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Applied Microbiology and Biotechnology (v.53, #4)

Alternative sources of natural rubber by H. Mooibroek; K. Cornish (pp. 355-365).

Rubber (cis-1,4-polyisoprene) is one of the most important polymers naturally produced by plants because it is a strategic raw material used in more than 40,000 products, including more than 400 medical devices. The sole commercial source, at present, is natural rubber harvested from the Brazilian rubber tree, Hevea brasiliensis. Primarily due to its molecular structure and high molecular weight (>1 million daltons) this rubber has high performance properties that cannot easily be mimicked by artificially produced polymers, such as those derived from, e.g., bacterial poly-hydroxy-alkanoates (PHAs). These high performance properties include resilience, elasticity, abrasion resistance, efficient heat dispersion (minimizing heat build-up under friction), and impact resistance. Medical rubber gloves need to fit well, be break-resistant, allow the wearer to retain fine tactile sensation, and provide an effective barrier against pathogens. The sum of all these characteristics cannot yet be achieved using synthetic gloves. The lack of biodiversity in natural rubber production renders continuity of supply insecure, because of the risk of crop failure, diminishing acreage, and other disadvantages outlined below. A search for alternative sources of natural rubber production has already resulted in a large number of interesting plants and prospects for immediate industrial exploitation of guayule (Parthenium argentatum) as a source of high quality latex. Metabolic engineering will permit the production of new crops designed to accumulate new types of valued isoprenoid metabolites, such as rubber and carotenoids, and new combinations extractable from the same crop. Currently, experiments are underway to genetically improve guayule rubber production strains in both quantitative and qualitative respects. Since the choice for gene activities to be introduced or changed is under debate, we have set up a complementary approach to guayule with yeast species, which may more quickly show the applicability and relevance of genes selected. Although economic considerations may prevent commercial exploitation of new rubber-producing microorganisms, transgenic yeasts and bacteria may yield intermediate or alternative (poly-)isoprenes suitable for specific applications.

Perspectives in the biological function and the technological application of polygalacturonases by C. Lang; H. Dörnenburg (pp. 366-375).

Polygalacturonases (PG) have evolved in the past years from a pectinase “simply” being used for food processing to an important parameter in plant–fungal interaction. PG-inhibiting proteins (PGIP) that are synthesised in plants as a specific response to PGs of pathogenic fungi, have become a focus as a possible target in resistance breeding, and PGIPs are also a concern as an inhibiting factor in food processing. Plant PGs have been identified as a major factor in fruit ripening, and PG-deficient transgenic plants have been bred. Mainly fungal PGs are used in industrial processes for juice clarification and the range of enzymes is being extended through new recombinant and non-recombinant fungal strains. Finally, novel fields of application can be envisaged for PGs in the production of oligogalacturonides as functional food components. Here we aim to highlight the various fields where PGs are encountered and where they are of biological or technological importance.

Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Saccharomyces cerevisiae by A. Eliasson; E. Boles; B. Johansson; M. Österberg; J. M. Thevelein; I. Spencer-Martins; H. Juhnke; B. Hahn-Hägerdal (pp. 376-382).

Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)⁻¹] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)⁻¹]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1Δ) in the PPP and deletion of trehalose 6-phosphate synthase (tps1Δ) together with trehalose 6-phosphate phosphatase (tps2Δ) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) – resulting in reduced enzyme activities – increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1Δ) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)⁻¹. Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)⁻¹ (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)⁻¹. Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol.

Axenic cultivation of anoxygenic phototrophic bacteria, cyanobacteria, and microalgae in a new closed tubular glass photobioreactor by T. Hai; H. Ahlers; V. Gorenflo; A. Steinbüchel (pp. 383-389).

A low-cost closed tubular glass photobioreactor allowing axenic cultivation of phototrophic microorganisms was constructed. Standard glass tubes were arranged in a helical array providing a working volume of 80 l. The glass tubes were connected with a degassing chamber, which also provided ports for measuring and regulating oxygen supply, pH, foam, and optical density and for adding substrates and antifoam agents as well as disposing of vent gas. A pump module allowed agitation of the medium in the bioreactor at a laminar flow rate of 1.5 m/s. Upstream of the pump module a gas inlet was located, allowing efficient mixing of the used gases with the medium. The temperature of the medium was controlled by a Pt-100 sensor and by a heat exchanger with an effective surface of 0.12 m² connected to an external thermostat. Irradiation was provided by three light panels each consisting of ten fluorescent tubes. The entire photobioreactor – apart from the light panels and motor – could be sterilized at 121 °C in an autoclave. In addition to a detailed description of this photobioreactor, we report on first experiments to cultivate the anoxygenic phototrophic bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum, the oxygenic phototrophic cyanobacterium Synechocystis sp. strain PCC6803, and the microalga Chlorella sp. in this photobioreactor.

Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase by M. Aoyama; M. Yasuda; K. Nakachi; N. Kobamoto; H. Oku; F. Kato (pp. 390-395).

A proteolytic enzyme from Bacillus pumilus strain TYO-67, which was able to coagulate the protein in soybean milk, was characterized enzymologically. The optimum pH and temperature for its activities were 9.0 and 50 °C, respectively. The enzyme was strongly believed to be a serine proteinase because it was completely inhibited by the addition of diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride. Hammerstein milk casein, cytochrome c and soybean protein were good substrates for the enzyme. Seven cleavages were detected using the oxidized insulin B-chain as peptide substrate for the proteolytic specificity test of the serine proteinase from B. pumilus. The bonds most susceptible to the action of the serine proteinase from B. pumilus were Leu-15–Tyr-16. The mode of action on soybean milk protein by the enzyme from B. pumilus was also investigated. The acidic subunit in glycinin and the α′-, α- and β-subunits in β-conglycinin were degraded during the enzyme reaction. However, the basic subunit in glycinin could not be degraded by the enzyme. The formation of coagula in soybean milk caused by the serine proteinase from B. pumilus was mainly due to the hydrophobic interaction.

Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase by P. D. Matthews; E. T. Wurtzel (pp. 396-400).

The recently discovered non-mevalonate pathway to isoprenoids, which uses glycolytic intermediates, has been modulated by overexpression of Escherichia coli d-1-deoxyxylulose 5-phosphate synthase (DXS) to increase deoxyxylulose 5-phosphate and, consequently, increase the isoprenoid precursor pool in E. coli. Carotenoids are a large class of biologically important compounds synthesized from isoprenoid precursors and of interest for metabolic engineering. However, carotenoids are not ordinarily present in E. coli. Co-overexpression of E. coli dxs with Erwinia uredovora gene clusters encoding carotenoid biosynthetic enzymes led to an increased accumulation of the carotenoids lycopene or zeaxanthin over controls not expressing DXS. Thus, rate-controlling enzymes encoded by the carotenogenic gene clusters are responsive to an increase in isoprenoid precursor pools. Levels of accumulated carotenoids were increased up to 10.8 times the levels of controls not overexpressing DXS. Lycopene accumulated to a level as high as 1333 μg/g dw and zeaxanthin accumulated to a level as high as 592 μg/g dw, when pigments were extracted from colonies. Zeaxanthin-producing colonies grew about twice as fast as lycopene-producing colonies throughout a time course of 11 days. Metabolic engineering of carbon flow from simple glucose metabolites to representatives of the largest class of natural products was demonstrated in this model system.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3 by H. Matsusaki; H. Abe; K. Taguchi; T. Fukui; Y. Doi (pp. 401-409).

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA_Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB_Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1 _Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB⁻4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1 _Ps gene only. In addition, recombinant strains of R. eutropha PHB⁻4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB⁻4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units.

His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening by S. A. Guerrero; J. A. Lopez; P. Steinert; M. Montemartini; H. M. Kalisz; W. Colli; M. Singh; M. J. M. Alves; L. Flohé (pp. 410-414).

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohé L (1997) Biol Chem 378: 827–836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 1 l fermentation broth of A ₆₀₀ 2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b(+)/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k _cat of 1.7 s⁻¹ and limiting K _m values of 51.8 μM and 1.7 μM for t-butyl hydroperoxide and CfTXN2H6, respectively.

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis by H. Visser; S. Vreugdenhil; J. A. M. de Bont; J. C. Verdoes (pp. 415-419).

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min⁻¹ (mg protein)⁻¹ towards 1,2-epoxyhexane.

Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems by T. Clemente; D. Shah; M. Tran; D. Stark; S. Padgette; D. Dennis; K. Brückener; A Steinbüchel; T. Mitsky (pp. 420-429).

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase.

Cloning of the cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. A2-5a and analysis of the raw starch-binding domain by K. Ohdan; T. Kuriki; H. Takata; S. Okada (pp. 430-434).

The cyclodextrin glucanotransferase (CGTase) gene of alkalophilic Bacillus sp. A2-5a was cloned and expressed in Bacillus subtilis ANA-1 as a host. The DNA region included an open reading frame encoding a 704-amino-acid polypeptide with a typical raw starch-binding motif in its C-terminal region. The CGTase purified from Bacillus sp. A2-5a bound to raw starch as strongly as porcine pancreas α-amylase, as expected from the sequence motif. A chromosomal region (a DNA fragment of about 14.1 kbp) including the CGTase gene was also cloned and the nucleotide sequence was determined. Possible cyclodextrinase and putative cyclodextrin-binding protein genes were found in the flanking region of the CGTase gene, which implied that the novel starch-degradation pathway postulated for a gram-negative bacterium [Klebsiella oxytoca; Fiedler et al. (1996) J Mol Biol 256: 279–291] also exists in a gram-positive bacterium i.e. Bacillus.

Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp.shermanii by E. H. Himmi; A. Bories; A. Boussaid; L. Hassani (pp. 435-440).

A comparative study was carried out in anaerobic batch cultures on 20 g/l of either glycerol or glucose using two propionibacteria strains, Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. In all cases, fermentation end-products were the same and consisted of propionic acid as the major product, acetic acid as the main by-product and two minor metabolites, n-propanol and succinic acid. Evidence was provided that greater production of propionic acid by propionibacteria was obtained with glycerol as carbon and energy sources. P. acidipropionici showed higher efficiency in glycerol conversion to propionic acid with a faster substrate consumption (0.64 g l⁻¹ h⁻¹) and a higher propionic acid production (0.42 g l⁻¹ h⁻¹ and 0.79 mol/mol). The almost exclusive production of propionic acid from glycerol by this bacterium suggested an homopropionic tendency of this fermentation. Acetic acid final concentration was two times lower on glycerol (2 g/l) than on glucose (4 g/l) for both micro-organisms. P. freudenreichii ssp. shermanii exhibited a glycerol fermentation pattern typical of non-associated glycerol-consumption-product formation. This could indicate a particular metabolism for P. freudenreichii ssp. shermanii oriented towards the production of other specific components. These results tend to show that glycerol could be an excellent alternative to conventional carbon sources such as carbohydrates for propionic acid production.

Formation and degradation of a synthetic humic acid derived from 3-fluorocatechol by U. Wunderwald; G. Kreisel; M. Braun; M. Schulz; C. Jäger; M. Hofrichter (pp. 441-446).

A synthetic fluorinated humic acid (FHA) was prepared by the spontaneous oxidative polymerization of 3-fluorocatechol. The ¹³C-solid-state NMR spectrum showed signals in the region for aromatic carbons with different substituents (aryl-H, aryl-C, aryl-O carbons) and for carboxyl-carbon. The latter indicated the formation of carboxylic groups, probably caused by ring cleavages during the polymerization process. An indication of the formation of carboxylic groups was also found in the infrared spectrum (band at 1715 cm⁻¹). The dissolved FHA was degraded with active mycelium of the agaric white-rot fungus Nematoloma frowardii as well as with its isolated manganese peroxidase. In both cases, decolorization of the brownish FHA solution and partial defluorination (45–60%) took place. Degradation proceeded via formation of lower-molecular-mass fulvic acid-like substances. The results demonstrate that halogenated humic substances, e.g., resulting from the humification of xenobiotic compounds (bound residues), can in principle be eliminated by ligninolytic fungi (e.g., soil colonizing litter decomposers) and their manganese peroxidase system.

Evidence for diverse oxidations in the catabolism of toluene by Rhodococcus rhodochrous strain OFS by L. A. Vanderberg; R. Krieger-Grumbine; M. N. Taylor (pp. 447-452).

Rhodococcus rhodochrous strain OFS grew on toluene as a sole source of carbon and energy with a maximum growth rate of 0.011 h⁻¹. Initial reaction products were extracted, derivatized and identified by GC-MS. Oxygen consumption studies indicated that OFS grown on an aliphatic substrate required an induction period before oxidizing toluene. OFS grown on toluene transformed an array of aromatic ground water pollutants including styrene, ethylbenzene and chlorobenzene. Products of these transformations were identified. The sole product of chlorobenzene biotransformation was 4-chlorophenol. Products from toluene oxidation included 3- and 4-methylcatechol as well as benzyl alcohol, p-cresol and cis-toluene dihydrodiol. The identification of these and the products of other aromatic substrate conversions affirm that oxidation occurred on the functional group as well as directly on the aromatic nucleus.

Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids by M. F. de Andrade Rodrigues; H. E. Valentin; P. A. Berger; M. Tran; J. Asrar; K. J. Gruys; A. Steinbüchel (pp. 453-460).

Burkholderia sp. accumulates polyhydroxyalkanoates (PHAs) containing 3-hydroxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media under limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source. Solvent fractionation and NMR spectroscopic characterization of these polyesters revealed the simultaneous accumulation of two homopolyesters rather than a co-polyester with random sequence distribution of the monomers [Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbüchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389–7395]. To understand the genetic requirements for such unusual polyester accumulation, we probed total genomic DNA from Burkholderia sp. by Southern hybridization experiments using phaC-specific probes. These experiments indicated the presence of more than one PHA synthase gene within the genome of Burkholderia sp. However, when total genomic DNA from Burkholderia sp. was used to complement a PHA-negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA synthase gene was obtained resembling the R. eutropha type of PHA synthases, based on amino acid sequence similarity. In addition to the PHA synthase gene, based on high sequence homology, genes encoding a β-ketothiolase and acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene. The arrangement of the three genes is quite similar to the R. eutropha poly-β-hydroxybutyrate biosynthesis operon.

Simultaneous production of high activities of thermostable endoglucanase and β-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus by I. Gomes; J. Gomes; D. J. Gomes; W. Steiner (pp. 461-468).

The culture-medium composition was optimised, on a shake-flask scale, for simultaneous production of high activities of endoglucanase and β-glucosidase by Thermoascus aurantiacus using statistical factorial designs. The optimised medium containing 40.2 g l⁻¹ Solka Floc as the carbon source and 9 g l⁻¹ soymeal as the organic nitrogen source yielded 1130 nkat ml⁻¹ endoglucanase and 116 nkat ml⁻¹β-glucosidase activities after 264 h as shake cultures. In addition, good levels of β-xylanase (3479 nkat ml⁻¹) and low levels of filter-paper cellulase, β-xylosidase, α-l-arabinofuranosidase, β-mannanase, β-mannosidase, α-galactosidase and β-galactosidase were detected. Batch fermentation in a 5-l laboratory fermentor using the optimised medium allowed the production of 940 nkat ml⁻¹ endoglucanase and 102 nkat ml⁻¹β-glucosidase in 192 h. Endoglucanase and β-glucosidase showed optimum activity at pH 4.5 and pH 5, respectively, and they displayed optimum activity at 75 °C. Endoglucanase and β-glucosidase showed good stability at pH values 4–8 and 4–7, respectively, after a prolonged incubation (48 h at 50 °C). Endoglucanase had half-lives of 98 h at 70 °C and 4.1 h at 75 °C, while β-glucosidase had half-lives of 23.5 h at 70 °C and 1.7 h at 75 °C. Alkali-treated bagasse, steam-treated wheat straw, Solka floc and Sigmacell 50 were 66, 48.5, 33.5 and 14.4% hydrolysed by a crude enzyme complex of T. aurantiacus in 50 h.

Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation by J. Pratap; G. Rajamohan; K. L. Dikshit (pp. 469-475).

Degradation of streptokinase (SK) has been frequently observed during large-scale protein production. An enhanced susceptibility of SK to degradation has been correlated with its existence in a partially unfolded state. The influence of the carbohydrate moiety on the stability and functional characteristics of SK has been examined by obtaining the glycoform of SK following its secretion through the methylotrophic yeast Pichia pastoris. Secretion of the protein product was achieved by replacing the native secretion signal codons of SK with those from α-factor leader peptide and expressing the fusion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P. pastoris after its integration into the host chromosome. Western blot and zymographic analysis of proteins secreted from the recombinant P. pastoris indicated that SK was glycosylated by the host cells, which resulted in the appearance of a SK species migrating slowly, corresponding to a 55-kDa protein product as compared to the 47-kDa native SK. The glycosylated SK retained a plasminogen activation capability identical to that of its unglycosylated counterpart. Glycoform SK exhibited an enhanced stability profile at 25 °C and 37 °C and improved resistance towards protease treatment compared to unglycosylated SK secreted through P. pastoris after tunicamycin treatment or that secreted from the recombinant Escherichia coli. The results presented thus illustrate that N-linked glycosylation of SK results in 30–40% enhancement of the protein stability and resistance towards degradation but does not interfere with its fibrinolytic function.

Combined action of redox potential and pH on heat resistance and growth recovery of sublethally heat-damaged Escherichia coli by C. Riondet; R. Cachon; Y. Waché; E. Sunyol i Bert; P. Gbaguidi; G. Alcaraz; C. Diviès (pp. 476-479).

The combined effect of redox potential (RP) (from −200 to 500 mV) and pH (from 5.0 to 7.0) on the heat resistance and growth recovery after heat treatment of Escherichia coli was tested. The effect of RP on heat resistance was very different depending on the pH. At pH 6.0, there was no significant difference, whereas at pH 5.0 and 7.0 maximum resistance was found in oxidizing conditions while it fell in reducing ones. In sublethally heat-damaged cells, low reducing and acid conditions allowed growth ability to be rapidly regained, but a decrease in the redox potential and pH brought about a longer lag phase and a slower exponential growth rate, and even led to growth failure (pH 5.0, ≤−100 mV).

APHE-3, a spore-associated antibiotic of Streptomyces griseocarneus NCIMB 40447 by R. Cruz; M. E. Arias; J. Soliveri (pp. 480-483).

Solid cultures of the producing strain grown on Bennett medium develop abundant mycelium and intense sporulation. Under these conditions biosynthesis of APHE antibiotics (APHE-1 to -3) is accomplished. Further studies show that APHE-3 is basically produced during spore formation and mostly present in spores, while APHE-1 and APHE-2 are the predominant antibiotics in the mycelium. APHE compounds are present in almost all streptomycetes tested, indicating a possible role in the life cycle of these microorganisms.

Dechlorination of polychlorinated methanes by a sequential methanogenic-denitrifying bioreactor system by Z. Yu; G. B. Smith (pp. 484-489).

A two-stage bioreactor has been developed to link dechlorination of halogenated methane compounds to the anaerobic processes of methanogenesis and denitrification. A digester methanogenic consortium was shown to dechlorinate chloroform (CF) and carbon tetrachloride (CT) to dichloromethane (DCM), and DCM was then mineralized by an acclimated denitrifying biological activated carbon consortium. Combining these two processes, a sequential methanogenic-denitrifying bioreactor (SMDB) system that completely degraded polychlorinated methanes including CT, CF, and DCM was developed. More than 95% of the added CT and CF was dechlorinated in the methanogenic bioreactor with methanol as the primary substrate, and the resultant DCM was biodegraded in the denitrifying bioreactor with nitrate as the electron acceptor. In the denitrifying bioreactor, the residual CF was completely removed, and the DCM removal efficiency was more than 95%. This novel bioreactor system eliminates the need for aeration and so avoids the air contamination associated with aerobic biotreatment of volatile chlorinated pollutants. This SMDB system provides an alternative to conventional biotreatment of wastewaters and other matrices contaminated with polychlorinated methanes and is, to our knowledge, the first report on such a sequential anoxic system.

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Applied Microbiology and Biotechnology (v.53, #4)

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Applied Microbiology and Biotechnology (v.53, #4)