Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli, and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87.

We characterized the membrane vesicle fraction (RD MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD MVs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the RD MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patches cells following the addition of the RD MV fraction. In the presence of the RD MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial Toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

In recent years, the number of patients with osteoporosis has increased as population grows older. Therefore, the chemoprevention of osteoporosis by better nutrition is important. White-rot fungi degrades milled wood lignin for growth and development. This degradation results in the formation of phenolic compounds such as syringic acid (SA) and vanillic acid (VA). In the artificial culture of edible mushrooms using a mushroom bed, the disposal of waste beds after mushroom cultivation is an important issue. The present study investigated the presence and amount of both SA and VA in the discarded waste beds after mushroom cultivation. The extracts from waste beds after cultivation of shiitake mushrooms, Lentinula edodes; buna shimeji, Hypsizygus marmoreus; maitake, Grifola frondosa; king trumpet mushrooms, Pleurotus eryngii; and butterscotch mushrooms, Pholiota microspora were analyzed using high performance liquid chromatography. Although the content of SA and VA was considerably different among the mushrooms, SA and VA were present in extracts obtained from all the waste beds. We also demonstrated that SA and VA exert their anti-osteoporotic effect independently of the estrogen receptor-mediated pathway using murine monocytic RAW264.7 cells, ovariectomized mice, and human breast cancer MCF-7 cells. Thus, these results suggest that the extracts are effective sources of SA and VA, which are effective in preventing osteoporosis.

Streptomyces similanensis 9X166 is known to be an antagonist of the black rot pathogen of orchids, Phytophthora palmivora. In this study, we investigated the production of highly viable S. similanensis 9X166 cells by solid state fermentation using agro-industrial substrates, and the shelf life of a S. similanensis 9X166 dried solid. Rice bran was found to be the most appropriate raw material for production of both viable cells and β-1,3-glucanase. A medium containing 12 g of rice bran and coconut husks at a ratio of 10:2, supplemented with 10 mL of mineral salts produced the highest number of viable cells and greatest level of β-1,3-glucanase. Ammonium sulfate was the most suitable nitrogen source, and an initial moisture content of 65% and a temperature of 30°C were found to be optimal conditions for the production of viable cells and β-1,3-glucanase. Storing the dried fermented solid under non-vacuum conditions resulted in the highest cell viability. The specific rate of degradation on viability increased as the temperature increased to 37°C, according to the Arrhenius equation. There was no difference between the storage time estimated by the Arrhenius equation from the specific rate of degradation compared to the validated storage time of S. similanensis 9X166 dried solids when maintained at the ambient temperature in Thailand. At 60 days, the product retained 10 6 CFU/g of S. similanensis 9X166 in dried solid, which was the minimal effective amount for 100% inhibition of P. palmivora in living orchids.

A total of 123 actinomycetes was isolated from 12 varieties of wild orchids and screened for potential antagonistic activity against Phytophthora, which causes black rot disease in orchids. In vitro and in vivo experimental results revealed that Streptomyces sp. strain 9X166 showed the highest antagonistic activity; its -1,3-glucanase production ability was a key mechanism for growth inhibition of the pathogen. PCR amplification and DNA sequencing of the 16S ribosomal RNA gene allowed the identification of this strain, with high similarity (99.93%) to the novel species Streptomyces similaensis. The glucanase enzyme, purified to homogeneity by anion exchange and gel filtration chromatography, showed a specific activity of 58Umg(-1) (a 3.9-fold increase) and yield of 6.4%. The molecular weight, as determined by SDS-PAGE and gel filtration, was approximately 99 and 80kDa, respectively, suggesting that the enzyme was a monomer. The purified enzyme showed the highest substrate specificity to laminarin, indicating that it was -1,3-glucanase. The hydrolyzed products of cello-oligosaccharides suggested that this enzyme was endo-type -1,3-glucanase. Streptomyces sp. 9X166 culture filtrate, possessing -1,3-glucanase activity, could degrade both freeze-dried and living mycelium. This is the first report on a -1,3-glucanase-producing Streptomyces sp. that could be an effective biocontrol agent for black rot disease in orchids.

In this study, it was shown for the first time that L-amino acid oxidase of Pseudomonas sp. AIU813, renamed as L-amino acid oxidase/monooxygenase (L-AAO/MOG), exhibits L-lysine 2-monooxygenase as well as oxidase activity. L-Lysine oxidase activity of L-AAO/MOG was increased in a p-chloromercuribenzoate (p-CMB) concentration-dependent manner to a final level that was fivefold higher than that of the non-treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying L-AAO/MOG C254I mutant enzyme, which showed five-times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high-resolution three-dimensional structure of L-AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of L-AAO/MOG, Cys-254, is located near the aromatic cage (Trp-418, Phe-473, and Trp-516). Although the location of Cys-254 indicates that it is not directly involved in the substrate binding, the chemical modification by p-CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp-516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

The purification and characterization of a neutral protease (PE-4) produced in mycelia during fruit-body formation in Hypsizygus marmoreus was carried out in the present study. The enzyme was purified to homogeneity using ammonium fractionation, ion exchange chromatography on DEAE-Toyopearl, hydrophobic exchange chromatography on Butyl-Toyopearl, and gel chromatography on Superdex 200. Protease activity was enhanced by Co^<2+>, in particular, and was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), aprotinin and Pefabloc SC, which are known serine protease inhibitors. Phosphoramidon did not inhibit the protease. The N-terminal amino acid sequence was identical with the serine protease from the fruit-body of H. marmoreus, hmsp. The same serine protease was present in both vegetative mycelia and the fruit-body in the growth stage. Protease activity activated in mycelia at fruit-body formation was inhibited by PMSF, EDTA, and phosphoramidon but not completely. These results also suggest that several neutral proteases besides PE-4 were present during kinkaki, which is removal of both the spawn and the uppermost layer of the medium, in the mature fruit-body in the mycelia.

Profiling of cellulases (endo-type cellulase, exo-type cellulase, and β-glucosidase) of Tremella fuciformis and Hypoxylon truncatum were carried out using crude enzymes from these two microorganisms cultured in sawdust-rice bran medium. Specific activity of the crude enzyme from T. fuciformis toward cellulose powder, Avicel, and p-nitrophenyl-β-D-glucopyranoside was 13.5, 35.9, and 47.6U/mg, respectively. The enzyme did not act on carboxymethyl cellulose (CMC). The crude enzyme from H. truncatum acted toward all substrates tested. Endo-type cellulase from H. truncatum was purified to homogeneity and its enzymatic properties were characterized. Following filtration and centrifugation, the enzyme solution was purified by ammonium sulfate fractionation, heat treatment, ion exchange chromatography, and gel chromatography. Its molecular weight was estimated as 46,000 by SDS-PAGE and 41,000 by gel chromatography, which suggested that the native enzyme is active as a monomer. The enzyme was stable at 60℃ and pH 5.0-6.0. The purified enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose, but did not degrade cellobiose or cellotriose. These results suggested that endo-type cellulase from H. truncatum can provide short cellooligosaccharides such as cellobiose, cellotriose, and cellotetraose to compensate for the slight or absent endo-cleavage of the T. fuciformis cellulase system during co-cultivation of these two fungi.

An L-amino acid oxidase was found from a newly isolated strain. Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with L-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with L-lysine as ligand. The enzyme oxidized L-lysine, L-ornithine and L-arginine, but not other L-amino acids and D-amino acids. The oxidase activity for L-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for L-ornithine and L-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a Flavoprotein and composed of two identical subunits with molecular mass of 54.5 kDa. The N-terminal amino acid sequence was similar to that of putative Flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of L-amino acid oxidases. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography-size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50A degrees C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50A degrees C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.

Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too. (c) 2011 Elsevier Inc. All rights reserved.

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.

Rhodococcus sp. strain Oct1 utilizing omega-octalactam as a sole source of carbon and nitrogen was isolated from soil. omega-Octalactam hydrolyzing enzyme was purified to homogeneity. The purified enzyme has a molecular weight of approximately 48,100 by SDS polyacrylamide gel electrophoresis and 99,1100 by gel filtration, indicating that the enzyme consists of 2 subunits. The purified enzyme catalyzed the hydrolysis of omega-octalactam to form 8-aminooctanoic acid at a rate of 3.95 U/mg. The purified enzyme also acted on omega-heptalactam, omega-laurolactam, nitroacetoanilide substitutions, and various aliphatic amides. The most suitable substrate was o-nitroacetanilide for the enzyme (11.6 U/mg). The enzyme belongs to aryl acylamidase. The gene for the enzyme was cloned and the deduced amino acid sequence showed similarity to omega-laurolactam hydrolase from Rhodococcus sp. U224 (51%) and putative aryl acylamidase from Nocardia farcinica IFM 10152 (98%), and N-terminal amino acid sequence (28 residues) of aryl acylamidase from Nocardia globerula IFO 13510 (92%). Aryl acylamidases and 6-aminohexanoate-cyclic-dimer hydrolases are in the same phylogenic lineage. These enzymes were mostly active toward non-natural amides. From phylogenic analysis, these enzymes were classified into amidase signature family. The enzyme was produced in a soluble form as a fusion protein (extension of 13 amino acids at C-terminal) in Escherichia coli. (C) 2009 Elsevier Inc. All rights reserved.

The genes encoding omega-laurolactam hydrolases from Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, and Sphingomonas sp. U238 were cloned and sequenced. Nucleotide and amino acid sequence analysis of the four genes indicated that the primary structures of these omega-laurolactam hydrolases are significantly similar to the 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12). These genes were expressed in Escherichia coli.. and the omega-laurolactam hydrolysing activity of the recombinant enzymes was compared with that of 6-aminohexanoate-cyclic-dimer hydrolase from Arthrobacter sp. K172. The enzyme from Acidovorax sp. T31 was most successfully expressed in E. coli. Cell-free extract of the recombinant strain was used for the synthesis of 12-aminolauric acid from omega-laurolactam by "enzymatic transcrystallization," because crystalline omega-laurolactam added into the enzyme solution was converted to crystalline 12-aminolauric acid (>= 97.3% yield). Under the optimum conditions, 208 g/l of 12-aminolauric acid was produced in 17 h. The resulting pure product was identical to authentic 12-aminolauric acid.

Several omega-laurolactam degrading microorganisms were isolated from soil samples. These strains were capable of growing in a medium containing omega-laurolactam as sole source of carbon and nitrogen. Among them, five strains (T7, T31, U124, U224, and U238) were identified as, Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, Rhodococcus sp. U224, and Sphingomonas sp. U238, respectively. The w-laurolactam hydrolyzing enzyme from Rhodococcus sp. U224 was purified to homogeneity, and its enzymatic properties were characterized. The enzyme acts on omega-octalactam and omega-laurolactam, but other lactam compounds, amides and amino acid amides, cannot be substrates. The enzyme, gene was cloned, and the deduced amino acid sequence showed high homology with 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12) from Arthrobacter sp. K172 and Pseudomonas sp. NK87. Enzymatic synthesis of 12-aminolauric acid was performed using partially purified w-laurolactam hydrolase from Rhodococcus sp. U224.

With the aim of establishing a transformation method for Tricholoma matsutake, research was conducted with the aim of isolation of monokaryon as a host strain, and developing genetic recombination technology using the protoplast-PEG method in the fungi. However, protoplasts could not be regenerated during the study period. Currently, we are using α-1,3-glucanase derived from the cell wall of the fungi component degradation microorganism isolated from nature to adjust protoplasts in an even larger amount, and continue regeneration experiments. On the other hand, as a genetic recombination experiment using the same basidiomycetes, Pleurotus salmoneostramineus, which can form fruiting bodies, was used in a model experiment. By the method established in this experiment, the expression of the pink colored protein gene (PsPCP) was suppressed, and white fruiting bodies were formed in the experiment.

In order to evaluate the mechanisms for gamma-aminobutyric acid (GABA) accumulation in edible mushroom, we purified and characterized the enzymes for GABA formation from Grifola frondosa, Lentinula edodes and Flammulina velutipes. The enzymes from G. frondosa, and F. velutipes enzyme used only L-glutamic acid as substrate for decarboxylation reaction so that these enzymes should belong to glutamic acid decarboxylase. However, the enzyme from L. edodes can use aspartic acid, other than glutamic acid, as substrate for the reaction. The internal amino acid sequences of this enzyme showed that the enzyme should be phosphatidylserine decarboxylase (PSD) of L. edodes. However, the reaction of PSD couldn’t be detected.

Fruit-body formation of Basidiomycetes was accelerated by the addition of carboxyl protease inhibitor (S-PI), and no occurrence of fruit-body by the addition of metallo protease inhibitors (Talopeptin, Phosphramidon, and EDTA).Several putative protease genes were selected using ForestGEN (L. edodes whole genome database). Among them, g876 (putative Eukaryotic aspartyl protease) showed high expression in mycelium stage of the fungus. In fruit-body (primordium)stage, g10056 (Eukaryotic aspartyl protease), g584, g1220 (Peptidase family 28) and g4055 (Peptidase family M13) were expressed higher than other putative protease genes. It seemed that g10056, g584, g1220 and g4055 have important role on fruit-body formation in L. edodes. On the other hand, phosphoramidon sensitive metalloprotease from Hypsizygus marmoreus was purified, and the gene was cloned. The protease gene sequence showed high similarity with Fungalysin (Peptidase M36 family).