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The yeast strains IPM32-16, ISM28-8sT, and IPM46-17, isolated from plant and soil samples from Iriomote Island, Japan, were explored in terms of lipid production during growth in a mixture of glucose and xylose. Phylogenetically, the strains were most closely related to Cystobasidium slooffiae, based on the sequences of the ITS regions and the D1/D2 domain of the LSU rRNA gene. The strains were oleaginous, accumulating lipids to levels > 20% dry cell weight. Moreover, kinetic analysis of the sugar-to-lipid conversion of a 1:1 glucose/xylose mixture showed that the strains consumed the two sugars simultaneously. IPM46-17 attained the highest lipid content (33%), mostly C16 and C18 fatty acids. Thus, the yeasts efficiently converted lignocellulosic sugars to lipids, aiding in biofuel production (which benefits the environment, promotes rural jobs, and strengthens fuel security). The strains constituted a novel species of Cystobasidium, for which we propose the name Cystobasidium iriomotense (type strain ISM28-8sT = JCM 24594T = CBS 15015T). [ABSTRACT FROM AUTHOR]

Thirty-four strains of docosahexaenoic acid (DHA)-producing microorganisms were newly isolated from brackish areas in Japan. These strains showing various compositions of fatty acids. Especially, the fatty acids produced by one of the strains, named D31, had a high DHA content (over 60 % of the total fatty acids) and the simple fatty acid composition (16:0, 18:0, 18:1 and DHA without any other polyunsaturated acids). Although most oleaginous microorganisms accumulate DHA as triacylglycerol, the strain D31 accumulated DHA mainly as a polar lipid (79.4 % of total DHA), especially as phosphatidylcholine (71.4 % of polar DHA). This strain D31 was identified as a related species of Crypthecodinium cohnii on the basis of phylogenetic analysis. Crypthecodinium sp. D31 showed high DHA productivity when cultivated in a medium containing glycerol as the carbon source and a mixture of yeast extract and polypeptone as the nitrogen sources, with a salinity that was equivalent to 50 % of that of seawater and a pH in the acidic range (

A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids. [ABSTRACT FROM AUTHOR]

Imidase, which preferably hydrolyzed cyclic imides to monoamidated dicarboxylates, was purified to homogeneity from a cell-free extract of Blastobacter sp. A17p-4. Cyclic imides are known to be hydrolyzed by mammalian dihydropyrimidinases. However. imidase was quite different from known dihydropyrimidinases in structure and substrate specificity. The enzyme has a relative molecular mass of 105000 and consists of three identical subunits. The purified enzyme showed higher activity and affinity toward cyclic imides, such as succinimide (Km = 0.94 mM: Vmax = 910 µmol -min-1 mg-1), glutarimide (Km, = 4.5 mM: Vmax = 1000 µmol . min-1 mg-1) and maleimide (Km = 0.34 mM: Vmax = 5800 µmol min-1 . mg-1), than toward cyclic urcides, which arc the substrates of dihydropyrimidinases, such as dihydrouracil and hydantoin. Sulfur-containing cyclic imides, such as 2.4-thiazolidinedione and rhodanine. were also hydrolyzed. The enzyme catalyzed the reverse reaction, cyclization, but with much lower activity and affinity. The enzyme was non-competitively inhibited by succinate, which was found to be a key compound in cyclic-imide transformation in relation with the tricarboxylic acid cycle in this bacterium, suggesting that the role of imidase is to catalyze the initial step of cyclic-imide degradation. [ABSTRACT FROM AUTHOR]

β-Ureidopropionase of aerobic bacterial origin was purified to homogeneity from Pseudomonas putida IFO 12996. The enzyme shows a broad substrate specificity. In addition to β-ureidopropionate (Km 3.74 mM, Vmax 4.12 U/mg). γ-ureido-n-butyrate (Km 11.6 mM, V max 19.4 U/mg), and several N-carbamoyl-α-amino acids, such as N-carbamoylglycine (Kmax 0.68 mM, Vmax 9.14x10-2U/mg). N-carbamoyl-L-alanine (Km 1.56 mM, Vmax 1,00 U/mg), N-carbamoyl-L-serine (Km 75.1 mM. Vmax 3.78 U/mg), and N-carbamoyl-DL-α-amino-n-butyrate (Km 2.81 mM. Vmax 1.08 U/mg), are also hydrolyzed. The hydrolysis of N-carbamoyl-α-amino acids is strictly L enantiomer specific. N-Formyl-c-alanine and N-acetyl-L-alanine are also hydrolyzed by the enzyme, but the rate of hydrolysis is lower than the rate for N-carbamoyl-L-alanine. The enzyme requires a divalent metal ion. such as Co2+, Ni2+ or Mn2+, for activity, and is significantly affected by sulfhydryl reagents. The enzyme consists of two polypeptide chains with identical relative molecular mass M1 45000. The broad substrate specificity and metal ion dependence of the enzyme show that the β-ureidopropionase of this aerobic bacterium is quite different from the β-ureidopropionases of mammals and anaerobic bacteria. [ABSTRACT FROM AUTHOR]

A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa-and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 μmol · min-1 · mg-1 at 30°C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiochotine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenyl-ethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+. [ABSTRACT FROM AUTHOR]