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Applied Microbiology and Biotechnology (v.53, #1)
Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus by J. Oddou; C. Stentelaire; L. Lesage-Meessen; M. Asther; B. Colonna Ceccaldi (pp. 1-6).
High-density cultures of Pycnoporus cinnabarinus were tested with a view to optimisation of ferulic acid bioconversion into vanillin. The dry weight was increased fourfold by using glucose, fructose or a mixture of glucose and phospholipids as carbon source instead of maltose, the carbon source previously used. 5 mmol l−1 vanillin, i.e. 760 mg l−1, was produced over 15 days with glucose-phospholipid medium. In contrast, formation of vanillin was lower using glucose or fructose compared to the maltose control. A bioreactor (2 l) with a glucose-phospholipid medium gave a molar yield of vanillin of 61% (4 mmol l−1). An alternative strategy was to grow the fungus on a glucose or fructose medium for 3 days, then switch to maltose during the bioconversion phase: this method allowed 3.3 mmol l−1 vanillin to be obtained in 10 days. Many by-products such as methoxyhydroquinone and vanillyl alcohol were also produced.
Resolution of 1,2-epoxyhexane by Rhodotorula glutinis using a two-phase membrane bioreactor by W. J. Choi; C. Y. Choi; J. A. M. De Bont; C. A. G. M. Weijers (pp. 7-11).
Large-scale resolution of epoxides by the yeast Rhodotorula glutinis was demonstrated in an aqueous/organic two-phase cascade membrane bioreactor. Due to the chemical instability and low solubility of epoxides in aqueous phases, an organic solvent was introduced into the reaction mixture in order to enhance the resolution of epoxide. A cascade hollow-fiber membrane bioreactor was used (1) to minimize the toxicity of organic solvents towards the epoxide hydrolase of R. glutinis, and (2) to remove inhibitory amounts of formed diol from the yeast cell containing aqueous phase. Dodecane was selected as a suitable solvent and 1,2-epoxyhexane as a model substrate. By use of this membrane bioreactor, highly concentrated (0.9 M in dodecane) enantiopure (> 98% ee) (S)-1,2-epoxyhexane (6.5 g, 30% yield) was obtained from the racemic mixture.
Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1 by P. Wahl; P. Walser-Volken; K. Laumen; M. Kittelmann; O. Ghisalba (pp. 12-18).
Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn2+ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure.
Inhibition of thermolysin by 3-trimethylsilylalanine derivatives by H. Ishikawa; H. Yamanaka; T. Kawamoto; A. Tanaka (pp. 19-22).
The inhibition of thermolysin by an optically active silicon-containing amino acid, 3-trimethylsilylalanine (TMS-Ala), and its derivatives was examined by considering the similarity of structure between TMS-Ala and leucine. Although free l- and d-TMS-Ala did not show the inhibition, several derivatives of l-TMS-Ala, especially Z-l-TMS-Ala and l-Leu-(l-TMS-Ala), exhibited a higher inhibitory activity toward thermolysin than did Z-l-Leu and l-Leu-l-Leu respectively. Effects of TMS-Ala on the activity of its derivatives and the mode of interaction between the derivatives of TMS-Ala and thermolysin are also discussed.
A set of Hansenula polymorpha integrative vectors to construct lacZ fusions by N. Brito; M. D. Pérez; G. Perdomo; C. González; P. García-Lugo; J. M. Siverio (pp. 23-29).
A set of YEp Saccharomyces cerevisiae-based, integrative Hansenula polymorpha plasmids was constructed to express lacZ gene under yeast gene promoters. The HpLEU2 and HpURA3 genes were used both as markers and to target the integration of plasmids into the corresponding H. polymorpha genome locus. The frequency of transformation reached with these plasmids linearised either in HpLEU2 or HpURA3 was around 100 transformants per microgram of plasmid DNA; in all transformants checked by Southern blotting the plasmid was integrated into the genome locus corresponding to the gene plasmid marker. PCR showed that about 50% of the transformants contained more than one plasmid copy per genome. Experiments carried out using the developed plasmids to determine the strength of the gene promoters involved in nitrate assimilation in H. polymorpha revealed that, in the presence of nitrate, the nitrate reductase gene promoter (YNR1) was the strongest, followed by nitrite reductase (YNI1) and nitrate transporter (YNT1).
Enhanced sterol-acyl transferase activity promotes sterol accumulation in Saccharomyces cerevisiae by T. Polakowski; R. Bastl; U. Stahl; C. Lang (pp. 30-35).
The sterol-acyl transferase encoded by the gene ARE2 was transcriptionally deregulated in the yeast Saccharomyces cerevisiae to understand its role in sterol storage and sterol enrichment. Our results show that sterols can indeed be enriched in yeast by enhancing the capacity of the cells to esterify sterols. ARE2 overexpression had no impact on the accumulation of the early sterols such as lanosterol, but influenced the later intermediates and the end product ergosterol. Thus an enhanced conversion of free sterols to their esterified counterparts may provide a tool to increase the overall sterol content of the yeast cell. We have previously shown that the overexpression of a truncated version of the key enzyme of the early sterol pathway, HMG-CoA reductase (HMG1), leads to an increase in the early sterols such as lanosterol and zymosterol. The simultaneous deregulation of both genes in one strain produces a cumulative effect in that both early and late sterols are enhanced. Karmellae-like structures can be detected when Are2p is overexpressed. Are2p therefore constitutes a new member of the karmellae-inducing protein family.
Nucleotide sequence and thermostability of pND324, a 3.6-kb plasmid from Lactococcus lactis by K. Duan; C. -Q. Liu; Y. -J. Liu; J. Ren; N. W. Dunn (pp. 36-42).
A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp. lactis LL57-1. Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE. The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal θ-type replicons of the pWV02/pCI305 family. pND324 was stable at both 30 °C and 37 °C, whereas derivatives that lack cisE were highly unstable at 37 °C, indicating that cisE is essential for thermostability. Sequences that are similar to orfX1 are commonly present in the lactococcal θ-type plasmids. The orfX2 product is homologous to TrfA, a 43-kDa protein of the E. coli theta-type plasmid RK2 required for replication and maintenance. Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324. Based on the minimal replication region of pND324, an integrative cloning vector, designated pND421, was constructed. In L. lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 °C for 40 generations. The integrated plasmid was totally stable for at least 100 generations without selection at 30 °C.
Metabolic approaches for the optimisation of recombinant fermentation processes by M. Cserjan-Puschmann; W. Kramer; E. Duerrschmid; G. Striedner; K. Bayer (pp. 43-50).
The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number.
Transient gene expression in mammalian and mosquito cells using a recombinant Semliki Forest virus expressing T7 RNA polymerase by A. Kohl; A. Billecocq; C. Préhaud; F. -Z. Yadani; M. Bouloy (pp. 51-56).
In this report, we describe a novel recombinant Semliki Forest virus (SFV) expressing T7 RNA polymerase (T7-RP), which was shown to drive transient expression of the chloramphenicol acetyltransferase (cat) gene in mammalian and mosquito cells after transfection of plasmids carrying the reporter gene under the control of the T7 promoter. To our knowledge, this is the first description of a T7-RP-based expression in mosquito cells. Expression of the cat gene was significantly enhanced in mammalian cells by inserting the sequence of the encephalomyocarditis virus internal ribosome entry site between the T7 promoter and the 5′ end of the cat gene. In mosquito cells, the level of chloramphenicol acetyltransferase activity was increased by flanking the cat gene with the Autographa californica baculovirus p10 gene 5′ and 3′ untranslated regions. SFV expressing T7-RP appears, therefore, to be an alternative to other virus-based gene-expression systems in mammalian cells and a powerful tool for protein expression in mosquito cells.
Molecular basis for enhanced biosynthesis of clavulanic acid by a redox-cycling agent, phenazine methosulfate, in Streptomyces clavuligerus by H. -J. Kwon; S. -U. Kim (pp. 57-62).
Phenazine methosulfate (PMS), a generator of superoxide, evoked the transcription of cas2 and cefF, ultimately resulting in the enhanced biosyntheses of clavulanic acid (CA) and cephamycin C (CMC) in Streptomyces clavuligerus. The transcriptional activation of cas2 and cefF was accompanied with that of ccaR, a regulatory gene for biosyntheses of CA and CMC. PMS or H2O2 in cell-free extract exerted a positive regulation on in vitro protein phosphorylation. The PMS-mediated activation of protein phosphorylation was significantly offset by butylated hydroxyanisole, a radical scavenger. Staurosporine, a protein kinase inhibitor, was shown to have a negative effect on PMS-promoted CA accumulation. Therefore, it is suggestive that PMS-activated transcription of cas2 and cefF is mediated by protein phosphorylation and the expression of a pathway- specific transcriptional activator as found in other streptomycetes. These experimental results present an example of the functional relationship between oxidative stimuli and secondary metabolite production in streptomycetes.
Biotransformation of selected monoterpenes under nitrate-reducing conditions by S. G. Pavlostathis; G. Misra (pp. 63-68).
Batch experiments were conducted to assess both the biotransformation potentials of one hydrocarbon (α-pinene) and four alcohol monoterpenes (arbanol, linalool, plinol, and α-terpineol) under nitrate-reducing conditions at 23 °C, as well as their effects on the nitrate-reducing process. A mixed, nitrate-reducing culture developed from a forest-soil extract was enriched using ethanol as the electron donor and used in this study. α-Pinene was not biotransformed under the conditions of this study and inhibited both ethanol and nitrate utilization. Partial transformation of the alcohol monoterpenes was observed and resulted in inhibition of the nitrate-reducing process and cessation of further utilization of the added monoterpenes. Accumulation of biotransformation products – mainly hydrocarbon monoterpenes such as camphene, β-myrcene, and d-limonene – was observed. The hydrocarbon monoterpenes formed may have been responsible for the observed inhibition of the nitrate-reducing process and lack of complete utilization of the alcohol monoterpenes. These results have significant implications for the expected rate and extent of biotransformation of monoterpenes under anoxic conditions as well as their effect on the nitrate-reducing process in both engineered and natural systems.
Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran by L. Micheli; D. Uccelletti; C. Palleschi; V. Crescenzi (pp. 69-74).
A capsular-polysaccharide-producing strain, LM-17, was isolated from kefir grains and was identified as a slime-forming, rod-shaped Lactobacillus. According to 1H- and 13C-NMR spectral data, the exopolysaccharide produced by the isolated bacterial strain is identical to the glucogalactan extracted from kefir grains and therefore known as kefiran. The kefiran produced was characterised by means of viscosity, optical rotatory power, circular dichroism and IR spectral measurements. A batch procedure was set up for the culture and extraction of the exopolysaccharide in laboratory conditions, resulting in a yield of 2 g/l purified kefiran from the culture supernatant of the LM-17 strain.
Metabolism of 2,4,6-trinitrotoluene by the white-rot fungus Bjerkandera adusta DSM 3375 depends on cytochrome P-450 by A. Eilers; E. Rüngeling; U. M. Stündl; G. Gottschalk (pp. 75-80).
Degradation of 2,4,6-trinitrotoluene (TNT) by the white-rot fungus Bjerkandera adusta DSM 3375 was studied in relation to extracellular ligninolytic activities. The Mn(II)-dependent peroxidase, the only ligninolytic enzyme detectable, reached a maximum activity of 600 ± 159 U/l after incubation in mineral medium with a sufficient nitrogen source. In contrast, the highest extent of [14C]TNT mineralization was detected in malt extract broth, so that the ability of B. adusta to mineralize TNT did not parallel ligninolytic activity. The microsomal fraction of cells grown in the presence of TNT was found to contain 11 pmol cytochrome P-450/mg protein. In cells grown without TNT, no microsomal cytochrome P-450 could be found. Instead, 14 pmol P-450/mg protein was present in the cytosolic fraction of these cells. Cytochrome P-450 apparently affected the TNT metabolism, as shown by inhibitory studies. Addition of the cytochrome P-450 inhibitor piperonyl butoxide diminished the 14CO2 release from 21% to 0.9%, as determined after 23 days of incubation, while 1-aminobenzotriazole and metyrapone decreased the mineralization to 8.6% and 6.3% respectively. Mass-balance analysis of TNT degradation in liquid cultures revealed that, by inhibition of cytochrome P-450, the TNT-derived radioactivity associated with biomass and with polar, water-soluble metabolites decreased from 93.9% to 15.0% and the fraction of radiolabelled metabolites extractable with organic solvents fell to 92.6%. The TNT metabolites of this fraction were identified as aminodinitrotoluenes, indicating that this initial transformation product of TNT may function as a substrate for cytochrome-P-450-dependent reactions in B. adusta.
Enhanced production of penicillin V acylase from Streptomyces lavendulae by R. Torres; F. Ramón; I. de la Mata; C. Acebal; M. P. Castillón (pp. 81-84).
At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.
Bioavailability of polycyclic aromatic hydrocarbons and formation of humic acid-like residues during bacterial PAH degradation by B. P. Ressler; H. Kneifel; J. Winter (pp. 85-91).
The degradation of single polycyclic aromatic hydrocarbons (PAHs: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene) and a mixture of all seven PAHs by a bacterial culture enriched from contaminated soil resulted in the formation of a dark-coloured residual fraction of dissolved (DOM) and particulate organic matter (POM). This fraction was highly resistant to bacterial degradation. Analysis of the DOM revealed a molecular-size-distribution similar to that of natural humic acids. A complete degradation of PAHs was apparently prevented by an irreversible incorporation of about 10% of the carbon from single PAHs or 20% of the carbon from the mixture of seven PAHs into the DOM- and POM- fraction. Some metabolites excreted during bacterial PAH-degradation were identified as known precursors for humification.
Oxygen-dependent xylitol metabolism in Pichia stipitis by H. Jeppsson; K. Holmgren; B. Hahn-Hägerdal (pp. 92-97).
Pichia stipitis CBS 6054 was cultivated in chemostat cultures under aerobic and oxygen-limited conditions with xylitol alone, a mixture of xylitol and glucose and a mixture of xylitol and xylose. Xylitol metabolism was strictly respiratory and no ethanol was formed. Simultaneous feeding of xylitol and glucose and xylitol and xylose to oxygen-limited xylitol-pregrown cells resulted in ethanol formation. In vitro both pyruvate decarboxylase activity and alcohol dehydrogenase activity were present in cells metabolising xylitol under oxygen-limited conditions; however, this did not result in ethanol formation. Glucose, xylose and xylitol utilisation, respectively, were compared under anaerobic conditions with regard to growth rate, carbon source and oxygenation level during pre-cultivation. Irrespective of pre-growth conditions, xylitol was not metabolised under anaerobic conditions, whereas ethanol was formed from both xylose and glucose. Anaerobic xylose utilisation required induction of a xylose-utilising metabolic pathway during pre-cultivation.
Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol by S. K. Samanta; A. K. Chakraborti; R. K. Jain (pp. 98-107).
Four polycyclic aromatic hydrocarbon (PAH)- degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp. HL4 and Pseudomonas sp. DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-14C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of 14CO2 was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to 14CO2 by RKJ4, P4-1, HL4 and DLC-P11, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in 14CO2 production from [9-14C]phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in 14CO2 production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-14C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in 14CO2 production. In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1. A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11. HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established.
A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products by M. Minekus; M. Smeets-Peeters; A. Bernalier; S. Marol-Bonnin; R. Havenaar; P. Marteau; M. Alric; G. Fonty; J. H. J. Huis in't Veld (pp. 108-114).
This paper introduces a new type of system to simulate conditions in the large intestine. This system combines removal of metabolites and water with peristaltic mixing to obtain and handle physiological concentrations of microorganisms, dry matter and microbial metabolites. The system has been designed to be complementary to the dynamic multi-compartmental system that simulates conditions in the stomach and small intestine described by Minekus et al. [Minekus M, Marteau P, Havenaar R, Huis in't Veld JHJ (1995) ATLA 23:197–209]. High densities of microorganisms, comparable to those found in the colon in vivo, were achieved by absorption of water and dialysis of metabolites through hollow-fibre membranes inside the reactor compartments. The dense chyme was mixed and transported by peristaltic movements. The potential of the system as a tool to study fermentation was demonstrated in experiments with pectin, fructo-oligosaccharide, lactulose and lactitol as substrates. Parameters such as total acid production and short-chain fatty acid (SCFA) patterns were determined with time to characterize the fermentation. The stability of the microflora in the system was tested after inoculation with fresh fecal samples and after inoculation with a microflora that was main-tained in a fermenter. Both approaches resulted in total anaerobic bacterial counts higher than 1010 colony-forming units/ml with physiological levels of Bifidobacterium, Lactobacillus, Enterobacteriaceae and Clostridium. The dry matter content was approximately 10%, while the total SCFA concentration was maintained at physiological concentrations with similar molar ratios for acetic acid, propionic acid and butyric acid as measured in vivo.
Nutritional requirements for the production of pyrazoloisoquinolinone antibiotics by Streptomyces griseocarneus NCIMB 40447 by R. Cruz; M. E. Arias; J. Soliveri (pp. 115-119).
This paper describes the effect of different nutrients on the production of pyrazoloisoquinolinone antibiotics (APHE) by Streptomyces griseocarneus. In a chemically defined medium with glucose as carbon and l-lysine as nitrogen source all APHE antibiotics (APHE-1 to -3) are produced, APHE-3 being the most abundant. Propionate and butyrate used as precursors with glucose as main carbon source increased the production of APHE-1 and -2, respectively. The presence of propionate or butyrate reduced the production of APHE-3. Results obtained in minimal medium supplemented with l-valine and l-histidine indicate a relationship between these amino acids and APHE biosynthesis. These data, together with those obtained in the presence of precursors of fatty acids, also show possible links with fatty acid biosynthesis. Different nutritional requirements were found for APHE-3 production in comparison with APHE-1 and APHE-2.
Isomerization of non-insecticidal α-hexachlorocyclohexane (HCH) to insecticidal γ-HCH by Pseudomonas strain Ptm+ by N. Ramesha; N. G. K. Karanth (pp. 120-126).
Pseudomonas strain Ptm+ grew on α-hexa-chlorocyclohexane (HCH, CAS no. 319846), using it as the sole source of carbon and energy. In a replacement-culture study, with the non-insecticidal α-HCH, γ-HCH (CAS no. 58899) was the first metabolite noticed at 6 h, and transient accumulation of insecticidal γ-HCH occurred for up to 18 h. Although delta- (CAS no. 319868) and beta-isomers (CAS no. 319857) were also detected, their concentrations were very low. By 18 h of incubation, about 23% of the α-HCH added was transformed into the gamma-isomer. Subsequently, the concentration of γ-HCH in the medium fell. Thin-layer chromatography, gas chromatography, gas chromatography-mass spectrometry and mosquito-larval bioassay analyses confirmed the formation of γ-HCH. This was associated with the formation of three more metabolites.
Screening of ectomycorrhizal fungi for degradation of polycyclic aromatic hydrocarbons by A. Braun-Lüllemann; A. Hüttermann; A. Majcherczyk (pp. 127-132).
Ectomycorrhizal fungi belonging to 16 species (27 strains) were tested for their ability to degrade polycyclic aromatic hydrocarbons (PAHs): phenanthrene, chrysene, pyrene and benzo[a]pyrene. Cultivated on a complex liquid medium, most of the fungi tested were able to metabolise these compounds. Approximately 50% of the benzo[a]pyrene was removed by strains of Amanita excelsa, Leccinum versipelle, Suillus grevillei, S. luteus, and S. variegatus during a 4-week incubation period. The same amount of phenanthrene was also metabolised by A. muscaria, Paxillus involutus, and S. grevillei. The degradation of the other two PAHs was, for the most part, less effective. Only S. grevillei was able to remove 50% of the pyrene, whereas Boletus edulis and A. muscaria removed 35% of the chrysene.
Degradation of proteins and amino acids by Caloramator proteoclasticus in pure culture and in coculture with Methanobacterium thermoformicicum Z245 by S. Tarlera; A. J. M. Stams (pp. 133-138).
This study investigated the degradation of proteins and amino acids by Caloramator proteoclasticus, an anaerobic thermophilic (55 °C) fermentative bacterium isolated from an anaerobic bioreactor. Experiments were performed in the presence and absence of Methanobacterium thermoformicicum Z245, a methanogen that can use both hydrogen and formate for growth. Higher production rates and yields of the principal fermentation products from gelatin were observed in methanogenic coculture. The specific proteolytic activity in coculture tripled the value obtained in pure culture. C. proteoclasticus fermented glutamate to acetate, formate, hydrogen and alanine. In methanogenic coculture, a shift towards higher amounts of acetate and hydrogen with no alanine production was observed. Extracts of glutamate-grown cells possessed high activities of β-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate. The presence of two enzymes (alanine-α-ketoglutarate aminotransferase and NADH-dependent alanine dehydrogenase) usually involved in the biosynthesis of alanine from pyruvate was also detected. The fermentation of amino acids known to be oxidatively deaminated (leucine and valine) was improved in the presence of both methanogenesis and glycine, a known electron acceptor in the Stickland reaction. Culture conditions seem to be very important in the way C. proteoclasticus disposes of reducing equivalents formed during the degradation of amino acids.