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1.
Microorganisms ; 10(8)2022 Jul 27.
Article in English | MEDLINE | ID: mdl-36013937

ABSTRACT

Four aerobic bacteria with bacteriolytic capabilities were isolated from the brackish water site Strait Uzynaral of Lake Balkhash in Kazakhstan. The morphology and physiology of the bacterial isolates have subsequently been analyzed. Using matrix assisted laser desorption ionization-time of flight mass spectrum and partial 16S rRNA gene sequence analyses, three of the isolates have been identified as Pseudomonas veronii and one as Paenibacillus apiarius. We determined the capability of both species to lyse pre-grown cells of the Gram-negative strains Pseudomonas putida SBUG 24 and Escherichia coli SBUG 13 as well as the Gram-positive strains Micrococcus luteus SBUG 16 and Arthrobacter citreus SBUG 321 on solid media. The bacteriolysis process was analyzed by creating growth curves and electron micrographs of co-cultures with the bacteriolytic isolates and the lysis sensitive strain Arthrobacter citreus SBUG 321 in nutrient-poor liquid media. One metabolite of Paenibacillus apiarius was isolated and structurally characterized by various chemical structure determination methods. It is a novel antibiotic substance.

3.
Appl Microbiol Biotechnol ; 105(1): 401-415, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33219393

ABSTRACT

The yeast strain Moniliella spathulata SBUG-Y 2180 was isolated from oil-contaminated soil at the Tengiz oil field in the Atyrau region of Kazakhstan on the basis of its unique ability to use crude oil and its components as the sole carbon and energy source. This yeast used a large number of hydrocarbons as substrates (more than 150), including n-alkanes with chain lengths ranging from C10 to C32, monomethyl- and monoethyl-substituted alkanes (C9-C23), and n-alkylcyclo alkanes with alkyl chain lengths from 3 to 24 carbon atoms as well as substituted monoaromatic and diaromatic hydrocarbons. Metabolism of this huge range of hydrocarbon substrates produced a very large number of aliphatic, alicyclic, and aromatic acids. Fifty-one of these were identified by GC/MS analyses. This is the first report of the degradation and formation of such a large number of compounds by a yeast. Inoculation of barley seeds with M. spathulata SBUG-Y 2180 had a positive effect on shoot and root development of plants grown in oil-contaminated sand, pointing toward potential applications of the yeast in bioremediation of polluted soils. KEY POINTS: • Moniliella spathulata an oil-degrading yeast • Increase of the growth of barley.


Subject(s)
Hordeum , Petroleum , Soil Pollutants , Basidiomycota , Biodegradation, Environmental , Hydrocarbons , Saccharomyces cerevisiae , Soil
4.
ACS Omega ; 5(24): 14324-14339, 2020 Jun 23.
Article in English | MEDLINE | ID: mdl-32596570

ABSTRACT

The green and environmentally friendly synthesis of highly valuable organic substances is one possibility for the utilization of laccases (EC 1.10.3.2). As reactants for the herein described syntheses, different o-substituted arylamines or arylthiols and 2,5-dihydroxybenzoic acid and its derivatives were used. In this way, the formation of phenothiazines, phenoxazines, and phenazines was achieved in aqueous solution mediated by the laccase of Pycnoporus cinnabarinus in the presence of oxygen. Two types of phenothiazines (3-hydroxy- and 3-oxo-phenothiazines) formed in one reaction assay were described for the first time. The cyclization reactions yielded C-N, C-S, or C-O bonds. The syntheses were investigated with regard to the substitution pattern of the reaction partners. Differences in C-S and C-N bond formations without cyclization are discussed.

5.
Appl Microbiol Biotechnol ; 104(8): 3569-3583, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32125477

ABSTRACT

Comparative analyses determined the relationship between the structure of bisphenol A (BPA) as well as of seven bisphenol analogues (bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), bisphenol F (BPF), bisphenol Z (BPZ), bisphenol AP (BPAP), bisphenol PH (BPPH)) and their biotransformability by the biphenyl-degrading bacterium Cupriavidus basilensis SBUG 290. All bisphenols were substrates for bacterial transformation with conversion rates ranging from 6 to 98% within 216 h and 36 different metabolites were characterized. Transformation by biphenyl-grown cells comprised four different pathways: (a) formation of ortho-hydroxylated bisphenols, hydroxylating either one or both phenols of the compounds; (b) ring fission; (c) transamination followed by acetylation or dimerization; and (d) oxidation of ring substituents, such as methyl groups and aromatic ring systems, present on the 3-position. However, the microbial attack of bisphenols by C. basilensis was limited to the phenol rings and its substituents, while substituents on the carbon bridge connecting the rings were not oxidized. All bisphenol analogues with modifications at the carbon bridge could be oxidized up to ring cleavage, while substituents at the 3-position of the phenol ring other than hydroxyl groups did not allow this reaction. Replacing one methyl group at the carbon bridge of BPA by a hydrophobic aromatic or alicyclic ring system inhibited both dimerization and transamination followed by acetylation. While most of the bisphenol analogues exhibited estrogenic activity, four biotransformation products tested were not estrogenically active.


Subject(s)
Benzhydryl Compounds/metabolism , Biotransformation , Cupriavidus/metabolism , Benzhydryl Compounds/classification , Cyclohexanes/metabolism , Phenols/metabolism , Soil Microbiology , Structure-Activity Relationship , Tandem Mass Spectrometry
6.
Appl Microbiol Biotechnol ; 103(17): 7261-7274, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31346684

ABSTRACT

Bacteria and fungi were isolated from eight different soil samples from different regions in Kazakhstan contaminated with oil or salt or aromatic compounds. For the isolation of the organisms, we used, on the one hand, typical hydrocarbons such as the well utilizable aliphatic alkane tetradecane, the hardly degradable multiple-branched alkane pristane, and the biaromatic compound biphenyl as enrichment substrates. On the other hand, we also used oxygenated derivatives of alicyclic and monoaromatic hydrocarbons, such as cyclohexanone and p-tert-amylphenol, which are known as problematic pollutants. Seventy-nine bacterial and fungal strains were isolated, and 32 of them that were clearly able to metabolize some of these substrates, as tested by HPLC-UV/Vis and GC-MS analyses, were characterized taxonomically by DNA sequencing. Sixty-two percent of the 32 isolated strains from 14 different genera belong to well-described hydrocarbon degraders like some Rhodococci as well as Acinetobacter, Pseudomonas, Fusarium, Candida, and Yarrowia species. However, species of the bacterial genus Curtobacterium, the yeast genera Lodderomyces and Pseudozyma, as well as the filamentous fungal genera Purpureocillium and Sarocladium, which have rarely been described as hydrocarbon degrading, were isolated and shown to be efficient tetradecane degraders, mostly via monoterminal oxidation. Pristane was exclusively degraded by Rhodococcus isolates. Candida parapsilosis, Fusarium oxysporum, Fusarium solani, and Rhodotorula mucilaginosa degraded cyclohexanone, and in doing so accumulate ε-caprolactone or hexanedioic acid as metabolites. Biphenyl was transformed by Pseudomonas/Stenotrophomonas isolates. When p-tert-amylphenol was used as growth substrate, none of the isolated strains were able to use it.


Subject(s)
Bacteria/metabolism , Fungi/metabolism , Hydrocarbons/metabolism , Petroleum/microbiology , Soil Microbiology , Soil Pollutants/metabolism , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Biodegradation, Environmental , Fungi/classification , Fungi/genetics , Fungi/isolation & purification
7.
Appl Microbiol Biotechnol ; 103(10): 4137-4151, 2019 May.
Article in English | MEDLINE | ID: mdl-30941461

ABSTRACT

The cycloalkanes, comprising up to 45% of the hydrocarbon fraction, occur in crude oil or refined oil products (e.g., gasoline) mainly as alkylated cyclohexane derivatives and have been increasingly found in environmental samples of soil and water. Furthermore, short-chain alkylated cycloalkanes are components of the so-called volatile organic compounds (VOCs). This study highlights the biotransformation of methyl- and ethylcyclohexane by the alkane-assimilating yeast Candida maltosa and the phenol- and benzoate-utilizing yeast Trichosporon mucoides under laboratory conditions. In the course of this biotransformation, we detected 25 different metabolites, which were analyzed by HPLC and GC-MS. The biotransformation process of methylcyclohexane in both yeasts involve (A) ring hydroxylation at different positions (C2, C3, and C4) and subsequent oxidation to ketones as well as (B) oxidation of the alkyl side chain to hydroxylated and acid products. The yeast T. mucoides additionally performs ring hydroxylation at the C1-position and (C) oxidative decarboxylation and (D) aromatization of cyclohexanecarboxylic acid. Both yeasts also oxidized the saturated ring system and the side chain of ethylcyclohexane. However, the cyclohexylacetic acid, which was formed, seemed not to be substrate for aromatization. This is the first report of several new transformation reactions of alkylated cycloalkanes for eukaryotic microorganisms.


Subject(s)
Candida/metabolism , Cyclohexanes/metabolism , Metabolic Networks and Pathways , Trichosporon/metabolism , Biotransformation , Chromatography, High Pressure Liquid , Environmental Pollutants/metabolism , Gas Chromatography-Mass Spectrometry
8.
AMB Express ; 8(1): 28, 2018 Feb 24.
Article in English | MEDLINE | ID: mdl-29478084

ABSTRACT

The inadequate removal of pharmaceuticals and other micropollutants in municipal wastewater treatment plants, as evidenced by their detection of these substances in the aquatic environment has led to the need for sustainable remediation strategies. Laccases possess a number of advantages including a broad substrate spectrum. To identify promoting or inhibitory effects of reaction partners in the remediation processes we tested not only single compounds-as has been described in most studies-but also mixtures of pollutants. The reaction of diclofenac (DCF) and flufenamic acid (FA), mediated by Trametes versicolor laccase resulted in the formation of products, which were more hydrophilic than the respective reactant (reactant concentration of 0.1 mM; laccase activity 0.5 U/ml). Analyses (HPLC, LC/MS) showed that the product 1a and 1b for DCF and FA, respectively, to be a para-benzoquinone imine derivative. The formation of 1a was enhanced by the addition of bisphenol A (BPA). After 6 days 97% more product was formed in the mixture of DCF and BPA compared with DCF tested alone. Product 1a was also detected in experiments with micropollutant-supplemented secondary effluent. Within 24 h 67% and 100% of DCF and BPA were transformed, respectively (25 U/ml). Experiments with a membrane reactor (volume 10 l; phosphate buffer, pH 7) were in good agreement with the results of the laboratory scale experiments (50 ml). EC50-values were also determined. The data support the use of laccases for the removal or detoxification of recalcitrant pollutants. Thus, the enzyme laccase may be a component of an additional environmentally friendly process for the treatment stage of wastewater remediation.

9.
Appl Microbiol Biotechnol ; 102(1): 345-354, 2018 Jan.
Article in English | MEDLINE | ID: mdl-29082419

ABSTRACT

The strain Phlebia tremellosa SBUG 1630 isolated from a thatched roof in Northern Germany is capable of colonizing and degrading effectively the water reed Phragmites communis. Within 96 h after inoculation, mycelia covered both the outer and the inner surface of reed shoot fragments as observed by scanning electron microscopy. Interestingly, top culm sections and culm edges were particularly susceptible towards fungal degradation. The weight loss of culms reached 20-73% depending on the environmental conditions applied during the incubation of 70 days. Reed degradation was stable at pH 4 to pH 8 and optimal between 25 and 30 °C. Short-term incubation at elevated temperatures (37 to 55 °C) affected the fungal reed degradation to only a minor extent, whereas > 18 h at 55 °C completely inhibited fungal growth and reed degradation. Supplementation with 43 mM NH4Cl enhanced the reed degradation up to 9%. In contrast, the addition of diammonium tartrate increased the weight loss of the samples considerably up to 16% at 344 mM. Furthermore, reed degradation by P. tremellosa was increased by supplementing the test medium with Mn (99 to 1584 µM), Cu (150 to 300 µM), and less significantly phosphate (4 mM), Zn (37 to 74 µM), and Ag (76 µM) after 70 days. In addition, activities of the ligninolytic enzymes laccase (max. 27.4 nmol ml-1 min-1) and lignin peroxidase (max. 22.8 nmol ml-1 min-1) were rather low in nitrogen-limited medium, whereas considerably higher levels of manganese peroxidase (max. 635.9 nmol ml-1 min-1) were observed.


Subject(s)
Poaceae/microbiology , Polyporales/physiology , Ammonium Chloride/pharmacology , Biodegradation, Environmental , Germany , Hydrogen-Ion Concentration , Laccase/metabolism , Lignin/metabolism , Microscopy, Electron, Scanning , Peroxidases/metabolism , Poaceae/drug effects , Poaceae/metabolism , Poaceae/ultrastructure , Polyporales/ultrastructure , Temperature , Water
10.
Front Microbiol ; 8: 1777, 2017.
Article in English | MEDLINE | ID: mdl-28966611

ABSTRACT

Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (Km -0.7 ± 0.2 mM, kcat -42.0 ± 8.2 s-1) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (Km -3.2 ± 0.2 mM, kcat -44.0 ± 3.2 s-1). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [Δagdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be not the only degradation pathway.

11.
Appl Microbiol Biotechnol ; 101(9): 3743-3758, 2017 May.
Article in English | MEDLINE | ID: mdl-28050635

ABSTRACT

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.


Subject(s)
Benzhydryl Compounds/metabolism , Cupriavidus/metabolism , Estrogens, Non-Steroidal/metabolism , Phenols/metabolism , Biotransformation , Carbon/metabolism , Cupriavidus/growth & development , Metabolic Networks and Pathways
12.
Microb Cell Fact ; 15(1): 175, 2016 Oct 12.
Article in English | MEDLINE | ID: mdl-27733155

ABSTRACT

BACKGROUND: The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation. RESULTS: In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs' (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000 U/L and 500 U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400 U/L and 200 U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and Δaadh2 mutant cultures had the effect of accelerating 1-butanol consumption. CONCLUSIONS: Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation.


Subject(s)
1-Butanol/metabolism , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Metabolic Networks and Pathways/genetics , Yeasts/enzymology , Alcohol Dehydrogenase/isolation & purification , Carbon/metabolism , Ethanol/metabolism , Gene Expression , Gene Knockout Techniques , NAD/metabolism , Yeasts/genetics , Yeasts/growth & development , Yeasts/metabolism
13.
J Basic Microbiol ; 56(11): 1252-1273, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27624187

ABSTRACT

Heavy contamination of soils by crude oil is omnipresent in areas of oil recovery and exploitation. Bioremediation by indigenous plants in cooperation with hydrocarbon degrading microorganisms is an economically and ecologically feasible means to reclaim contaminated soils. To study the effects of indigenous soil bacteria capable of utilizing oil hydrocarbons on biomass production of plants growing in oil-contaminated soils eight bacterial strains were isolated from contaminated soils in Kazakhstan and characterized for their abilities to degrade oil components. Four of them, identified as species of Gordonia and Rhodococcus turned out to be effective degraders. They produced a variety of organic acids from oil components, of which 59 were identified and 7 of them are hitherto unknown acidic oil metabolites. One of them, Rhodococcus erythropolis SBUG 2054, utilized more than 140 oil components. Inoculating barley seeds together with different combinations of these bacterial strains restored normal growth of the plants on contaminated soils, demonstrating the power of this approach for bioremediation. Furthermore, we suggest that the plant promoting effect of these bacteria is not only due to the elimination of toxic oil hydrocarbons but possibly also to the accumulation of a variety of organic acids which modulate the barley's rhizosphere environment.


Subject(s)
Hordeum/growth & development , Petroleum Pollution , Petroleum/metabolism , Soil Microbiology , Biodegradation, Environmental , Biomass , Environmental Pollutants , Gordonia Bacterium/isolation & purification , Gordonia Bacterium/metabolism , Hydrocarbons/metabolism , Kazakhstan , Petroleum/microbiology , Plant Roots/microbiology , Rhizosphere , Rhodococcus/isolation & purification , Rhodococcus/metabolism , Seeds/growth & development , Seeds/microbiology , Soil Pollutants/chemistry
14.
Chemosphere ; 162: 208-21, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27497351

ABSTRACT

Adding clay to marine oil pollution represents a promising approach to enhance bacterial hydrocarbon degradation in nutrient poor waters. In this study, three types of regionally available clays (Ca-bentonite, Fuller's Earth and kaolin) were tested to stimulate the biodegradation of source and weathered oil collected from the Deepwater Horizon spill. The weathered oil showed little biodegradation prior to experimentation and was extensively degraded by bacteria in the laboratory in a similar way as the alkane-rich source oil. For both oils, the addition of natural clay-flakes showed minor enhancement of oil biodegradation compared to the non-clay bearing control, but the clay-oil films did limit evaporation. Only alkanes of a molecular weight (MW) > 420 showed significant reduction by enhanced biodegradation following natural clay treatment. In contrast, all fertilized clay flakes showed major bacterial degradation of the oil, with a 6-10 times reduction in alkane content, and an up to 8 fold increase in the rate of O2 consumption. Compared to the control, such treatment showed particular reduction of longer chained alkanes (MW > 226). The application of natural and fertilized clay flakes also showed selective reduction of PAHs, mainly in the MW range of 200-300, but without significant change in the toxicity indices measured. These results imply that a large variety of clays may be used to boost oil biodegradation by aiding attachment of fertilizing nutrients to the oil.


Subject(s)
Aluminum Silicates/pharmacology , Bacteria/drug effects , Bacteria/metabolism , Hydrocarbons/metabolism , Petroleum Pollution , Biodegradation, Environmental/drug effects , Clay , Gulf of Mexico , Hydrocarbons/isolation & purification , Petroleum/analysis , Petroleum Pollution/analysis , Weather
15.
FEMS Yeast Res ; 16(3)2016 May.
Article in English | MEDLINE | ID: mdl-26912215

ABSTRACT

In this study, alcohol dehydrogenase 1 from Arxula adeninivorans (Aadh1p) was identified and characterized. Aadh1p showed activity with short and medium chain length primary alcohols in the forward reaction and their aldehydes in the reverse reaction. Aadh1p has 64% identity with Saccharomyces cerevisiae Adh1p, is localized in the cytoplasm and uses NAD(+) as cofactor. Gene expression analysis showed a low level increase in AADH1 gene expression with ethanol, pyruvate or xylose as the carbon source. Deletion of the AADH1 gene affects growth of the cells with 1-butanol, ethanol and glucose as the carbon source, and a strain which overexpressed the AADH1 gene metabolized 1-butanol more rapidly. An ADH activity assay indicated that Aadh1p is a major enzyme for the synthesis of ethanol and the degradation of 1-butanol in A. adeninivorans.


Subject(s)
1-Butanol/metabolism , Alcohol Dehydrogenase/metabolism , Ethanol/metabolism , Saccharomycetales/enzymology , Alcohol Dehydrogenase/genetics , Amino Acid Sequence , Carbon/metabolism , Coenzymes/metabolism , Cytoplasm/chemistry , Gene Expression Profiling , Molecular Sequence Data , NAD/metabolism , Pyruvic Acid/metabolism , Saccharomycetales/genetics , Sequence Homology, Amino Acid , Xylose/metabolism
16.
Appl Environ Microbiol ; 81(16): 5497-510, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-26048925

ABSTRACT

The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6× His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6× His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml(-1) could be achieved even with a nonoptimized cultivation procedure.


Subject(s)
Carboxylic Ester Hydrolases/isolation & purification , Carboxylic Ester Hydrolases/metabolism , Saccharomycetales/enzymology , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/genetics , Catalytic Domain , Chromatography, Affinity , Enzyme Stability/drug effects , Enzyme Stability/radiation effects , Gene Expression , Hydrogen-Ion Concentration , Industrial Waste , Malus/microbiology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Solvents , Substrate Specificity , Temperature
17.
Mar Biotechnol (NY) ; 17(3): 290-304, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25678259

ABSTRACT

The marine isolate Bacillus pumilus SBUG 1800 is able to lyse living cells of Arthrobacter citreus on solid media as well as pasteurized A. citreus cells in liquid mineral salt medium. The cultivation of B. pumilus in the presence of pasteurized A. citreus is accompanied by an enhanced production of 2,5-diketopiperazines (DKPs). DKPs inhibit bacterial growth, but do not seem to cause bacteriolysis. This study shows that B. pumilus also lyses living cells of A. citreus in co-culture experiments as an intraguild predator, even if the inoculum of B. pumilus is low. In order to characterize the bacteriolytic process, more precisely changes in the extracellular metabolome and proteome have been analyzed under different culture conditions. Besides the known DKPs, a number of different pumilacidins and bacteriolytic enzymes are produced. Two lipopeptides with [M + H](+) = 1008 and [M + H](+) = 1022 were detected and are proposed to be pumilacidin H and I. While the lipopeptides lyse living bacterial cells in lysis test assays, a set of extracellular enzymes degrades the dead cell material. Two of the cell wall hydrolases involved have been identified as N-acetylmuramoyl-L-alanine amidase and beta-N-acetylglucosaminidase. These findings together with electron microscopic and cell growth monitoring during co-culture experiments give a detailed view on the bacteriolytic process.


Subject(s)
Acetylglucosaminidase/isolation & purification , Anti-Bacterial Agents/biosynthesis , Arthrobacter/drug effects , Bacillus/metabolism , Bacteriolysis , N-Acetylmuramoyl-L-alanine Amidase/isolation & purification , Acetylglucosaminidase/biosynthesis , Acetylglucosaminidase/genetics , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Antibiosis , Arthrobacter/chemistry , Bacillus/genetics , Bacillus/pathogenicity , Bacillus/ultrastructure , Diketopiperazines/isolation & purification , Diketopiperazines/metabolism , Diketopiperazines/pharmacology , Gene Expression , Lipopeptides/biosynthesis , Lipopeptides/isolation & purification , Lipopeptides/pharmacology , Metabolome , N-Acetylmuramoyl-L-alanine Amidase/biosynthesis , N-Acetylmuramoyl-L-alanine Amidase/genetics , Peptides, Cyclic/biosynthesis , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Proteome/isolation & purification
18.
Appl Microbiol Biotechnol ; 99(9): 4071-84, 2015 May.
Article in English | MEDLINE | ID: mdl-25592733

ABSTRACT

Three microbial strains were isolated from the rhizosphere of alfalfa (Medicago sativa), grass mixture (Festuca rubra, 75 %; Lolium perenne, 20 %; Poa pratensis, 10 %), and rape (Brassica napus) on the basis of their high capacity to use crude oil as the sole carbon and energy source. These isolates used an unusually wide spectrum of hydrocarbons as substrates (more than 80), including n-alkanes with chain lengths ranging from C12 to C32, monomethyl- and monoethyl-substituted alkanes (C12-C23), n-alkylcyclo alkanes with alkyl chain lengths from 4 to 18 carbon atoms, as well as substituted monoaromatic and diaromatic hydrocarbons. These three strains were identified as Gordonia rubripertincta and Rhodococcus sp. SBUG 1968. During their transformation of this wide range of hydrocarbon substrates, a very large number of aliphatic, alicyclic, and aromatic acids was detected, 44 of them were identified by GC/MS analyses, and 4 of them are described as metabolites for the first time. Inoculation of plant seeds with these highly potent bacteria had a beneficial effect on shoot and root development of plants which were grown on oil-contaminated sand.


Subject(s)
Carboxylic Acids/metabolism , Gordonia Bacterium/metabolism , Petroleum/metabolism , Rhizosphere , Rhodococcus/metabolism , Soil Microbiology , Soil Pollutants/metabolism , Carbon/metabolism , Energy Metabolism , Environmental Pollution , Gas Chromatography-Mass Spectrometry , Gordonia Bacterium/classification , Gordonia Bacterium/isolation & purification , Kazakhstan , Plant Roots/microbiology , Rhodococcus/classification , Rhodococcus/isolation & purification
19.
AMB Express ; 4: 75, 2014.
Article in English | MEDLINE | ID: mdl-25309846

ABSTRACT

Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.

20.
Mar Biotechnol (NY) ; 16(4): 385-95, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24449388

ABSTRACT

We report the detection by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry analyses of the secreted 2,5-diketopiperazines (DKPs) cyclo(-Ala-Pro), cyclo(-Gly-Pro), cyclo(-Val-Pro), cyclo(-Ile-Pro), cyclo(-Leu-Pro), cyclo(-Pro-Pro), cyclo(-HyP-Pro), cyclo(-Met-Pro), and cyclo(-Phe-Pro) produced by Bacillus pumilus. The study focuses on a marine isolate and a laboratory test strain of B. pumilus with capabilities to lyse pregrown living cell lawns of different bacterial species, among them Arthrobacter citreus. Chromatographic methods were used to analyze induced bioactive compounds. At least 13 different DKPs are produced by B. pumilus. Both strains respond with an increased production of the DKPs cyclo(-Gly-Pro), cyclo(-Ala-Pro), and cyclo(-Val-Pro) to the presence of pasteurized A. citreus cells after 4 h in a nutrient-poor liquid medium. In agar diffusion assays, these DKPs did not cause lysis zones in living cell lawns, but they did inhibit further growth of several pregrown test bacteria in microplates even at concentrations as low as 1 µg ml(-1). Antibiotic substances produced by B. pumilus after 20 h of cultivation in a special lysis medium showed lytic activity in cell-free extracts of B. pumilus culture supernatants.


Subject(s)
Arthrobacter/physiology , Bacillus/metabolism , Bacteriolysis/physiology , Cell Communication/physiology , Diketopiperazines/chemistry , Diketopiperazines/metabolism , Aquatic Organisms/microbiology , Diketopiperazines/isolation & purification
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