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Applied Microbiology and Biotechnology (v.55, #2)
The production of cytotoxic lignans by plant cell cultures by Maike Petersen; Wilhelm A. Alfermann (pp. 135-142).
Cytotoxic lignans derived from podophyllotoxin are currently used in cancer chemotherapy. Podophyllotoxin for semi-synthetic derivatization is isolated from the rhizomes of Podophyllum plants growing wild, some of which are counted as endangered species. An alternative source for podophyllotoxin or related lignans may in future be cell cultures derived from different plant species, such as Podophyllum spp or Linum spp. These cell cultures were shown to accumulate considerable amounts of podophyllotoxin or 5-methoxypodophyllotoxin. Optimization of the cell cultivation regime might lead to a renewable source of cytotoxic lignans for medicinal uses. This Mini-Review summarizes the attempts to establish plant cell cultures for the production of podophyllotoxin and related lignans and their optimization towards high levels of these target compounds. It also summarizes the results of studies on the biosynthesis of podophyllotoxin and 5-methoxypodophyllotoxin.
Plate screening methods for the detection of polysaccharase-producing microorganisms by H. Ruijssenaars; S. Hartmans (pp. 143-149).
Polysaccharide-degrading enzymes (polysaccharases) are widely applied in industry. One of the sources of these enzymes are polysaccharide-degrading microorganisms. To obtain such microorganisms from enrichment cultures, strain collections or gene libraries, efficient plate screening methods are required that discriminate between intact and degraded polysaccharide. This can be achieved by making use of specific physicochemical properties of the polysaccharide, such as complex formation with dyes and gelling capacity, or by the application of dye-labelled polysaccharides. This review presents a survey of plate methods based on these principles. Both theoretical and practical aspects of the methods are discussed.
Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus equi A4 by Irena Přepechalová; Ludmila Martínková; Andreas Stolz; Mária Ovesná; Karel Bezouška; Jan Kopecký; Vladimír Křen (pp. 150-156).
The nitrile hydratase from Rhodococcus equi A4 consisted of two kinds of subunits which slightly differed in molecular weight (both approximately 25 kDa) and showed a significant similarity in the N-terminal amino acid sequences to those of the nitrile hydratase from Rhodococcus sp. N-774. The enzyme preferentially hydrated the S-isomers of racemic 2-(2-, 4-methoxyphenyl)propionitrile, 2-(4-chlorophenyl)propionitrile and 2-(6-methoxynaphthyl)propionitrile (naproxennitrile) with E-values of 5–15. The enzyme functioned in the presence of 5–98% (v/v) of different hydrocarbons, alcohols or diisopropyl ether. The addition of 5% (v/v) of n-hexane, n-heptane, isooctane, n-hexadecane, pristane and methanol increased the E-value for the enzymatic hydration of 2-(6-methoxynaphthyl)propionitrile.
β-Glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance by C. Decker; J. Visser; P. Schreier (pp. 157-163).
From Aspergillus tubingensis CBS 643.92 four distinct β-glucosidases (I–IV) were purified by a four-step purification procedure. SDS-PAGE revealed molecular masses of 131, 126, 54 and 54 kDa, respectively, and their isoelectric points were determined to be 4.2, 3.9, 3.7 and 3.6, respectively. The β-glucosidases exhibited high diversity with respect to pH and temperature optima and stability, as well as to substrate specificity and glucose tolerance. The major β-glucosidase (I) preferentially hydrolysed oligosaccharides. The acid-stable and heat-tolerant β-glucosidase II hydrolysed aryl and terpenyl β-D-glucosides as well as 1-O-trans-cinnamoyl β-D-glucoside. In contrast to β-glucosidases I and II, the minor β-glucosidases III and IV were found to be glucose-tolerant; inhibition constants of 470 and 600 mM, respectively, were determined.
Enhanced anthocyanin production by repeated-batch culture of strawberry cells with medium shift by W. Zhang; M. Jin; X. Yu; Q. Yuan (pp. 164-169).
Repeated-batch cultures of strawberry cells (Fragaria ananassa cv. Shikinari) subjected to four medium-shift procedures (constant LS medium, constant B5 medium, alternation between LS and B5 starting from LS and alternation between LS and B5 starting from B5) were investigated for the enhanced anthocyanin productivity. To determine the optimum period for repeated batch cultures, two medium-shift periods of 9 and 14 days were studied, which represent the end of the exponential growth phase and the stationary phase. By comparison with the corresponding batch cultures, higher anthocyanin productivity was achieved for all the repeated-batch cultures at a 9-day medium-shift period. The average anthocyanin productivity was enhanced 1.7- and 1.76-fold by repeated-batch cultures in constant LS and constant B5 medium at a 9-day shift period for 45 days, respectively. No further improvement was observed when the medium was alternated between LS (the growth medium) and B5 (the production medium). Anthocyanin production was unstable at a 14-day shift period regardless of the medium-shift procedures. The results show that it is feasible to improve anthocyanin production by a repeated-batch culture of strawberry cells.
Characteristics and N-terminal amino acid sequence of manganese peroxidase from solid substrate cultures of Agaricus bisporus by Pauliina V. Lankinen; Alice M. Bonnen; Lori H. Anton; David A. Wood; Nisse Kalkkinen; Annele Hatakka; Christopher F. Thurston (pp. 170-176).
Extracellular manganese peroxidase (MnP) was purified from the compost extract of Agaricus bisporus using anion exchange chromatography, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two forms (MnP1 and MnP2) were separated by isoelectric focusing and their isoelectric points were determined to be 3.25 (MnP1) and 3.3 (MnP2). Both forms had a molecular mass of 40 kDa. The first 25 amino acids of the N-terminal end of MnP1 sequence was found to share 68% identity with a Pleurotus ostreatus and a P. eryngii MnP. Lignin peroxidase was not detected during any of the steps in the purification process. In liquid cultures with both soluble and insoluble carbon sources in defined medium (d-glucose, glycerol, Whatman CC-41 microcrystalline cellulose or Solka-floc cellulose) MnP protein was detected in culture fluid by Western blot, but no MnP activity could be detected. A. bisporus appears to be in the group of ligninolytic fungi which do not produce lignin peroxidase.
Chemo-enzymatically induced copolymerization of phenolics with acrylate compounds by Carsten Mai; Wiebke Schormann; Aloys Hüttermann (pp. 177-186).
Initiation of copolymerization of lignin-like phenolic and acrylic compounds by the phenoloxidase laccase (EC 1.10.3.2) and a peroxide species (t-butylhydroperoxide, t-BHP) was compared to a Fenton-like system (ferrous ion, t-BHP). Initially, the relative activity of laccase towards different phenolic compounds and the optimum pH of some characteristic phenolics were determined. The polymer yield and the average molecular weight $$left( {ar M_w }
ight)$$ of chemo-enzymatically produced polymers were dependent both on the type of each phenolic tested and on the phenol/monomer ratio. Furthermore, the success of copolymerization of the phenolics was dependent both on their redox potential and on the type of acrylic monomer applied. The extent of phenol incorporation into the polymer chain was enhanced by the presence of laccase in the reaction mixture and was significantly higher than in polymerization initiated by a Fenton-like reaction.
High-level production of heme-containing holoproteins in Escherichia coli by Yongwon Jung; Juhyoun Kwak; Younghoon Lee (pp. 187-191).
The expression of recombinant protein is essential for the investigation of the functions and properties of heme-containing protein as an electron carrier. For the expression of fully active recombinant protein, conversion of the expressed apoprotein into holoprotein is the most important and difficult problem. In this study, a system was developed for the production of heme-containing protein in a pure, recombinant holoprotein form, using the bovine cytochrome b5 tryptic fragment and Escherichia coli bacterioferritin as heterologous and homologous heme-containing model proteins, respectively. This system is based on the slow synthesis of recombinant apoprotein, which can maintain the balanced consumption of amino acids between protein synthesis and heme synthesis, so that the synthesized apoprotein continues to act as a heme sink. From a 1-l culture, 15 mg of cytochrome b5 and 40 mg of bacterioferritin were purified as pure holoprotein forms. Our expression system provides a rapid and simple method for obtaining large quantities of the active holo-form of heme-containing proteins.
Identification of an intragenic integration site for foreign gene expression in recombinant Streptococcus gordonii strains by Tove C. Bolken; Christine A. Franke; Gloria O. Zeller; Dennis E. Hruby (pp. 192-197).
A new intragenic chromosomal integration site within the lacG gene of the lac operon has been identified in Streptococcus gordonii for use in the expression of foreign genes. Introduction of a portion of the Streptococcus pyogenes emm6 gene into the lacG locus resulted in the lactose-inducible surface expression of the S. pyogenes M6 protein. This result demonstrates the ability to modulate the in vitro or in vivo expression of a foreign gene in a S. gordonii recombinant using a biosynthetic metabolite.
Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones by Petra C. Traub; Claudia Schmidt-Dannert; Jutta Schmitt; Rolf D. Schmid (pp. 198-204).
Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.
Heterologous expression of the acyl–acyl carrier protein thioesterase gene from the plant Umbellularia californica mediates polyhydroxyalkanoate biosynthesis in recombinant Escherichia coli by Bernd H. Rehm; Alexander Steinbüchel (pp. 205-209).
The acyl–acyl carrier protein (ACP) thioesterase cDNA from the plant Umbellularia californica was functionally expressed in various recombinant Escherichia coli strains in order to establish a new metabolic route toward medium-chain-length polyhydroxyalkanoate (PHAMCL) biosynthesis from non-related carbon sources. Coexpression of the PHA synthase genes from Ralstonia eutropha and Pseudomonas aeruginosa, or only the PHA synthase gene from P. aeruginosa, respectively, showed PHAMCL accumulation when the type II PHA synthase from P. aeruginosa was produced. Both wild-type E. coli and various fad mutants were investigated; and only when the β-oxidation pathway was impaired PHAMCL accumulation from gluconate was observed, contributing to about 6% of cellular dry weight. Thus coexpression of type II PHA synthase gene with cDNA encoding the medium-chain acyl-ACP thioesterase from U. californica established a new PHAMCL biosynthesis pathway, connecting fatty acid de novo biosynthesis with fatty acid β-oxidation, using a non-related carbon source.
Screening of micro-organisms for decolorization of melanins produced by bluestain fungi by M. Rättö; M. Chatani; A.-C. Ritschkoff; L. Viikari (pp. 210-213).
A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate. On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747. The four fungi were more efficient on nitrogen-limited medium than on complete medium. The melanin-decolorizing activity of G. geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.
Improved osmotolerance of recombinant Escherichia coli by de novo glycine betaine biosynthesis by N. von Weymarn; A. Nyyssölä; T. Reinikainen; M. Leisola; H. Ojamo (pp. 214-218).
The genes from the extreme halophile Ectothiorhodospira halochloris encoding the biosynthesis of glycine betaine from glycine were cloned into Escherichia coli. The accumulation of glycine betaine and its effect on osmotolerance of the cells were studied. In mineral medium with NaCl concentrations from 0.15 to 0.5 M, the accumulation of both endogenously synthesized and exogenously provided glycine betaine stimulated the growth of E. coli. The intracellular levels of glycine betaine and the cellular yields were clearly higher for cells receiving glycine betaine exogenously than for cells synthesizing it. The lower level of glycine betaine accumulation in cells synthesizing it is most likely a consequence of the limited availability of precursors (e.g. S-adenosylmethionine) rather than the result of a low expression level of the genes. Glycine betaine also stimulated the growth of E. coli and decreased acetate formation in mineral medium with high sucrose concentrations (up to 200 g·l–1).
Studies on the possibility of histidine biosynthesis from histidinol, imidazolepyruvic acid, imidazoleacetic acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro by S. Wadud; R. Onodera; M. Or-Rashid (pp. 219-225).
The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39°C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 µmol/g MN per hour), followed by BP (33.13 µmol/g MN per hour) and then P (18.6 µmol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 µmol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 µmol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4–6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.
Influence of glucose on glycerol metabolism by wild-type and mutant strains of Clostridium butyricum E5 grown in chemostat culture by Hassiba Malaoui; Régis Marczak (pp. 226-233).
In order to improve the yield of 1,3-propanediol (1,3-PPD) in Clostridium butyricum E5, we carried out cofermentation experiments on glucose/glycerol mixtures in chemostat culture. The results showed the influence of the ratio of the two carbon substrates on the production of the required diol. The progressive increase of glucose in culture medium containing a given concentration of glycerol made it possible to highlight the deviation of carbon flow from the oxidative towards the reducing pathway, in order to maintain the oxidation/reduction balance in the cell. The conversion of glycerol into 1,3-PPD thus increased from 0.63 mol mol–1, without the addition of glucose, to a maximum of 0.89 mol mol–1 for a molar glucose/glycerol ratio of 0.2 for the wild-type strain. The same experiments carried out with the mutant MD strain, which is resistant to allyl alcohol, led to similar results but with a maximum of 0.84 mol mol–1 for a glucose/glycerol molar ratio of 0.1. Beyond a molar ratio of 0.2, the biosynthesis of enzymes for the glycerol metabolism was less subject to catabolic repression by glucose in the mutant MD strain than in the wild-type strain.
A calorimetrically based method to convert toxic compounds into poly-3-hydroxybutyrate and to determine the efficiency and velocity of conversion by T. Maskow; W. Babel (pp. 234-238).
A fed-batch method for converting toxic substrates into poly-3-hydroxybutyrate is presented. The method involves a series of batch-growth processes, regulated by adding small amounts of carbon substrate, during the course of which the concentration of the nitrogen source decreases and controls the distribution of the substrate-carbon assimilated. The addition of carbon substrate is controlled, and the small changes that occur in the growth pattern are interpreted using high-resolution reaction calorimetry. The method was tested with Ralstonia eutropha DSM 4058 growing on phenol, and Variovorax paradoxus DSM 4065 growing on sodium benzoate. The maximum carbon conversion efficiencies (CCEs) obtained, 23% and 27% respectively,were compared with the theoretically possible values.
Dehalogenation of dichloroethene in a contaminated soil: fatty acids and alcohols as electron donors and an apparent requirement for tetrachloroethene by Nelson R. Villarante; Piero M. Armenante; Titos A. Quibuyen; Fabio Fava; David Kafkewitz (pp. 239-247).
Environmental soil contamination at an industrial site in Marion, Ohio (USA) with tetrachloroethene (perchloroethene, PCE) resulted in residual cis-1, 2-dichloroethene (DCE) contamination that had not declined after more than 15 years. Microcosm slurries containing 2.6% soil from this site were supplemented with different electron donors, i.e., individual fatty acids or alcohols. None of the microcosms supported complete DCE dechlorination, unless PCE was added to the microcosm at initiation. The addition of fresh PCE resulted in the dehalogenation of PCE to DCE in the microcosms supplemented with fatty acids having an even number of carbon atoms (acetate, butyrate, and caproate), but not in those with an odd number of carbon atoms (formate, propionate, and valerate), where negligible or no activity was detected. No significant further DCE degradation was observed in any of the microcosms supplied with fatty acids as electron donors. Microcosms supplemented with freshly added PCE bioconverted PCE to DCE and completely dehalogenated both the ex-novo and soil-supplied DCE within 60 days, but only if alcohols having an even number of carbon atoms (ethanol or butanol) were also added as electron donors. Odd-numbered alcohols either did not produce dehalogenation (as with methanol) or only dehalogenated PCE to DCE (as with propanol).
Diversity in kinetics of trichloroethylene-degrading activities exhibited by phenol-degrading bacteria by Hiroyuki Futamata; Shigeaki Harayama; Kazuya Watanabe (pp. 248-253).
Whole-cell kinetics of phenol- and trichloroethylene (TCE)-degrading activities expressed by 13 phenol-degrading bacteria were analyzed. The K s (apparent affinity constant in Haldane's equation) values for TCE were unexpectedly diverse, ranging from 11 µM to over 800 µM. The V max/K s values for phenol were three orders of magnitude higher than the values for TCE in all bacteria analyzed, suggesting that these bacteria preferentially degrade phenol rather than TCE. A positive correlation between K s for phenol and K s for TCE was found, i.e., bacteria exhibiting high K s values for phenol showed high K s values for TCE, and vice versa. A comparison of the K s values allowed grouping of these bacteria into three types, i.e., low-, moderate- and high-K s types. Pseudo-first-order degradation-rate constants for TCE at 3.8 µM were found to be adequate to rapidly discriminate among the three types of bacteria. When bacteria were grown on phenol at the initial concentration of 2 mM, Comamonas testosteroni strain R5, a representative of low-K s bacteria, completely degraded TCE at 3.8 µM, while strain P-8, a representative of high-K s bacteria, did not. A mixed culture of these two bacteria poorly degraded TCE under the same conditions, where P-8 outgrew R5. These results suggest that low-K s bacteria should be selectively grown for effective bioremediation of TCE-contaminated groundwater.
Parameters affecting performance and modeling of biofilters treating alkylbenzene-polluted air by M. Veiga; C. Kennes (pp. 254-258).
Both short-term and long-term biofiltration experiments were undertaken with a biofilter inoculated with a defined microbial consortium and treating an alkylbenzene mixture. The results obtained with such a biofilter in short-term experiments were very similar to those obtained with a biofilter inoculated with a non-defined mixed culture, in terms of maximum elimination capacities (70–72 g m–3 h–1) and the corresponding removal efficiencies (>95%). However, in long-term experiments, a better performance was reached, with a maximum elimination capacity of 120 g m–3 h–1, corresponding to a removal efficiency >99% after 2 years of operation. Inoculation proved to be useful for shortening the start-up period. In the long term, it appeared that biomass distribution was not homogenous along the biofilter, which in some cases resulted in a bad fit between simple model equations and experimental data.