but not glyoxylic acid (as is the case for native OcAOD), whereas the Km and Vmax values for glycolic acid and the pH stability were higher than those of native OcAOD. A consensus sequence for the twin-arginine translocation (Tat) pathway was identified in the N-terminal region of the precursor for the ß-subunit, and the active form of OcAOD was localized in the periplasm of recombinant E. coli, which indicated that OcAOD would be transported from the cytoplasm to the periplasm by the hitchhiker mechanism through the Tat pathway. The OcAOD productivity of the recombinant E. coli was 24-fold higher than that of Ochrobactrum sp. AIU 033, and it was further enhanced by 1.2 times by the co-expression of additional tatABC from E. coli BL21(DE3). Our findings thus suggest a function of the ß-subunit of OcAOD in membrane translocation, and that the recombinant OcAOD has characteristics that are suitable for the enzymatic synthesis of glyoxylic acid as well as native OcAOD.

Meiotic chromosomal recombination defect in sake yeasts.

Sake yeast strains are classified into Saccharomyces cerevisiae and have a heterothallic life cycle. This feature allows cross hybridization between two haploids to breed new strains with superior characteristics. However, cross hybridization of sake yeast is very difficult because only a few spores develop in a sporulation medium, and most of these spores do not germinate. We hypothesized that these features are attributable to chromosome recombination defect in meiosis, which leads to chromosome loss. To test this hypothesis, we examined meiotic recombination of sake yeast Kyokai no. 7 (K7) using the following three methods: (i) analysis of the segregation patterns of two heterozygous sites in the same chromosome in 100 haploid K7 strains; (ii) sequencing of the whole genomes of four haploid K7 strains and comparison of the bases derived from the heterozygosities; and (iii) construction of double heterozygous disruptants of CAN1 and URA3 on the chromosome V of K7 and the examination of the genotypes of haploids after sporulation. We could not detect any recombinant segregants in any of the experiments, which indicated defect in meiotic recombination in K7. Analyses after sporulation of the same double heterozygous disruptants of K6, K9, and K10 also indicated meiotic recombination defect in these strains. Although rapamycin treatment increased the sporulation efficiency of K7, it did not increase the meiotic recombination of the double heterozygous K7. Moreover, the spo13 disruptant of the K7 derivative produced two spore asci without meiotic recombination. These results suggest that sake yeasts have defects in meiotic recombination machinery.

An enzymatic method for the production of 6-oxohexanoic acid from 6-aminohexanoic acid by an enzyme oxidizing ω-amino compounds from Phialemonium sp. AIU 274.

An enzymatic method for 6-oxohexanoic acid production was developed using 6-aminohexanoic acid and an ω-amino group-oxidizing enzyme (ω-AOX) from Phialemonium sp. AIU 274. 6-Oxohexanoic acid was produced from 6-aminohexanoic acid with 100% yield by incubation with 0.3 U of the ω-AOX and 20 U of catalase at 30 °C for 30 h in 0.1 M potassium phosphate buffer (pH 7.0).

Sucrose supplementation suppressed the growth inhibition in polyhydroxyalkanoate-producing plants.

Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA, phaB, and phaC (collectively referred to as phaABC). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC-overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites.

New alkalophilic ß-galactosidase with high activity in alkaline pH region from Teratosphaeria acidotherma AIU BGA-1.

A ß-d-galactosidase exhibiting high activity in the alkaline pH region was purified from Teratosphaeria acidotherma AIU BGA-1, which we previously isolated as a unique fungal producer of three acidophilic and one alkalophilic ß-d-galactosidases (Isobe et al., J. Biosci. Bioeng., 116, 171-174, 2013). The enzyme was stable in the pH range 7.5-10.0 and exhibited optimal activity at pH 8.0 and 60°C. The enzyme hydrolyzed 2-nitrophenyl ß-d-galactopyranoside, 4-nitrophenyl ß-d-galactopyranoside, and lactose, and the Km values were estimated to be 0.349 mM, 0.488 mM, and 701 mM, respectively. Chelating reagents (EDTA and o-phenanthroline) and metals (Cu2+and Ni2+) inhibited the enzyme activity, and Mn2+ was a good activator. The enzyme also exhibited transgalactosylation activity for lactose. The enzyme's molecular mass was estimated to be 180 kDa, and its structure was monomeric. Thus, the enzymatic and physicochemical characteristics of the alkalophilic ß-galactosidase in this study clearly differed from those of the previously known alkalophilic ß-d-galactosidases.

Novel acidophilic ß-galactosidase with high activity at extremely acidic pH region from Teratosphaeria acidotherma AIU BGA-1.

A ß-galactosidase exhibiting maximal activity at pH 1.0 was purified from Teratosphaeria acidotherma AIU BGA-1. The enzyme had a molecular mass of 180 kDa and consisted of two heterosubunits of 120 kDa and 66 kDa. The N-terminal amino acid sequence of the large subunit was found to be SPNLQDIVTVDGESY. These physicochemical properties differed from those of other microbial ß-galactosidases. At pH values of 1.5 and pH 4.5, the enzyme exhibited its highest activity at temperatures of 70°C and 80°C, respectively. Thus, the enzyme exhibited the lowest optimal pH and highest optimal temperature among the microbial ß-galactosidases thus reported. The enzyme retained more than 80% of its original activity in the pH range from 2.0 to 8.0 by incubation at 50°C for 30 min. The enzyme hydrolyzed 4-nitrophenyl-ß-D-fucopyranoside, 2-nitrophenyl-ß-D-galactopyranoside, and 4-nitrophenyl-ß-D-galacto-pyranoside at relative reaction rates of 100, 59, and 24, respectively, at pH 1.5, and its affinity for ß-D-galactopyranosides was higher than that for ß-D-fucopyranosides. The enzyme also efficiently hydrolyzed lactose in milk and whey from yoghurt at pH 1.5.

Characterization of two amine oxidases from Aspergillus carbonarius AIU 205.

We have reported that Aspergillus carbonarius AIU 205, which was isolated by our group, produced three enzymes exhibiting oxidase activity for 4-aminobutanamide (4-ABAD) (J. Biosci. Bioeng., 117, 263-268, 2014). Among three enzymes, characteristics of enzyme I have been revealed, but those of the other two enzymes have not. In this study, we purified enzymes II and III, and compared their characteristics with those of enzyme I. Enzymes II and III also oxidized aliphatic monoamines, aromatic amines, and aliphatic aminoalcohols. In addition, the oxidase activity of both enzymes was strongly inhibited by carbonyl reagents and specific inhibitors for copper-containing amine oxidases. Thus, enzymes II and III were also classified into the copper-containing amine oxidase group (EC 1.4.3.6) along with enzyme I. However, these three enzymes differed from each other in their enzymatic, kinetic, and physicochemical properties. The N-terminal amino acid sequences also differed from each other; that of enzyme I was modified, that of enzyme II was similar to those of peroxisomal copper-containing amine oxidases, and that of enzyme III was similar to those of copper-containing amine oxidases from the genus Aspergillus. Therefore, we concluded that A. carbonarius AIU 205 produced three different types of amine oxidase in the mycelia.

A new aldehyde oxidase catalyzing the conversion of glycolaldehyde to glycolate from Burkholderia sp. AIU 129.

We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (αßγ structure), in which the α, ß, and γ subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (α,α'-dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50°C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30°C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate.

New enzymatic methods for selective assay of L-lysine using an L-lysine specific decarboxylase/oxidase from Burkholderia sp. AIU 395.

We developed new enzymatic methods for the selective assay of L-lysine by utilizing an oxidase reaction and a decarboxylation reaction by the L-lysine-specific decarboxylase/oxidase (L-Lys-DC/OD) from Burkholderia sp. AIU 395. The method utilizing the oxidase reaction of this enzyme was useful for determination of high concentrations of L-lysine. The method utilizing the decarboxylase reaction, which proceeded via the combination of the L-Lys-DC/OD and putrescine oxidase (PUO) from Micrococcus rubens, was effective for determination of low concentrations of L-lysine. Both methods showed good linearity, and neither was affected by other amino acids or amines. In addition, the within-assay and between-assay precisions of both methods were within the allowable range. The coupling of L-Lys-DC/OD with PUO was also useful for the differential assay of L-lysine and cadaverine. These newly developed methods were applied to the assay of L-lysine in biological samples and found to be effective.

Using drawing tests to explore the multidimensional psychological aspects of children with cancer.

In seeking to understand how life-threatening illness affects children psychologically, projective testing may be beneficial, particularly when attempting to identify psychological problems in younger adolescents. The advantages of projective testing are that it does not depend on patients' verbal ability and is not invasive. Three cancer inpatients from Kyoto Prefectural University of Medicine, aged between 10 and 16 years old, participated in the study. Projective testing was used to measure the depth of participants' distress and included a tree-drawing test, a person-drawing test and a free drawing test. Results from the tree- and person-drawing tests indicated energy loss, anxiety and a sense of emptiness. However, results from the free drawing test suggested that the children had hope and a desire to recover from their illness. The combination of drawing tests in this study may increase the understanding of the internal psychological difficulties faced by children hospitalized with cancer.

Characterization of a pyridoxal-5'-phosphate-dependent l-lysine decarboxylase/oxidase from Burkholderia sp. AIU 395.

A novel enzyme, which catalyzed decarboxylation of l-lysine into cadaverine with release of carbon dioxide and oxidative deamination of l-lysine into l-2-aminoadipic 5-semialdehyde with release of ammonia and hydrogen peroxide, was found from a newly isolated Burkholderia sp. AIU 395. The enzyme was specific to l-lysine and did not exhibit enzyme activities for other l-amino acids, l-lysine derivatives, d-amino acids, and amines. The apparent Km values for l-lysine in the oxidation and decarboxylation reactions were estimated to be 0.44 mM and 0.84 mM, respectively. The molecular mass was estimated to be 150 kDa, which was composed of two identical subunits with molecular mass of 76.5 kDa. The enzyme contained one mol of pyridoxal 5'-phosphate per subunit as a prosthetic group. The enzyme exhibiting decarboxylase and oxidase activities for l-lysine was first reported here, while the deduced amino acid sequence was homologous to that of putative lysine decarboxylases from the genus Burkholderia.

Characterization of a novel l-amino acid oxidase with protein oxidizing activity from Penicillium steckii AIU 027.

An enzyme exhibiting oxidase activity for ß-lactoglobulin, myoglobin, and l-lysine-containing peptides was found from a newly isolated fungal strain, Penicillium steckii AIU 027. The enzyme also oxidized l-amino acids, N(α)-benzyloxycarbonyl-l-lysine (N(α)-Z-l-lysine) and N(ε)-Z-l-lysine, but not d-amino acids and amines. Thus, the enzyme was classified into a group of l-amino acid oxidases (l-AAOs). However, characteristics of this l-AAO were significantly different from those of other l-AAOs as follows. The l-AAO from P. steckii AIU 027 oxidized both the α-amino group and the ε-amino group in l-amino acids and l-lysine-containing peptides, and the Km values for l-lysine-containing polypeptides were lower than those for N(α)-Z-l-lysine and l-lysine-containing dipeptides. The enzyme contained flavin and iron, and composed of four identical subunits with molecular mass of 75.3 kDa. The N-terminal amino acid sequence, ENIADVADAMGPWFDGVAYMKSKKN, was different from that of other l-AAOs. Thus, the l-AAO with protein oxidase activity was first reported here from P. steckii AIU 027.