Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

On the biosynthetic origin of methoxymalonyl-acyl carrier protein, the substrate for incorporation of "glycolate" units into ansamitocin and soraphen A

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.     

The phenol efficient degradation using co-culture coupled with enhanced electricity generation capability

The co-culture of strain Citrobacter sp. RDC and Geobacter sulfurreducens PCA was used in this study and it was found that the co-culture using 200 mg/L phenol as carbon source exhibited higher maximum current density than using the single strain RDC and G. sulfurreducens PCA, respectively. Meanwhile, the co-culture was used to generate electricity by degrading phenol with the current density of 699.07 μA/cm² by using 200 mg/L phenol as the sole carbon source, which was higher than that only using G. sulfurreducens PCA (236.20 μA/cm²). Especially, the degradation efficiency of 200 mg/L phenol by co-culture can reach 55.16 % within 36 h being 4.16-fold higher than the single strain G. sulfurreducens PCA. Furthermore, the component ratio of two strains was optimized for increasing electricity generation using 500 mg/L phenol as carbon source. The maximum current density was 501.54 μA/cm² under the ratio of 3 : 1 for strain RDC to G. sulfurreducens PCA. These results highlight that phenol is good carbon source for co-culture to produce electricity. The co-culture system provides a promising application pathway for phenol degradation treatment coupled with electricity generation in the future.     

Detection of phenolic compounds by thick film sensors based on Pseudomonas putida

Amperometric biosensors using bacterial cells were developed for the determination of phenolic compounds and the measurement was based on the respiratory activity of the cells. For this purpose, Pseudomonas putida DSM 50026 which is one of the well-known phenol degrading organisms, was used as a biological component. The cells were grown in the presence of phenol as the sole source of organic carbon. As well as phenol adapted cells, the bacterium which used the glucose as the major carbon source, was also used to obtain another type of biosensor for the comparison of the responses and specificities towards different xenobiotics. The commercial oxygen electrode was used as a transducer to test the sensor responses for both induced and non-induced cells. Our results showed that the adaptation step enable us to obtain biosensor devices with different substrate specificity. Moreover, P. putida was immobilized on the surface of thick film working electrodes made of gold by using gelatin membrane cross-linked with glutaraldehyde. The biosensors were calibrated for different phenolic substances. Furthermore, phenol detection was performed in synthetic wastewater samples.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Degradation bactérienne de l'acide dehydroabiétique par Flavobacterium resinovorum

Flavobacterium Resinovorum was grown on dehydroabietic acid 1a as sole carbon and energy source. Slowly growing cultures were obtained either by omission of nitrogen, Fe²⁺ and Mg²⁺ ions or in the presence of an inhibitor (α,α′-dipyridyle). Under these conditions, different intermediates of the metabolic pathway were accumulated. Diphenol diketone 6a has been isolated. The unique behaviour of bacterial attack at C-3 before the degradation of the cyclic system is confirmed.     

Desymmetrization of meso 1,3- and 1,4-diols with a dinuclear zinc asymmetric catalyst

A dinuclear asymmetric zinc catalyst generated by mixing a 2:1 ratio of diethylzinc and 2,6-bis[5-2-diarylhydroxy methyl-1-pyrrolidinyl]-4-methylphenol has been contrasted with enzymes for the desymmetrization of some meso diols. The best ligand has a p-biphenylyl group as the aromatic substituent defining the chiral space. A series of 2-substituted propanediols were examined. The best acyl transfer agent proved to be vinyl benzoate. Diacylation normally did not occur. The phenyl substituted substrate gave 91-95% ee which compares favorably with the best ee of 92% reported for an enzymatic desymmetrization. The methyl substituted substrate gave significantly better results with the dinuclear zinc catalyst (89% yield, 82% ee) as compared to the best enzymatic esterification (70% yield, 60% ee). One case of a 1,4-diol, cis-1,2-bis(hydroxymethyl) cyclohexane, also gave much better results with the dinuclear zinc catalysts (93% yield, 91% ee) as compared to the reported enzymatic process (44% yield, 7% ee). A model to rationalize the results is presented.     

Insights into the Role of Glucose and Glycerol as a Mixed Carbon Source in the Improvement of ε-Poly-l-Lysine Productivity

Using glucose–glycerol mixed carbon source has proved to be an effective strategy for ε-poly-l-lysine (ε-PL) production with rapid cell growth and much higher ε-PL productivity. In this study, we attempt to focus on key enzymes and intracellular energy cofactors to reveal the underlying mechanisms involved in such significant improvements. The activities of key enzymes involved in the pentose phosphate pathway, TCA cycle, anaplerotic pathway and the aspartate family amino acid biosynthesis pathway as well as ε-PL synthetase showed overall enhancement with the mixed carbon source, especially in the late stages of fermentation, compared with those in either glucose or glycerol single carbon sources. Moreover, the intracellular cofactors in terms of NADH and ATP kept higher formation and consumption rates in the mixed carbon source, respectively, throughout batch fermentation. As a result, Streptomyces sp. M-Z18 could be accelerated in cell growth and precursor l-lysine biosynthesis in the mixed carbon source, thus finally shortening fermentation time and enhancing ε-PL productivity. Understanding this process will provide information for the rational regulation of the metabolism network of the quantative production of ε-PL by metabolic engineering.     

Lactase Production by Solid-state Cultivation of Kluyveromyces marxianus CDBBL 278 on an Inert Support: Effect of Inoculum, Buffer, and Nitrogen Source

A study was carried out to select the conditions for cultivation of Kluyveromyces marxianus CDBBL 278 in solid-state culture (SSC) using polyurethane foam (PUF) as an inert support. PUF was impregnated with culture media containing lactose (50 g/L) as the carbon and energy source. Evaluation of culture parameters during different growth phases was carried out by respirometry. The effect of inoculum level, buffer capacity of the medium, and nitrogen source upon the yield of biomass on lactose (Yx/s) and production of lactase and inulinase was investigated. The highest lactase titre was achieved with an inoculum level of 1 x 10(7) cells per gram of wet matter (gwm) and 20% of the total nitrogen source provided as urea. The best biomass yield (0.37) was obtained when less than 40% of the total nitrogen was provided as urea. Using potassium phosphate allowed 90% substrate consumption in 30 h. In the best conditions, intracellular lactase and extracellular inulinase activities of 1147.7 IU/gX and 241.6 IU/gX were obtained, respectively, with a lag phase of 13.8 h and a rate of respiratory activity (microCO2) of 0.23 +/- 0.01 h(-1). To our knowledge, this is the first report on lactase production by K. marxianus CDBBL 278 in SSC. This study gives basic information about biomass yield and enzyme production using lactose as the sole carbon source in SSC on an inert support.     

Structural and spectroscopic properties of the peroxodiferric intermediate of Ricinus communis soluble Δ9 desaturase

Large-scale quantum and molecular mechanical methods (QM/MM) and QM calculations were carried out on the soluble Δ(9) desaturase (Δ(9)D) to investigate various structural models of the spectroscopically defined peroxodiferric (P) intermediate. This allowed us to formulate a consistent mechanistic picture for the initial stages of the reaction mechanism of Δ(9)D, an important diferrous nonheme iron enzyme that cleaves the C-H bonds in alkane chains resulting in the highly specific insertion of double bonds. The methods (density functional theory (DFT), time-dependent DFT (TD-DFT), QM(DFT)/MM, and TD-DFT with electrostatic embedding) were benchmarked by demonstrating that the known spectroscopic effects and structural perturbation caused by substrate binding to diferrous Δ(9)D can be qualitatively reproduced. We show that structural models whose spectroscopic (absorption, circular dichroism (CD), vibrational and M?ssbauer) characteristics correlate best with experimental data for the P intermediate correspond to the μ-1,2-O(2)(2-) binding mode. Coordination of Glu196 to one of the iron centers (Fe(B)) is demonstrated to be flexible, with the monodentate binding providing better agreement with spectroscopic data, and the bidentate structure being slightly favored energetically (1-10 kJ mol(-1)). Further possible structures, containing an additional proton or water molecule are also evaluated in connection with the possible activation of the P intermediate. Specifically, we suggest that protonation of the peroxide moiety, possibly preceded by water binding in the Fe(A) coordination sphere, could be responsible for the conversion of the P intermediate in Δ(9)D into a form capable of hydrogen abstraction. Finally, results are compared with recent findings on the related ribonucleotide reductase and toluene/methane monooxygenase enzymes.     

Synthesis gas as substrate for the biological production of fuels and chemicals

Synthesis gas provides a simple substrate for the production of fuels and chemicals. Synthesis gas can be produced via established technologies from a variety of feedstocks including coal, wood, and agricultural and municipal wastes. The gasification is thermally efficient and results in complete conversion of the feedstock to fermentable substrate.Clostridium ljungdahlii grows on the synthesis gas components, carbon monoxide, hydrogen, and carbon dioxide. Production of acetic acid and ethanol accompanies growth with synthesis gas as sole source of energy and carbon. Rate and yield parameters are compared forC. ljungdahlii grown on carbon monoxide, or hydrogen with carbon dioxide.

     

Microbial reclamation of squid pens and shrimp shells

Byproducts generated in high levels by marine processes have been recognized for their value as recyclable or reclaimable waste. Among marine byproducts, shrimp shells, crab shells, and squid pens have the highest chitin content. The chemical treatments of these chitin-containing byproducts for preparing chitin and chitosan create waste disposal problems because neutralization and detoxification of the discharged wastewater are necessary. Therefore, the cost of chitin and chitosan preparations was far higher than those of their raw materials, marine chitin-containing byproducts. Chitin and chitosan have been widely used as the major carbon source of bacteria for producing chitinolytic enzymes. In 1997, the bifunctional chitinase/lysozymes from Pseudomonas aeruginosa K-187 using shrimp and crab shells as the sole carbon/nitrogen (C/N) source was first reported. Thereafter, the use of squid pens as the only C/N source for producing enzymes and bioactive materials had also been studied. The use of shellfish chitin waste as the sole C/N source not only solves environmental problems, it decreases the production costs for microbial conversion. This review summarizes our recent research of microbial reclamation of these marine byproducts for producing enzymes and bioactive materials; the characterization and applications of these products were also studied.     

Metabolic Behavior and Enzymatic Aspects of Denitrifying EBPR Sludge in a Continuous-Flow Anaerobic–Anoxic System

The metabolic aspects of enhanced biological phosphorus removal (EBPR) were investigated for the first time in a continuous-flow anaerobic–anoxic plant fed with acetate, propionate, or substrates which are involved in the tricarboxylic acid and/or glyoxylate cycle, i.e., fumarate, malate, or oxaloacetate, as the sole carbon source. Although the polyphosphate-accumulating organisms (PAOs) population remained stable with any carbon source examined, no typical EBPR metabolism was observed during fumarate, malate, or oxaloacetate utilization. Specific enzymatic activities related to EBPR were determined in activated sludge homogenates and directly correlated with the nutrient metabolic rates. The experimental results indicated the direct involvement of alkaline phosphatase, pyrophosphatase, and exopolyphosphatase in the denitrifying EBPR process. Metabolic aspects of glyoxylate cycle enzymes are discussed with regard to the biomass anaerobic and anoxic activity. Process performance was highly influenced by the kind of substrate utilized, indicating that specific metabolic pathways should be followed to favor efficient EBPR.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

The conformational free-energy landscape of β-D-mannopyranose: evidence for a (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary in β-mannosidases

The mechanism of glycosidic bond cleavage by glycosidases involves substrate ring distortions in the Michaelis complex that favor catalysis. Retaining β-mannosidases bind the substrate in a (1)S(5) conformation, and recent experiments have proposed an unusual substrate conformational pathway ((1)S(5) → B(2,5) → (O)S(2)) for the hydrolysis reaction. By means of Car-Parrinello metadynamics simulations, we have obtained the conformational free-energy surface (FES) of a β-d-mannopyranose molecule associated with the ideal Stoddart conformational diagram. We have found that (1)S(5) is among the most stable conformers and simultaneously is the most preactivated conformation in terms of elongation/shortening of the C1-O1/C1-O5 bonds, C1-O1 orientation, and charge development at the anomeric carbon. Analysis of the computed FES gives support to the proposed (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary, showing that the degree of preactivation of the substrate in glycoside hydrolases (GHs) is related to the properties of an isolated sugar ring. We introduce a simple preactivation index integrating several structural, electronic, and energetic properties that can be used to predict the conformation of the substrate in the Michaelis complex of any GH.     

Degradation of Organic Cyanides byPseudomonas aeruginosa

A bacterium capable of utilizing acetonitrile (methyl cyanide) as the sole source of carbon and nitrogen was isolated from soil and identified asPseudomonas aeruginosa. This bacterium could also utilize and oxidize numerous lower-mol-wt nitrile compounds and their corresponding amides as growth substrates. A metabolite of acetonitrile in the culture medium was determined to be ammonia. The accumulation of ammonia in the culture medium was proportional to the concentration of the substrate and the inoculum. Cell extracts of the bacterium contained activities corresponding to nitrile aminohydrolase (E C 3.5.5.1) and amidase (E C 3.5.1.4), which regulate the degradation of acetonitrile. Both enzymes were inducible and hydrolyzed a wide range of substrates, and it was determined that the specific activity of amidase was far greater than the activity of nitrile aminohydrolase.     

Thermodynamics of Inter- and Intramolecular Hydrogen Bonding in (Diphosphine)platinum(II) Diolate Complexes and Its Role in Carbohydrate Complexation Regioselectivity

Thermodynamic data are reported for intermolecular hydrogen-bonding association of 1 and 2 equiv of phenol with [1,3-bis(diphenylphosphino)propane](phenylethane-1,2-diolato)platinum(II) ((dppp)Pt(Ped)) in dichloromethane solution: = -7.0 +/- 0.1 kcal/mol, = -7.7 +/- 0.4 kcal/mol, = -11.3 +/- 0.4 eu, and = -17.8 +/- 1.2 eu. For comparison, the thermodynamics for hydrogen bonding of phenol to triphenylphosphine oxide in dichloromethane were also determined: DeltaH degrees = -5.1 +/- 0.3 kcal/mol; DeltaS degrees = -8.8 +/- 1.0 eu. Competitive coordination exchange reactions have been used to determine the apparent intramolecular hydrogen bond strengths in (dppp)Pt(1,2-O,O'-glycerolate) and (dppp)Pt(1,2-O,O'-butane-1,2,4-triolate) in both dichloromethane (DeltaG(313) = -2.05 +/- 0.05 and -2.52 +/- 0.06 kcal/mol, respectively) and pyridine (DeltaG(313) = -0.62 +/- 0.03 and -0.82 +/- 0.03 kcal/mol, respectively). Based on these findings, the role of hydrogen-bonding interactions in determining the regioselectivities of complexation of carbohydrates to diphosphine Pt(II) is discussed.     

Geminal 2JCCH spin-spin coupling constants as probes of the phi glycosidic torsion angle in oligosaccharides

Two-bond (13)C-(1)H NMR spin-spin coupling constants ((2)J(CCH)) between C2 and H1 of aldopyranosyl rings depend not only on the relative orientation of electronegative substituents on the C1-C2 fragment but also on the C-O torsions involving the same carbons. The latter dependencies were elucidated theoretically using density functional theory and appropriate model pyranosyl rings representing the four relative configurations at C1 and C2, and a 2-deoxy derivative, to probe the relationship between (2)J(C2,H1) magnitude and sign and the C1-O1 (phi, phi) and C2-O2 (alpha) torsion angles. Related calculations were also conducted for the reverse coupling pathway, (2)J(C1,H2). Computed J-couplings were validated by comparison to experimentally measured couplings. (2)J(CCH) displays a primary dependence on the C-O torsion involving the carbon bearing the coupled proton and a secondary dependence on the C-O torsion involving the coupled carbon. These dependencies appear to be caused mainly by the effects of oxygen lone pairs on the C-H and C-C bond lengths along the C-C-H coupling pathway. New parameterized equations are proposed to interpret (2)J(C1,H2) and (2)J(C2,H1) in aldopyranosyl rings. The equation for (2)J(C2,H1) has particular value as a potential NMR structure constraint for the C1-O1 torsion angle (phi) comprising the glycosidic linkages of oligosaccharides.     

Products from enzyme-catalyzed oxidations of norcarenes

Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPDelta2E1, CYPDelta2E1 T303A, and CYPDelta2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn- and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations.