Production and properties of ∝-mannosidase from aspergillus sp.

Summary Aspergillus sp NCIM 508 produced 22 U/L of extracellular -mannosidase activity in a medium containing 8 % brewer's yeast cells. The optimum period and pH range for maximum production of the enzyme were 7 days and 4.0–6.0, respectively. The optimum pH and temperature for enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable for 24 h at 28°C, in the pH range 6.0–7.0. The enzyme retained 100 and 65 % of its original activity after heating for 15 min at 45 and 55°C, respectively. The Km and V_max for p-nitrophenyl--D- mannoside (PNPM) were 71M and 7.5 × 10^–2 moles/min/mg, respectively. The enzyme was strongly inhibited by 1 mM Hg⁺⁺ and Cu⁺⁺ and partially by Co.⁺⁺ (NCL Communication No.; 5780)     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

Purification and properties ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524

Summary Purification and properties of two -fructofuranosidases, which produce 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, fromAureobasidium sp. ATCC 20524 are reported. The enzymes were purified to homogeneity by fractionations involving ethanol, calcium acetate and ammonium sulfate and DEAE-Cellulofine and Sephadex G-200 chromatography. Molecular weights of the enzymes were estimated to be about 318000 (P-1) and 346000 (P-2) daltons by gel filtration. The enzymes were glycoproteins that contained about 30% (w/v) (P-1) and 53% (w/v) (P-2) carbohydrate. The optimum pH for the enzymatic reactions were 4.5–5.5 (P-1) and 4.5–6 (P-2). The enzymes were stable over a wide pH range (4–9). The optimum reaction temperatures for both enzymes were 50–55°C and they retained more than 94% (P-1) and 98% (P-2) activities at 50°C after 15 min. TheK _m values for sucrose were 0.47 M (P-1) and 0.65 M (P-2). The enzymes were inhibited by mercury, copper and lead ions as well asp-chloromercuribenzoate.     

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. “FVHLFEW” was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Cold-adapted yeasts from Antarctica and the Italian Alps—description of three novel species: Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov.

Worldwide glaciers are annually retreating due to global overheating and this phenomenon determines the potential lost of microbial diversity represented by psychrophilic microbial population sharing these peculiar habitats. In this context, yeast strains, all unable to grow above 20°C, consisting of 42 strains from Antarctic soil and 14 strains isolated from Alpine Glacier, were isolated and grouped together based on similar morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal DNA confirmed the previous analyses and demonstrated that the strains belong to unknown species. Three new species are proposed: Mrakia robertii sp. nov. (type strain CBS 8912), Mrakia blollopis sp. nov. (type strain CBS 8921) and a related anamorphic species Mrakiella niccombsii sp. nov. (type strain CBS 8917). Phylogenetic analysis of the ITS region revealed that the new proposed species were closely related to each other within the Mrakia clade in the order Cystofilobasidiales, class Tremellomycetes. The Mrakia clade now contains 8 sub-clades. Teliospores were observed in all strains except CBS 8918 and for the Mrakiella niccombsii strains.     

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid.

The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color.

The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.     

Cloning of the Gene Encoding α-Methylserine Hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 and Characterization of the Recombinant Enzyme

Genes encoding α-methylserine hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 were cloned and expressed in Escherichia coli. The purified enzymes were homodimers with a 46-kDa subunit and contained 1 mol/mol-subunit of pyridoxal 5′-phosphate. The V _max of these enzymes catalyzing the conversion of α-methyl-L-serine to D-alanine via tetrahydrofolate was 22.1 U/mg (AJ110403) and 15.4 U/mg (AJ110404).     

Kinds of glycosidase and properties of β-N-acetylglucosaminidase bound to mycelia of streptomyces sp.

An extract liberated from mycelia of the L-13 strain of Streptomyces by 0.2 M phosphate buffer, pH 6.5, contained β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase and a little α-glucosidase. On chromatofocusin, the isoelectric point of β-N-acetylglucosaminidase was around pH 8.1. The enzyme prepared thus was homogeneous, and had both β-N-acetylglucosaminidase and β-N-acetlygalactosaminidase activities. The β-N-acetylglucosaminidase was most active at pH 6.0 and stable between pH 4 to 8. The K_m value for p-nitrophenyl β-N-acetylglucosaminide was 0.25 mM. N-Acetylglucosaminolactone was the most potent inhibitor tested.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

Neokomagataea gen. nov., with descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., osmotolerant acetic acid bacteria of the α-Proteobacteria

Isolates AH11(T) and AH13(T) were isolated from flowers of lantana and candle bush respectively collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates formed an independent cluster, which was then connected to the type strain of Saccharibacter floricola. The calculated pair-wise 16S rRNA gene sequence similarities of isolate AH11(T) were 95.7-92.3% to the type strains of the type species of the 12 genera of acetic acid bacteria. The DNA base composition was from 51.2 to 56.8 mol % G+C, with a range of 5.6 mol %. When isolate AH11(T) was labeled, DNA-DNA similarities were 100, 12, 4, 5, and 4% respectively to isolates AH11(T) and AH13(T) and the type strains of Saccharibacter floricola, Gluconobacter oxydans, and Acetobacter aceti. The two isolates were non-motile and did not oxidize either acetate or lactate. No growth was found in the presence of 0.35% acetic acid w/v. The two isolates were not osmophilic but osmotolerant, produced 2,5-diketo-D-gluconate from D-glucose, and did not oxidize lactate, thus differing from strains of Saccharibacter floricola, which showed weak lactate oxidation. The two isolates contained unsaturated C(18:1)ω7c fatty acid as the major fatty acid, and were unique in the presence of a considerable amount of straight-chain C(18:1)2OH fatty acid. Q-10 was present as the major isoprenoid quinone. Neokomagataea gen. nov. was proposed with the two species, Neokomagataea thailandica sp. nov. for isolate AH11(T) (=BCC 25710(T)=NBRC 106555(T)), which has 56.8 mol % G+C, and Neokomagataea tanensis sp. nov. for isolate AH13(T) (=BCC 25711(T)=NBRC 106556(T)), which has 51.2 mol % G+C.     

Neokomagataea gen. nov., with Descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., Osmotolerant Acetic Acid Bacteria of the α-Proteobacteria

Isolates AH11^T and AH13^T were isolated from flowers of lantana and candle bush respectively collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates formed an independent cluster, which was then connected to the type strain of Saccharibacter floricola. The calculated pair-wise 16S rRNA gene sequence similarities of isolate AH11^T were 95.7–92.3% to the type strains of the type species of the 12 genera of acetic acid bacteria. The DNA base composition was from 51.2 to 56.8 mol % G+C, with a range of 5.6 mol %. When isolate AH11^T was labeled, DNA-DNA similarities were 100, 12, 4, 5, and 4% respectively to isolates AH11^T and AH13^T and the type strains of Saccharibacter floricola, Gluconobacter oxydans, and Acetobacter aceti. The two isolates were non-motile and did not oxidize either acetate or lactate. No growth was found in the presence of 0.35% acetic acid w/v. The two isolates were not osmophilic but osmotolerant, produced 2,5-diketo-D-gluconate from D-glucose, and did not oxidize lactate, thus differing from strains of Saccharibacter floricola, which showed weak lactate oxidation. The two isolates contained unsaturated C_18:1ω7c fatty acid as the major fatty acid, and were unique in the presence of a considerable amount of straight-chain C_18:12OH fatty acid. Q-10 was present as the major isoprenoid quinone. Neokomagataea gen. nov. was proposed with the two species, Neokomagataea thailandica sp. nov. for isolate AH11^T (=BCC 25710 ^T =NBRC 106555^T), which has 56.8 mol % G+C, and Neokomagataea tanensis sp. nov. for isolate AH13^T (=BCC 25711^T=NBRC 106556^T), which has 51.2 mol % G+C.     

Tropical bloom-forming mesoalgae Cladophoropsis sp. and Laurencia sp.—responses to ammonium enrichment and a simulated heatwave

Algal blooms are increasing worldwide, driven by elevated nutrient inputs. However, it is still unknown how tropical benthic algae will respond to heatwaves, which are expected to be more frequent under global warming. In the present study, a multifactorial experiment was carried out to investigate the potential synergistic effects of increased ammonium inputs (25 μM, control at 2.5 μM) and a heatwave (31°C, control at 25°C) on the growth and physiology (e.g., ammonium uptake, nutrient assimilation, photosynthetic performance, and pigment concentrations) of two bloom-forming algal species, Cladophoropsis sp. and Laurencia sp. Both algae positively responded to elevated ammonium concentrations with higher growth and chlorophyll a and lutein concentrations. Increased temperature was generally a less important driver, interacting with elevated ammonium by decreasing the algaes' %N content and N:P ratios. Interestingly, this stress response was not captured by the photosynthetic yield (Fv/Fm) nor by the carbon assimilation (%C), which increased for both algae at higher temperatures. The negative effects of higher temperature were, however, buffered by nutrient inputs, showing an antagonistic response in the combined treatment for the concentration of VAZ (violaxanthin, antheraxanthin, zeaxanthin) and thalli growth. Ammonium uptake was initially higher for Cladophoropsis sp. and increased for Laurencia sp. over experimental time, showing an acclimation capacity even in a short time interval. This experiment shows that both algae benefited from increased ammonium pulses and were able to overcome the otherwise detrimental stress of increasingly emerging temperature anomalies, which provide them a strong competitive advantage and might support their further expansions in tropical marine systems.     

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.     

Purification and Characterization of a Novel α-Agarase from a Thalassomonas sp.

An agar-degrading Thalassomonas bacterium, strain JAMB-A33, was isolated from the sediment off Noma Point, Japan, at a depth of 230 m. A novel -agarase from the isolate was purified to homogeneity from cultures containing agar as a carbon source. The molecular mass of the purified enzyme, designated as agaraseA33, was 85 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that it is a monomer. The optimal pH and temperature for activity were about 8.5 and 45°C, respectively. The enzyme had a specific activity of 40.7 U/mg protein. The pattern of agarose hydrolysis showed that the enzyme is an endo-type -agarase, and the final main product was agarotetraose. The enzyme degraded not only agarose but also agarohexaose, neoagarohexaose, and porphyran.     

Description of Ulvella elegans sp. nov. and U. islandica sp. nov. (Ulvellaceae,Ulvophyceae) from Iceland – a study based on morphology of species in culture and tufA gene sequences

Two new Ulvella species, U. elegans R. Nielsen & K. Gunnarsson and U. islandica R. Nielsen & K. Gunnarsson are described. These microfilamentous marine green algae were found in the sublittoral zone in northern Iceland, epiphytic on Euthora cristata and associated with a calcareous polychaete tube, respectively. Unialgal cultures were established from field-collected material for morphological observations. In culture, Ulvella elegans was characterized by rosettes of monostromatic pseudoparenchyma consisting of radiating filaments with a margin of mutually free filaments. Each cell had one pyrenoid. Hairs were not observed. Ulvella islandica had a heterotrichous morphology, consisting of dense tufts of upright broad branches and much narrower, rhizoid-like branches. Acrochaete-type hairs occurred; these are hyaline non-septate merocytic extensions from a more or less bulbous base, which may be separated from the vegetative cell below. Most cells had one pyrenoid except for a few broad cells which had two or three. In a phylogenetic reconstruction based on the chloroplast-encoded tufA gene, the sequences for the two species were clearly distinct from any other Ulvella sequence available for this gene. Ulvella islandica was placed in a clade together with U. lens, U. wittrockii, U. reticulata and U. pseudorepens. Ulvella elegans occupied a branch deep in the phylogeny but the position was poorly supported.     

Morphology and molecular phylogeny of Nitzschia bizertensis sp. nov.—A new domoic acid-producer

A new toxin-producing marine diatom, Nitzschia bizertensis sp. nov., isolated from the Bizerte Lagoon (Tunisia, Southwest Mediterranean Sea) is, based on studies on eight different strains, characterized morphologically by light microscopy, transmission and scanning electron microscopy, and phylogenetically using the nuclear rDNA regions: SSU, ITS1, 5.8S, ITS2 and D1–D3 of the LSU. The species belongs to the sections Lanceolatae or Lineares as defined by Cleve and Grunow (1880). These sections are characterized by species having linear-lanceolate valves with an eccentric raphe where the fibulae does not extend into the valve, and are otherwise famous for the lack of characters useful for delineation of species. Nitzschia bizertensis differs from most other species in these sections by having a high density of interstriae. The morphological and phylogenetic studies and comparisons with previously described Nitzschia species showed Nitzschia bizertensis sp. nov. to be a new species. Batch culture experiments were conducted for estimations of maximum growth rate and production of domoic acid (DA). Maximum cellular DA content of the examined strains ranged from 2 × 10⁻⁴ to 3.6 × 10⁻² pg cells⁻¹. The total DA concentration (pg mL⁻¹) was high already in exponential growth phase maybe due to reinoculation of “old” stationary phase cells, and increased into stationary growth phase where it reached a stationary level varying among the strains from ca. 4500 to 9500 pg mL⁻¹. Nitzschia bizertensis represents a new domoic acid-producing diatom and is the second toxin producing Nitzschia species. The resolution of Nitzschia bizertensis and Nitzschia navis-varingica in different parts of the LSU phylogenetic tree, and the recovery of the Pseudo-nitzschia species phylogenetically distant from those two species suggests that the ability to produce DA either evolved multiple times independently or was lost multiple times.     

Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

Comparative paleohistology in osteoderms of Pleistocene Panochthus sp. Burmeister, 1886 and Neuryurus sp. (Xenarthra,Glyptodontidae)

Xenarthran osteoderms are integumentary bones with high fossilization potential presenting a high degree of morphological and histological diversity. Here, new data on the osteoderms histology of two glyptodonts, Panochthus and Neuryurus are presented. The poor spatial organization of the mineralized fibers and a large trabecular area in the middle zone identified in Neuryurus indicate a different bone pattern than the one found in Panochthus, which is mainly characterized by a middle zone with less spongiosa. Through the Bone Profiler program, the degree of compactness of the specimens was obtained, with about 70% for Neuryurus sp. and approximately 90% for Panochthus sp., showing the difference in bone pattern. These values confirm the visible difference in the histological patterns of these taxa, especially in the middle zone. This work demonstrates the microstructural variation studied in osteoderms and shows the importance of paleohistology as a starting point for a better understanding of extinct taxa.