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Applied Microbiology and Biotechnology (v.77, #6)
Formation of volatile sulfur compounds and metabolism of methionine and other sulfur compounds in fermented food by Sophie Landaud; Sandra Helinck; Pascal Bonnarme (pp. 1191-1205).
The formation of volatile sulfur compounds (VSC) in fermented food is a subject of interest. Such compounds are essential for the aroma of many food products like cheeses or fermented beverages, in which they can play an attractive or a repulsive role, depending on their identity and their concentration. VSC essentially arise from common sulfur-bearing precursors, methionine being the most commonly found. In the first section of this paper, the main VSC found in cheese, wine, and beer are reviewed. It is shown that a wide variety of VSC has been evidenced in these food products. Because of their low odor threshold and flavor notes, these compounds impart essential sensorial properties to the final product. In the second section of this review, the main (bio)chemical pathways leading to VSC synthesis are presented. Attention is focused on the microbial/enzymatic phenomena—which initiate sulfur bearing precursors degradation—leading to VSC production. Although chemical reactions could also play an important role in this process, this aspect is not fully developed in our review. The main catabolic pathways leading to VSC from the precursor methionine are presented.
Keywords: Volatile sulfur compounds; Fermented food; Biochemical pathway
Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by gacA-inactivated Pseudomonas sp. M18G using response surface method by Yaqian Li; Haixia Jiang; Yuquan Xu; Xuehong Zhang (pp. 1207-1217).
The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l−1 of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l−1), glucose (12.7 g l−1), soy peptone (10.9 g l−1), and ethanol (13.8 ml l−1) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l−1, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l−1 PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.
Keywords: Phenazine-1-carboxylic acid; Pseudomonas sp. M18G; Plackett–Burman design; Response surface method; Nutrient component; Optimization
Production of isopropanol by metabolically engineered Escherichia coli by Toru Jojima; Masayuki Inui; Hideaki Yukawa (pp. 1219-1224).
A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.
Keywords: Isopropanol ; Escherichia coli ; Clostridium acetobutylicum ; Clostridium beijerinckii ; Metabolic engineering
Improvement in enzymatic desizing of starched cotton cloth using yeast codisplaying glucoamylase and cellulose-binding domain by Takeshi Fukuda; Michiko Kato-Murai; Kouichi Kuroda; Mitsuyoshi Ueda; Shin-ichiro Suye (pp. 1225-1232).
To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry.
Keywords: Yeast cell surface engineering; Rhizopus oryzae glucoamylase; Trichoderma reesei cellulose-binding domain; Enzymatic desizing
High-cell-density cultivation for co-production of ergosterol and reduced glutathione by Saccharomyces cerevisiae by Fei Shang; Zheng Wang; Tianwei Tan (pp. 1233-1240).
Two different high-cell-density cultivation processes based on the mutant Saccharomyces cerevisiae GE-2 for simultaneous production of glutathione and ergosterol were investigated. Compared with keeping the ethanol volumetric concentration at a constant low level, feedback control of glucose feeding rate (F) by keeping the descending rate of ethanol volumetric concentration (ΔE/Δt) between −0.1% and 0.15% per hour was much more efficient to achieve a high glutathione and ergosterol productivity. This bioprocess overcomes some disadvantages of traditional S. cerevisiae-based cultivation process, especially shortening cultivation period and making the cultivation process steady-going. A classical on or off controller was used to manipulate F to maintain ΔE/Δt at its set point. The dry cell weight, glutathione yield and ergosterol yield reached 110.0 ± 2.6 g/l, 2,280 ± 76 mg/l, and 1,510 ± 28 mg/l in 32 h, respectively.
Keywords: High cell density; Saccharomyces cerevisiae ; Ergosterol; Reduced glutathione; Ethanol
Novel peroxidases of Marasmius scorodonius degrade β-carotene by Manuela Scheibner; Bärbel Hülsdau; Kateryna Zelena; Manfred Nimtz; Lex de Boer; Ralf G. Berger; Holger Zorn (pp. 1241-1250).
Two extracellular enzymes (MsP1 and MsP2) capable of efficient β-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of ~150 and ~120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of ~23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).
Keywords: Basidiomycetes; Carotenoid degradation; DyP-type peroxidase
Functional expression, purification, and characterization of the recombinant Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700 by Anne Völker; Anett Kirschner; Uwe T. Bornscheuer; Josef Altenbuchner (pp. 1251-1260).
For the investigation of the NADPH-dependent Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700, the encoding gene mekA with a C-terminal strep-tag was cloned and expressed under the control of a l-rhamnose inducible promoter from Escherichia coli. The mekA gene was found by analyzing the methylethylketone (MEK) degradation pathway by Onaca et al. J Bacteriol 189:3759–3767, 2007. Sequence analysis of the corresponding protein, which catalyzes the Baeyer-Villiger oxidation of MEK to ethyl acetate, showed two binding sites (Rossman-fold motifs) for cofactors NAD(P)H and FAD. Although expression of mekA resulted in large amounts of inclusion bodies compared to soluble protein, high amounts of purified and active MekA were obtained by affinity chromatography. The substrate spectrum of MekA was investigated with purified enzyme and whole cells using a variety of aliphatic, aromatic, and cyclic ketones including four chiral substrates. The specific activity of MekA with MEK as substrate was determined to be 1.1 U/mg protein. K M values were determined for MEK and the cofactors NADPH and NADH to be 6, 11, and 29 μM, respectively.
Keywords: Baeyer-Villiger monooxygenase; Ketones; Ester; Affinity tag purification; Methylethylketone
Marine lysozyme from a marine bacterium that inhibits angiogenesis and tumor growth by Junli Ye; Chunbo Wang; Xuehong Chen; Shenbo Guo; Mi Sun (pp. 1261-1267).
Recent studies suggest that lysozyme, rich in hen egg, has an antitumor function. In the present study, we investigated the antitumor and antiangiogenesis effects of a newly isolated marine lysozyme both in vitro and in vivo. First, we showed that this marine-derived lysozyme specifically inhibits the proliferation of endothelial cells (ECV304) in a dose-dependent manner with no cytotoxicity (IC50 = 3.64 μM). Second, we showed that this marine lysozyme directly suppresses neovascularization in chicken embryos using chorioallantoic membrane assay. Third, we demonstrated that this marine lysozyme markedly inhibits tumor growth in mice bearing either sarcoma 180 or hepatoma 22. Unexpectedly, hen egg lysozyme has no effects on the proliferation of endothelial cells in vitro or neovascularization in chicken embryos or tumor growth in nude mice at the same dosage range. Taken together, our studies clearly show that the newly identified marine lysozyme is a potent antitumor molecule, which may inhibit tumor growth and inhibit angiogenesis. We believe that this marine lysozyme may have a therapeutic value in antitumor drug development.
Keywords: Lysozyme; Antiangiogenesis; Endothelial cells; CAM assay; Antitumor; Marine lysozyme
Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity by S. Raimondi; D. Uccelletti; D. Matteuzzi; U. M. Pagnoni; M. Rossi; C. Palleschi (pp. 1269-1277).
Superoxide dismutase (SOD) activity is one major defense line against oxidative stress for all of the aerobic organisms, and industrial production of this enzyme is highly demanded. The Cu/Zn superoxide dismutase gene (KmSOD1) of Kluyveromyces marxianus L3 was cloned and characterized. The deduced KmSod1p protein shares 86% and 71% of identity with Kluyveromyces lactis and Saccharomyces cerevisiae Sod1p, respectively. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc and in enzymatic function were conserved. To the aim of developing a microbial production of Cu/Zn superoxide dismutase, we engineered the K. marxianus L3 strain with the multicopy plasmid YG-KmSOD1 harboring the KmSOD1 gene. The production of KmSOD1p in K. marxianus L3 and K. marxianus L3 (pYG-KmSOD1) in response to different compositions of the culture medium was evaluated. The highest specific activity (472 USOD mgprot −1) and the highest volumetric yield (8.8 × 105 USOD l−1) were obtained by the recombinant strain overexpressing KmSOD1 in the presence of Cu2+ and Zn2+ supplements to the culture media. The best performing culture conditions were positively applied to a laboratory scale fed-batch process reaching a volumetric yield of 1.4 × 106 USOD l−1.
Keywords: Kluyveromyces marxianus ; Cu/Zn superoxide dismutase; Expression; Productivity
Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii by Takuya Koseki; Yuichiro Mese; Shinya Fushinobu; Kazuo Masaki; Tsutomu Fujii; Kiyoshi Ito; Yoshihito Shiono; Tetsuya Murayama; Haruyuki Iefuji (pp. 1279-1285).
The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61ΔCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61ΔCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61ΔCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61ΔCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl β-d-glucoside, p-nitrophenyl β-d-cellobioside, and p-nitrophenyl β-d-cellotrioside.
Keywords: Endoglucanase; Family 61 glycoside hydrolase; Cellulose-binding domain; Aspergillus kawachii
Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene by L. B. Pinheiro; M. D. Gibbs; G. Vesey; J. J. Smith; P. L. Bergquist (pp. 1287-1295).
Fluorescent reference strains of bacteria carrying a stable chromosomally integrated single copy of the gfp gene have been developed. A modified version of the gfp gene has been generated by mutagenesis and expressed under the control of the bacteriophage lambda promoter PL. A cassette comprising bacteriophage Mu transposon arms flanking the modified gfp gene and regulatory regions was irreversibly integrated as an in-vitro-assembled transposition complex into the genomes of Escherichia coli and Salmonella spp. The modified green fluorescent protein (GFP) protein retained the fluorescence excitation and emission wavelengths of wild-type GFP. However, it fluoresced more brightly in E. coli and Salmonella compared to wild-type GFP, presumably due to improved protein maturation. Fluorescent E. coli and Salmonella strains carrying the gfp gene cassette were easily differentiated from their respective non-fluorescent parental strains on various growth media by visualization under UV light. The bacterial strains produced by this method remained viable and stably fluorescent when incorporated into a matrix for delivery of exact numbers of viable bacterial cells for use as quality control agents in microbiological procedures.
Microbial production of l-glutamate and l-glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb by Qian Liu; Jiao Zhang; Xiao-Xing Wei; Shao-Ping Ouyang; Qiong Wu; Guo-Qiang Chen (pp. 1297-1304).
Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more l-glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more l-glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA′ was able to produce l-glutamine effectively. Co-expression of vgb and glnA′ genes in C. glutamicum produced 17 g/l l-glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA′ gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l l-glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, l-glutamate, and l-glutamine production by recombinant C. glutamicum.
Keywords: Corynebacterium glutamicum ; l-glutamate; l-glutamine; Vitreoscilla hemoglobin; vgb
Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli by Masayuki Inui; Masako Suda; Sakurako Kimura; Kaori Yasuda; Hiroaki Suzuki; Hiroshi Toda; Shogo Yamamoto; Shohei Okino; Nobuaki Suzuki; Hideaki Yukawa (pp. 1305-1316).
A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.
Keywords: Butanol production; Clostridium acetobutylicum ; Escherichia coli ; Biofuel
Transfer of megaplasmid pKB1 from the rubber-degrading bacterium Gordonia westfalica strain Kb1 to related bacteria and its modification by Daniel Bröker; Matthias Arenskötter; Alexander Steinbüchel (pp. 1317-1327).
Because engineering of the 101.016-bp megaplasmid pKB1 of Gordonia westfalica Kb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation from G. westfalica Kb1 to a kanamycin-resistant mutant of Rhodococcus opacus PD630 at a frequency of about 6.2 × 10−8 events per recipient cell. Furthermore, pKB1 was transferred to G. polyisoprenivorans strains VH2 and Y2K and to Mycobacterium smegmatis by electroporation at frequencies of 5.5 × 103, 1.9 × 103, and 8.3 × 102 transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance gene cadA was used for selection in these experiments. Recombinant pKB1-containing G. polyisoprenivorans VH2 and M. smegmatis were then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encoded cadA gene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination between cadA on suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer into Gordonia and related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box for Gordonia.
Keywords: Gordonia ; Megaplasmid; Conjugational transfer; Electroporation; Heavy metal resistance; Cadmium; Genetic manipulation; Suicide plasmid
Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis by Vincenza Faraco; Carmine Ercole; Giovanna Festa; Paola Giardina; Alessandra Piscitelli; Giovanni Sannia (pp. 1329-1335).
Among the laccases produced by the white-rot fungus Pleurotus ostreatus, there are two closely related atypical isoenzymes, POXA3a and POXA3b. These isoenzymes are endowed with quaternary structure, consisting of two subunits very different in size. The POXA3 large subunit is clearly homologous to other known laccases, while the small subunit does not show significant homology with any protein in data banks. To investigate on the singular structure of the POXA3 complex, a new system for recombinant expression of heterodimer proteins in the yeast Kluyveromyces lactis has been set up. A unique expression vector has been used and the cDNAs encoding the two subunits have been cloned under the control of the same bi-directionally acting promoter. Expression of the large subunit alone and co-expression of both subunits in the same host have been demonstrated and the properties of the recombinant proteins have been compared. Clones expressing the large subunit alone exhibited always notably lower activity than those expressing both subunits. In addition to the activity increase, the presence of the small subunit led to a significant increase of laccase stability. Therefore, a role of the small subunit in POXA3 stabilisation is suggested.
Keywords: Fungus; Recombinant laccase; Co-expression; Yeast
Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures by Pablo I. Nikel; M. Julia Pettinari; Miguel A. Galvagno; Beatriz S. Méndez (pp. 1337-1343).
Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the ‘one-factor-at-a-time’ technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett–Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box–Wilson design. Under such optimized conditions (22.02 g l−1 glycerol, 1.78 g l−1 CAS, and 1.83 g l−1 inoculum) microaerobic batch cultures gave rise to 8.37 g l−1 CDW and 3.52 g l−1 PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l−1. After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l−1, respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures.
Keywords: Poly(3-hydroxybutyrate); Escherichia coli ; arcA mutant; Microaerobic cultivation; Fed-batch culture
Microbial transformation of ginsenoside Rb1 by Acremonium strictum by Guang-Tong Chen; Min Yang; Yan Song; Zhi-Qiang Lu; Jin-Qiang Zhang; Hui-Lian Huang; Li-Jun Wu; De-An Guo (pp. 1345-1350).
Preparative-scale fermentation of ginsenoside Rb1 (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg3 (4), ginsenoside F2 (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb1 in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.
Keywords: Ginsenoside Rb1 ; Biotransformation; Acremonium strictum
Simultaneous mineralization of glyphosate and diuron by a consortium of three bacteria as free- and/or immobilized-cells formulations by S. Bazot; T. Lebeau (pp. 1351-1358).
A bacterial consortium able to mineralize two herbicides, glyphosate (Pseudomonas 4ASW) and diuron (Arthrobacter sp. N4 and Delftia acidovorans), was cultivated in both a synthetic culture medium without phosphate and a sediment extract medium. In the aim at optimizing glyphosate and diuron mineralization, all the combinations, i.e., free and/or immobilized cells in Ca-alginate beads were tested. With the synthetic medium, the simultaneous mineralization of glyphosate and diuron required at least the immobilization of Pseudomonas 4ASW. Conversely, with the sediment extract medium, only the mineralization of diuron was observed, most probably, because of both nutrient deficiency and phosphate in the sediment extract medium.
Keywords: Arthrobacter sp.; Co-culture; Delftia Acidovorans ; Herbicides; Immobilization; Pseudomonas
Pathway, inhibition and regulation of methyl tertiary butyl ether oxidation in a filamentous fungus, Graphium sp. by Kristin M. Skinner; Adriana Martinez-Prado; Michael R. Hyman; Kenneth J. Williamson; Lynda M. Ciuffetti (pp. 1359-1365).
The filamentous fungus Graphium sp. (ATCC 58400) co-metabolically oxidizes the gasoline oxygenate methyl tertiary butyl ether (MTBE) after growth on gaseous n-alkanes. In this study, the enzymology and regulation of MTBE oxidation by propane-grown mycelia of Graphium sp. were further investigated and defined. The trends observed during MTBE oxidation closely resembled those described for propane-grown cells of the bacterium Mycobacterium vaccae JOB5. Propane-grown mycelia initially oxidized the majority (∼95%) of MTBE to tertiary butyl formate (TBF), and this ester was biotically hydrolyzed to tertiary butyl alcohol (TBA). However, unlike M. vaccae JOB5, our results collectively suggest that propane-grown mycelia only have a limited capacity to degrade TBA. None of the products of MTBE exerted a physiologically relevant regulatory effect on the rate of MTBE or propane oxidation, and no significant effect of TBA was observed on the rate of TBF hydrolysis. Together, these results suggest that the regulatory effects of MTBE oxidation intermediates proposed for MTBE-degrading organisms such as Mycobacterium austroafricanum are not universally relevant mechanisms for MTBE-degrading organisms. The results of this study are discussed in terms of their impact on our understanding of the diversity of aerobic MTBE-degrading organisms and pathways and enzymes involved in these processes.
Keywords: Methyl tertiary butyl alcohol; Cometabolism; Graphium sp. ; Tertiary butyl alcohol; Tertiary butyl formate; Alkane monooxygenase
EDTA-dependent assimilation of glucose and organic acids by an EDTA-degrading bacterium by E. G. Dedyukhina; T. I. Chistyakova; D. N. Badrutdinova; E. I. Yudina; I. G. Minkevich; M. B. Vainshtein (pp. 1367-1370).
Bacterial strain VKM B-2445 is characterized by ethylenediaminetetraacetate (EDTA) requirement for cell growth. This strain could not grow on glucose and organic acids as the sole sources of carbon and energy, but it was able to metabolize these substrates added to EDTA medium. EDTA initiated assimilation of glucose, succinate, fumarate, malate, and citrate and supplied nitrogen for the biomass production from these substrates. Utilization of primarily nongrowth substrates by strain VKM B-2445 started when EDTA was exhausted or at least considerably degraded.
Keywords: EDTA; Degradation; Bacteria; Growth; Substrate
Degradation of 2,4-DB in Argentinean agricultural soils with high humic matter content by Virginia Cuadrado; Luciano J. Merini; Cecilia G. Flocco; Ana M. Giulietti (pp. 1371-1378).
The dissipation of 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) in high-humic-matter-containing soils from agricultural fields of the Argentinean Humid Pampa region was studied, employing soil microcosms under different experimental conditions. The added herbicide was dissipated almost completely by soils with and without history of herbicide use by day 28. At 500 ppm, both soils showed the same degradation rates; but at 5-ppm concentration, the chronically exposed soil demonstrated a faster degradation of the herbicide. 2,4-DB addition produced increases in herbicide-degrading bacteria of three and 1.5 orders of magnitude in soils with and without history of herbicide use, respectively, in microcosms with 5 ppm. At 500-ppm concentration, the increase in 2,4-DB degraders was five orders of magnitude after 14 days, independent of the history of herbicide use. No differences were observed in either 2,4-DB degradation rates or in degrader bacteria numbers in the presence and absence of alfalfa plants, in spite of some differential characteristics in patterns of 2,4-DB metabolite accumulation. The main factor affecting 2,4-DB degradation rate would be the history of herbicide use, as a consequence of the adaptation of the indigenous microflora to the presence of herbicides in the field.
Keywords: Argentinean Humid Pampa region; Humic acids; Microcosms; 2,4-DB; Biodegradation; Soil microflora; Alfalfa rhizosphere
Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications by L. Cao-Hoang; F. Dumont; P. A. Marechal; M. Le-Thanh; P. Gervais (pp. 1379-1387).
The effects of slow chilling (2°C min−1) and rapid chilling (2,000°C min−1) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.
Keywords: Chilling rate; Rapid cold shock; Cell inactivation; Membrane fluidity; Membrane modifications