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Applied Microbiology and Biotechnology (v.52, #3)

Microbial production of 1,3-propanediol by H. Biebl; K. Menzel; A.-P. Zeng; W.-D. Deckwer (pp. 289-297).

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO₂, H₂, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH₂, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a “green” polyester is a more cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism.

Biosynthesis of gibberellins in Gibberella fujikuroi: biomolecular aspects by B. Tudzynski (pp. 298-310).

Gibberellins (GAs) are a large family of isoprenoid plant hormones, some of which are bioactive growth regulators, controlling seed germination, stem elongation, and flowering. The rice pathogen Gibberella fujikuroi (mating population C) is able to produce large amounts of GAs, especially the bioactive compounds gibberellic acid (GA₃) and its precursors, GA₄ and GA₇. The main steps of the biosynthetic pathway have long been established from the identification of intermediates in wild-type G. fujikuroi and mutant strains. However, the genetics of the fungus have been rather under-developed, and molecular genetic studies of the GA pathway started just recently. The progress in researching GA biosynthesis in the last 2 years resulted primarily from development of the molecular tools, e.g. transformation systems for the fungus, and cloning the genes encoding GA biosynthesis enzymes, such as the bifunctional ent-copalyl diphosphate/kaurene synthase and several cytochrome P450 monooxygenases. The availability of these genes opened new horizons both for detailed study of the pathway and the regulation mechanisms at the molecular level, and for modern strain improvement programs. This review gives a short overview of the well-known physiological and biochemical studies and concentrates mainly on the new molecular genetic data from GA research, including new information on the regulation of GA biosynthesis.

Yeast cells as tools for target-oriented screening by T. Munder; A. Hinnen (pp. 311-320).

Information about biomolecular interaction networks is crucial for understanding cellular functions and the development of disease processes. Many diseases are known to be based on aberrations of DNA sequences encoding proteins with key functions in the cellular metabolism. Alterations in the respective proteins often lead to disturbances in biomolecular interactions caused by unbalanced stoichiometries, and thus result in alterations of molecule fluxes, cell architecture and signalling pathways. Drug discovery programmes have been designed to find promising chemical lead structures with the help of target-oriented bioassay systems. These are, in most cases, based upon the interaction of small molecules to specific macromolecular targets in vivo or in vitro, as exemplified by enzyme assays or small-ligand-based receptor systems. In addition, interactions between large biomolecules, such as proteins or nucleic acids, offer a huge arsenal of potential drug targets that can be addressed by small chemical compounds. This latter approach is gaining considerable attention because many potential target structures are becoming available through genomic research. Funnelling these new targets into high-throughput screening programs represents a major challenge for today's pharmaceutical research. An important outcome of the ongoing genome projects is the fact that the basic cellular structures, pathways and signalling principles show a high degree of conservation. Model organisms that are easily approachable by genetic, biochemical and physiological means can thus play an important role in the design of target-oriented screening systems. They offer the possibility to express individual proteins, nucleic acids or even more complex aggregates of biomolecules such as protein-interaction networks or transcription-initiation complexes, which can be addressed by small effector molecules in vivo. Combining these targets with biological signalling systems is an attractive way of creating robust cellular assay systems.

Production of 2,3-butanediol by newly isolated Enterobacter cloacae by B. C. Saha; R. J. Bothast (pp. 321-326).

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of l-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars).

Production of (R)-3-pentyn-2-ol through stereoinversion of racemic 3-pentyn-2-ol by Nocardia fusca AKU 2123 by S.-X. Xie; J. Ogawa; S. Shimizu (pp. 327-331).

Wet cells of Nocardia fusca AKU 2123 are good catalysts for the production of (R)-3-pentyn-2-ol (PYOH) from (RS)-PYOH through a stereoinversion reaction. Under optimal conditions (350 mM potassium phosphate buffer, pH 8.0, 30% (w/v) wet cells, 0.12% NADPH, 10% glucose, and 30 U/ml glucose dehydrogenase) (R)-PYOH of high optical purity (98.7% e.e.) was produced from 2% (v/v) (RS)-PYOH with a yield of 70.4% by 140 h incubation.

Succinoglycan production by solid-state fermentation with Agrobacterium tumefaciens by M. Stredansky; E. Conti (pp. 332-337).

Succinoglycan was produced by cultivating Agrobacterium tumefaciens on various solid substrates, including agar medium, spent malt grains, ivory nut shavings, and grated carrots, impregnated with a nutrient solution. Fermentations were performed on a laboratory scale, both under static conditions and with agitation, using bottles and a prototype horizontal bioreactor. Several fermentation parameters were examined and optimized, including carbon and nitrogen composition, water content and layer thickness of the substrate. The yields and rheological properties of the polymers obtained under different fermentation conditions were compared. The highest succinoglycan yield was achieved in static cultivation, reaching 42 g/l of impregnating solution, corresponding to 30 g/kg of wet substrate. The polymer production in the horizontal bioreactor was faster, but the final yield was lower (29 g/l of impregnating solution).

Improved process for production of recombinant yeast-derived monomeric human G-CSF by C. S. Bae; D. S. Yang; J. Lee; Y.-H. Park (pp. 338-344).

The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction. It was hardly monomerized even by urea at high concentration. This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process. When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secretion efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5). Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography. A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents.

An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells by C. Burger; M. J. T. Carrondo; H. Cruz; M. Cuffe; E. Dias; J. B. Griffiths; K. Hayes; H. Hauser; D. Looby; C. Mielke; J.-L. Moreira; E. Rieke; A. V. Savage; G. N. Stacey; T. Welge (pp. 345-353).

Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. “mono-”, “bi-” and “tri-cistronic” vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in anti-tumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation – time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals.

Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor by L. D. Collins; A. J. Daugulis (pp. 354-359).

A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-l sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l⁻¹ h⁻¹. This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH.

Benzene/toluene/p-xylene degradation. Part II. Effect of substrate interactions and feeding strategies in toluene/benzene and toluene/p-xylene fermentations in a partitioning bioreactor by L. D. Collins; A. J. Daugulis (pp. 360-365).

A two-phase aqueous/organic partitioning bioreactor scheme was used to degrade mixtures of toluene and benzene, and toluene and p-xylene, using simultaneous and sequential feeding strategies. The aqueous phase of the partitioning bioreactor contained Pseudomonas sp. ATCC 55595, an organism able to degrade benzene, toluene and p-xylene simultaneously. An industrial grade of oleyl alcohol served as the organic phase. In each experiment, the organic phase of the bioreactor was loaded with 10.15 g toluene, and either 2.0 g benzene or 2.1 g p-xylene. The resulting aqueous phase concentrations were 50 mg/l, 25 mg/l and 8 mg/l toluene, benzene and p-xylene respectively. The simultaneous fermentation of benzene and toluene consumed these compounds at volumetric rates of 0.024 g l⁻¹ h⁻¹ and 0.067 g l⁻¹ h⁻¹, respectively. The simultaneous fermentation of toluene and p-xylene consumed these xenobiotics at volumetric rates of 0.066 g l⁻¹ h⁻¹ and 0.018 g l⁻¹ h⁻¹, respectively. A sequential feeding strategy was employed in which toluene was added initially, but the benzene or p-xylene aliquot was added only after the cells had consumed half of the initial toluene concentration. This strategy was shown to improve overall degradation rates, and to reduce the stress on the microorganisms. In the sequential fermentation of benzene and toluene, the volumetric degradation rates were 0.056 g l⁻¹ h⁻¹ and 0.079 g l⁻¹ h⁻¹, respectively. In the toluene/p-xylene sequential fermentation, the initial toluene load was consumed before the p-xylene aliquot was consumed. After 12 h in which no p-xylene degradation was observed, a 4.0-g toluene aliquot was added, and p-xylene degradation resumed. Excluding that 12-h period, the microbes consumed toluene and p-xylene at volumetric rates of 0.074 g l⁻¹ h⁻¹ and 0.025 g l⁻¹ h⁻¹, respectively. Oxygen limitation occurred in all fermentations during the rapid growth phase.

Comparative assessment of chelated spent mushroom substrates as casing material for the production of Agaricus bisporus by H. S. S. Sharma; A. Furlan; G. Lyons (pp. 366-372).

Spent mushroom substrate (SMS) leached with water or treated with chelating agents to remove metal cations, pasteurised to remove any harmful micro-organisms and mixed with peat has potential as a casing material for mushroom production. The microbial and chemical changes in SMS after treatment with citric acid, ethylene diaminetetraacetic acid (EDTA) and water were compared; treatment with the chelating agents resulted in lower ash content, conductivity and minerals, higher fibre fractions, carbon, hydrogen and nitrogen. The microbial and chemical changes in the materials after treatment with the two chelators and water were compared. Blending peat with the heat-treated materials at a ratio of 1:1 resulted in improved physical properties. The casings prepared from the test materials and the control, consisting of 100% peat, were compared after neutralising with lime for their productivity in a mushroom yield trial. As expected, the compost bags cased with the control were the most productive compared to the other casings. Of the three treated materials, casing prepared from SMS treated with EDTA blended with peat was the most productive. Dry matter of harvested mushrooms from chelated-SMS casings was significantly higher than the control casing. Comparison of the main components of peat and chelated SMS revealed that the major differences were in the proportions of ash, lipid, lignin and fibre fractions. The stability of some of these components, when complexed with metal cations present in lime may play an important role in determining the composition of the cell wall in fruiting bodies leading to high dry matter content.

Immunoaffinity layering of enzymes by M. Farooqi; P. Sosnitza; M. Saleemuddin; R. Ulber; T. Scheper (pp. 373-379).

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion by M. J. van der Werf; R. V. A. Orru; K. M. Overkamp; H. J. Swarts; I. Osprian; A. Steinreiber; J. A. M. de Bont; K. Faber (pp. 380-385).

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1R,2S)- and then the (1S,2R)-isomer was observed. The hydrolysis of (4R)- and (4S)-limonene-1,2-epoxide resulted in, respectively, (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities (E ratios between 34 and 3).

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10 by J.-C. Wang; M. Sakakibara; J.-Q. Liu; T. Dairi; N. Itoh (pp. 386-392).

The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20–29% identity with zinc-containing, NAD⁺-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD⁺- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity.

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature by P. Cassland; L. J. Jönsson (pp. 393-400).

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance.

Genetic transformation of a Rhizomucor pusillus mutant defective in asparagine-linked glycosylation: production of a milk-clotting enzyme in a less-glycosylated form by H. Yamazaki; Y. Ohnishi; K. Takeuchi; N. Mori; N. Shiraishi; Y. Sakata; H. Suzuki; S. Horinouchi (pp. 401-409).

Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura⁺ transformants with 1 μg pRPPyr4 DNA and 1 × 10⁷ viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man_0∼1GlcNAc₂ at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.

Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris by Y. Sakuma; M. Kimura; T. Takabatake; K. Takeshima; H. Fujimura (pp. 410-414).

We have successfully secreted the amino-terminal functional domain of mouse sonic hedgehog protein (SHH) into culture fluid using a yeast Pichiapastoris expression system. A cDNA fragment encoding the amino-terminal domain of mouse SHH was inserted downstream of the Saccharomyces cerevisiaeα-mating factor secretion signal. The DNA fragment was introduced into the host genome by the spheroplast transformation method. Transformants were selected based on their resistance to G418. His⁺ transformants which showed resistance to over 8 mg G418/ml were selected and analyzed for determination of the plasmid copy number. One His⁺ clone which has eight copies of the expression cassette per genome was cultured in minimal medium deficient for histidine, and further cultured in buffered medium supplemented with methanol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficient expression and secretion of mouse SHH into culture fluid. The yield of secreted SHH was estimated to be 50 μg/ml. Purified protein was assayed for biological activity and found to activate the transcription of the Patched genes (Ptc-1 and Ptc-2) encoding receptors for SHH.

β-Poly(l-malate) production by Physarum polycephalum by B.-S. Lee; T. Maurer; H. R. Kalbitzer; E. Holler (pp. 415-420).

β-Poly(l-malate) (PMLA) production in Physarum polycephalum has been followed by using d-[1-¹³C]glucose and Ca¹³CO₃. Nuclear magnetic resonance studies of PMLA showed that the ¹³C label from [1-¹³C]glucose was incorporated in the presence of CaCO₃ into positions C-3 (-CH₂-) and C-4 (-CO-) of the l-malate repeating unit of PMLA. The ¹³C label from Ca¹³CO₃ was incorporated into position C-4 and indicated that not only the endogenous CO₂ but also the exogenous CO₂ from CaCO₃ served significantly as a carbon source for PMLA production. In the absence of CaCO₃, the ¹³C labeling pattern of PMLA from d-[1-¹³C]glucose was almost indistinguishable from that for the natural abundance ¹³C-NMR spectrum of the polymer. These results indicated that l-malate used for PMLA production is synthesized either via carboxylation of pyruvate and reduction of oxaloacetate in the presence of CaCO₃ or via the oxidative tricarboxylic acid (TCA) cycle in the absence of CaCO₃. Avidin strongly inhibited the formation of l-malate via carboxylation; the ¹³C labeling pattern of PMLA in the presence of CaCO₃ was almost identical with that for the natural abundance spectrum when avidin was added, indicating that l-malate utilized for PMLA production was supplied under this condition by the oxidative TCA cycle.

Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture by K. Kirimura; S. Yusa; S. Rugsaseel; H. Nakagawa; M. Osumi; S. Usami (pp. 421-428).

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like α-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semi-solid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 μg/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electron-microscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.

Endogenous elicitor-like effects of alginate on physiological activities of plant cells by C. Akimoto; H. Aoyagi; H. Tanaka (pp. 429-436).

The effects of alginate on the physiological activities of plant cells were studied. Addition of alginate oligomer (AO) to the suspension culture of Catharanthus roseus L. or Wasabia japonica cells promoted the production of antibiotic enzymes such as 5′-phosphodiesterase or chitinase respectively. Ajmalicine (a secondary metabolite) production by C. roseus CP3 cells was also promoted when AO was added to the suspension culture. On the basis of these results, we assumed that alginate is an elicitor-like substance. We therefore compared the effect of AO on C. roseus L. and W. japonica cells with those of chitosan oligomer (CO) and oligo-galacturonic acid (OGA), which are well known as an exogenous elicitor and endogenous elicitor respectively. The effects of various concentrations of AO, OGA, and CO on the physiological activities, membrane permeability and protoplast formation of C. roseus L. or W. japonica cells were investigated. AO and OGA showed similar physiological effects, which were quite different from those of CO. Since alginate appeared to have similar effects to galacturonic acid, we concluded that alginate acts as an endogenous elicitor. Both alginate and galacturonic acid are uronic acids, and we considered their structural similarity. The effects of esterification of the carboxylic groups of alginate by propylene oxide were also studied. The greater the degree of esterification, the less the secretion of 5′-phosphodiesterase. Hence we assumed that carboxylic groups have an important role in the initiation of the elicitation reaction in plant cells, as shown in the case of galacturonic acid.

Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity by M. A. Kacena; E. E. Smith; P. Todd (pp. 437-439).

The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

Continuous degradation of mixtures of 4-nitrobenzoate and 4-aminobenzoate by immobilized cells of Burkholderia cepacia strain PB4 by C. M. Peres; B. Van Aken; H. Naveau; S. N. Agathos (pp. 440-445).

Although isolated on 4-aminobenzoate, Burkholderia cepacia strain PB4 is also able to grow on 4-nitrobenzoate. Degradation of an equimolar mixture of the nitroaromatic compound 4-nitrobenzoate and its corresponding aminoaromatic derivative 4-aminobenzoate by this strain was investigated. Batch experiments showed that, irrespective of preculturing conditions, both compounds were degraded simultaneously. The mixture-degrading ability of B. cepacia strain PB4 was subsequently tested in continuous packed bed reactors (PBR) with the strain immobilized on Celite grade R-633 or R-635. Higher degradation rates were achieved with the larger particles of Celite R-635. Maximum simultaneous degradation rates per liter of packed bed of 0.925 mmol l⁻¹ h⁻¹ 4-nitrobenzoate and 4-aminobenzoate were obtained for an applied loading rate of the same value (0.925 mmol l⁻¹ h⁻¹ of each compound). Even when the applied load was not removed in its entirety, neither of the two compounds was degraded preferentially but a percentage of both of them was mineralized. The present study shows the possibility for a pure strain to biodegrade not only a nitroaromatic compound (4-nitrobenzoate) but also its corresponding amino derivative (4-aminobenzoate) continuously and simultaneously.

Bioavailability of water-polluting sulfonoaromatic compounds by J. Ruff; T. Hitzler; U. Rein; A. Ritter; A. M. Cook (pp. 446-450).

Highly substituted arenesulfonates are chemically stable compounds with a range of industrial applications, and they are widely regarded as being poorly degradable. We did enrichment cultures for bacteria able to utilise the sulfonate moiety of 14 compounds, and we obtained mixed cultures that were able to desulfonate each compound. The products formed were usually identified as the corresponding phenol, but because we could not obtain pure cultures, we followed up these findings with quantitative work in pure cultures of, e.g., Pseudomonas putida S-313, which generated the same phenols from the compounds studied. Many of these phenols are known to be biodegradable, or to be subject to binding to soil components. We thus presume that the capacity to degrade aromatic sulfonates extensively is widespread in the environment, even though the degradative capacity is spread over several organisms and conditions.

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Applied Microbiology and Biotechnology (v.52, #3)

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Applied Microbiology and Biotechnology (v.52, #3)