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Applied Microbiology and Biotechnology (v.59, #6)
Enzymatic production of cyclodextrins by A. Biwer; G. Antranikian; E. Heinzle (pp. 609-617).
Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly α-amylases. Due to its low solubility in water, separation and purification of β-CD is relatively easy compared to α- and γ-CD. In recent years more economic processes for γ-CD and especially α-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of α-CD production processes is presented.
A review of the production of ethanol from softwood by M. Galbe; G. Zacchi (pp. 618-628).
Ethanol produced from various lignocellulosic materials such as wood, agricultural and forest residues has the potential to be a valuable substitute for, or complement to, gasoline. One of the major resources in the Northern hemisphere is softwood. This paper reviews the current status of the technology for ethanol production from softwood, with focus on hemicellulose and cellulose hydrolysis, which is the major problem in the overall process. Other issues of importance, e.g. overall process configurations and process economics are also considered.
Biofouling in water systems – cases, causes and countermeasures by H.-C. Flemming (pp. 629-640).
Biofouling is referred to as the unwanted deposition and growth of biofilms. This phenomenon can occur in an extremely wide range of situations, from the colonisation of medical devices to the production of ultra-pure, drinking and process water and the fouling of ship hulls, pipelines and reservoirs. Although biofouling occurs in such different areas, it has a common cause, which is the biofilm. Biofilms are the most successful form of life on Earth and tolerate high amounts of biocides. For a sustainable anti-fouling strategy, an integrated approach is suggested which includes the analysis of the fouling situation, a selection of suitable components from the anti-fouling menu and an effective and representative monitoring of biofilm development.
An improved stereoselective reduction of a bicyclic diketone by Saccharomyces cerevisiae combining process optimization and strain engineering by M. Katz; I. Sarvary; T. Frejd; B. Hahn-Hägerdal; M. F. Gorwa-Grauslund (pp. 641-648).
The stereoselective reduction of the bicyclic diketone bicyclo[2.2.2]octane-2,6-dione, to the ketoalcohol (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one, was used as a model reduction to optimize parameters involved in NADPH-dependent reductions in Saccharomyces cerevisiae with glucose as co-substrate. The co-substrate yield (ketoalcohol formed/glucose consumed) was affected by the initial concentration of bicyclic diketone, the ratio of yeast to glucose, the medium composition, and the pH. The reduction of 5 g l–1 bicyclic diketone was completed in less than 20 h in complex medium (pH 5.5) under oxygen limitation with an initial concentration of 200 g l–1 glucose and 5 g l–1 yeast. The co-substrate yield was further enhanced by genetically engineered strains with reduced phosphoglucose isomerase activity and with the gene encoding alcohol dehydrogenase deleted. Co-substrate yields were increased 2.3-fold and 2.4-fold, respectively, in these strains.
Flavin reductase coupling with two monooxygenases involved in dibenzothiophene desulfurization: purification and characterization from a non-desulfurizing bacterium, Paenibacillus polymyxa A-1 by T. Ohshiro; Y. Aoi; K. Torii; Y. Izumi (pp. 649-657).
The dibenzothiophene (DBT) desulfurizing bacterium metabolizes DBT to form 2-hydroxybiphenyl without breaking the carbon skeleton. Of the DBT desulfurization enzymes, DszC and DszA catalyze monooxygenation reactions, both requiring flavin reductase. We searched for non-DBT-desulfurizing microorganisms producing a flavin reductase that couples more efficiently with DszC than that produced by the DBT desulfurizing bacterium Rhodococcus erythropolis D-1, and found Paenibacillus polymyxa A-1 to be a promising strain. The enzyme was purified to complete homogeneity. K m values for FMN and NADH were 2.1 µM and 0.57 mM, respectively. Flavin compounds were good substrates, some nitroaromatic compounds were also active, and regarding the electron donor, the activity for NADPH was about 1.5 times that for NADH. In the coupling assay with DszC, only FMN or riboflavin acted as the electron acceptor. The coupling reactions of P. polymyxa A-1 flavin reductase with DszC and DszA proceeded more efficiently (3.5- and 5-fold, respectively) than those of R. erythropolis D-1 flavin reductase when identical enzyme activities of each flavin reductase were added to the reaction mixture. The result of the coupling reaction suggested that, in the microbial DBT desulfurization, flavin reductase from the non-DBT-desulfurizing bacterium was superior to that from the DBT-desulfurizing bacterium.
Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor by H. Ichinose; H. Wariishi; H. Tanaka (pp. 658-664).
A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.
The mannitol dehydrogenase gene (mdh) from Leuconostoc mesenteroides is distinct from other known bacterial mdh genes by J. Aarnikunnas; K. Rönnholm; A. Palva (pp. 665-671).
The N-terminal amino acid sequences of the intact protein and three tryptic peptides from a 41 kDa protein purified from a commercial mannitol dehydrogenase (MDH) enzyme preparation of Leuconostoc mesenteroides ATCC-9135 were determined. Oligonucleotides deduced from these peptide sequences were used to isolate the putative mdh gene from L. mesenteroides using the Vectorette system. Nucleotide sequence analysis revealed an open reading frame (ORF1) of 1,014 bp encoding a putative MDH protein of 338 amino acids, and another open reading frame (ORF2) encoding an unknown protein of 245 amino acids. In Northern blots, a transcript of approximately 2.2-kb was detected with an mdh-specific probe. Mapping of the 5′-end of the 2.2-kb transcript indicated that mdh was the first gene of the operon. After fusion of six histidine codons to the 3′-end of the mdh gene and expression in Escherichia coli M15, active MDH was isolated using HisTrap purification. The overexpressed enzyme showed high specificity for mannitol and fructose. In dot blot hybridisation, the L. mesenteroides mdh-specific probe bound strongly to chromosomal DNA of Leuconostoc pseudomesenteroides and weakly to DNA of some heterofermentative Lactobacillus strains, whereas no hybridisation signals were obtained with DNA derived from strains carrying characterised mdh genes. Furthermore, the amino acid sequence similarity between L. mesenteroides MDH and other known MDHs was very low, suggesting that MDHs from heterofermentative lactic acid bacteria form a structurally and functionally separate enzyme group. Interestingly, L. mesenteroides MDH shared significant sequence similarity with the medium-chain dehydrogenase/reductase protein family.
Redox-mediated decolorization of synthetic dyes by fungal laccases by H. Claus; G. Faber; H. König (pp. 672-678).
Laccases from the lignin-degrading basidiomycetes Trametes versicolor, Polyporus pinisitus and the ascomycete Myceliophthora thermophila were found to decolorize synthetic dyes to different extents. Differences were attributed to the specific catalytic properties of the individual enzymes and to the structure of the dyes. Due to their higher oxidative capacities, the laccases from the two basidiomycetes decolorized dyes more efficiently than that of the ascomycete. The azo dye Direct Red 28, the indigoid Acid Blue 74 and anthraquinonic dyes were directly enzymatically decolorized within 16 h. The addition of 2 mM of the redox-mediator 1-hydroxybenzotriazole further improved and facilitated the decolorization of all nine dyes investigated. Laccases decolorized dyes both individually and in complex mixtures in the presence of bentonite or immobilized in alginate beads. Our data suggest that laccase/mediator systems are effective biocatalysts for the treatment of effluents from textile, dye or printing industries.
Biotransformation of p-xylene and 2,6-dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate by M. Bramucci; M. Singh; V. Nagarajan (pp. 679-684).
Sphingomonas strain ASU1 was isolated from an industrial wastewater bioreactor and grew on 2,6-dimethylnaphthalene (2,6-DMN) as the sole carbon/energy source. The genes for a xylene monooxygenase were cloned from strain ASU1. Expression of the ASU1 xylene monooxygenase was compared to expression of the pWWO xylene monooxygenase in Escherichia coli. Both monooxygenases transformed p-xylene and 2,6-DMN by initially hydroxylating one methyl group. In addition, the ASU1 monooxygenase also hydroxylated the second methyl group on p-xylene and 2,6-DMN whereas the pWWO monooxygenase hydroxylated the second methyl group only on p-xylene. Endogenous E. coli enzymes contributed to further oxidation of the resulting aromatic alcohols to form aromatic carboxylates.
Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors by G. Miksch; S. Kleist; K. Friehs; E. Flaschel (pp. 685-694).
The gene for phytase from Escherichia coli was sequenced and compared with the appA gene. It was found to be a mutant derivative of the appA gene. After fusion with a C-terminal His-tag, phytase was purified by affinity chromatography and the enzymatic properties were analyzed. To develop a system for overexpression and extracellular production of phytase in E. coli, factors affecting the expression and secretion such as promoter type, host strain and selection pressure were analyzed. Using a secretion system based on the controlled expression of the kil gene, the expression of phytase was improved and the enzyme was released into the culture medium at a high level. An effective fermentation strategy based on fed-batch operation was developed.
A polymerase chain reaction-based test for Verticillium fungicola causing dry bubble disease on the cultivated mushroom, Agaricus bisporus by C. P. Romaine; B. Schlagnhaufer; M. Stone (pp. 695-699).
A polymerase chain reaction (PCR)-based test is described for the specific detection of Verticillium fungicola var. aleophilum (Vfa), the fungal pathogen causing dry bubble disease on the cultivated button mushroom, Agaricus bisporus. PCR primers were tailored to target a 162-bp arbitrary sequence in the Vfa genome. In PCR amplifications using the primer pair, all of 20 isolates of Vfa that had been collected during a 29-year period at commercial mushroom operations located primarily in North America were found to generate the diagnostic 162-bp DNA product. Conversely, the primers failed to produce the specific amplicon with DNA from isolates representing 5 other species of Verticillium, the pathogenic subspecies V. fungicola var. fungicola from Europe, and 12 other fungal species commonly inhabiting mushroom compost. A protocol was designed enabling a confirmed diagnosis of dry bubble disease in less than 3 h. The PCR-based test should find application for the rapid diagnosis and detection of the fungal pathogen in disease management programs and, potentially, in screening for on-the-farm sources of infection.
Use of the pAL5000 replicon in PAH-degrading mycobacteria: application for strain labelling and promoter probing by P. Wattiau; D. Springael; S. N. Agathos; S. Wuertz (pp. 700-705).
Three environmental Mycobacterium strains (LB501T, LB307T and VM552) able to degrade anthracene, phenanthrene or pyrene, respectively, were successfully electroporated with pAL5000-based plasmids containing the green fluorescent protein (gfp) gene of Aequoria victoria under the control of the hsp60 promoter of Mycobacterium bovis following a slightly modified standard procedure. Transformants showed irregular gfp expression profiles. Four plasmid derivatives were constructed that contained gene promoters isolated from, and adapted to, gene expression in polycyclic aromatic hydrocarbon (PAH)-degrading mycobacteria. One derivative directed strong and homogeneous expression of GFP, allowing dual analysis of both GFP- and PAH-derived fluorescence as assessed by confocal laser scanning microscopy. The results reported here demonstrate the suitability of the pAL5000 replicon for the development of recombinant DNA-based studies in PAH-degrading Mycobacterium spp.
Monitoring of morphological development of the arachidonic-acid-producing filamentous microorganism Mortierella alpina by E. Y. Park; T. Hamanaka; K. Higashiyama; S. Fujikawa (pp. 706-712).
Morphological parameters, such as hyphal growth rate, tip formation rate, tip extension rate and branch formation rate, of Mortierella alpina have been measured using a flow-through chamber under 25 different combinations of carbon and nitrogen concentrations. Morphological parameters were influenced not by C/N ratio but by carbon concentration in the medium. Specific rates of hyphal growth and tip formation both remained constant at a low carbon concentration of 5 g/l. Tip extension rate from one tip was 60 µm tip–1 h–1 at a carbon concentration below 15 g/l, and the branching formation rate was independent of carbon concentration. Tip extension rate was a function of specific hyphal growth rate, which in turn was linearly proportional to the specific tip formation rate, demonstrating that tip extension rate was exponentially proportional to the specific tip formation rate. Branch formation rate per hyphal element remained unchanged even at tip extension rates lower than 60 µm tip–1 h–1 and at specific hyphal growth rates lower than 0.83 h–1, but decreased drastically at higher rates of tip extension and hyphal growth.
Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F by V. M. Gonçalves; T. C. Zangirolami; R. L. C. Giordano; I. Raw; M. M. Tanizaki; R. C. Giordano (pp. 713-717).
The influence of medium composition and culture conditions on Streptococcus pneumoniae serotype 23F cultivation was investigated in order to develop an industrial method for polysaccharide (PS) production. Acid-hydrolyzed casein (AHC) and dialyzed enzymatically hydrolyzed soybean meal (EHS) were investigated as nitrogen sources, and the vitamin solution of Hoeprich's medium and dialyzed yeast extract as vitamin sources. The influence of initial glucose concentration was also evaluated. In flask experiments, the best nitrogen source for PS production was AHC; EHS yielded small amounts of PS without interfering with bacterial growth. Dialyzed yeast extract provided an approximately 2-fold increase in PS production when compared to Hoeprich's vitamin solution. In a 5-l bioreactor, it was observed that the pneumococcus did not grow under aerobic conditions, CO2 did not increase PS yield, glucose was inhibitory above 30 g l–1, and the main glucose catabolism product was lactate, which had an inhibitory effect on cell growth. When anaerobic cultivation was performed under N2 flow using the optimized medium, 240 mg l–1 of soluble PS was obtained, which represents a 3-fold increase in yield as compared to that described in the published patent [Yavordios and Cousin (1983) European Patent 0 071515 A1]. Application of these results would considerably simplify upstream and downstream processes for PS production.
Role of electrostatic interactions in cohesion of bacterial biofilms by X. Chen; P. S. Stewart (pp. 718-720).
Significant decreases in the apparent viscosity of a bacterial biofilm suspension were measured following addition of sodium, potassium, magnesium, or calcium salts, whereas iron salts increased the viscosity. Electrostatic interactions contribute to biofilm cohesion and iron cations are potent crosslinkers of the biofilm matrix.
Isolation and characterization of a Trichoderma strain capable of fermenting cellulose to ethanol by D. M. Stevenson; P. J. Weimer (pp. 721-726).
The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l–1. This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol. Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l–1 when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol, >5.0 g l–1, were obtained by the fermentation of glucose. Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobically) a wide range of carbon sources. In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.
Saccharomyces bayanus var. uvarum in Tokaj wine-making of Slovakia and Hungary by G. I. Naumov; E. S. Naumova; Z. Antunovics; M. Sipiczki (pp. 727-730).
Using genetic hybridisation analysis and molecular karyotyping we revealed an association of Saccharomyces bayanus var. uvarum species with Tokaj wine-making. Along with identification of Saccharomyces strains isolated by E. Minárik in Slovakia, the composition of Tokaj populations in Hungary was studied. Twenty-eight Hungarian Saccharomyces strains were analysed in terms of karyotype. The majority of strains belong to S. bayanus var. uvarum. Two non-identified Saccharomyces strains were found to be polyploid according to their complex karyotype patterns.
Quantitative and rapid detection of the trichloroethylene-degrading bacterium Methylocystis sp. M in groundwater by real-time PCR by T. Kikuchi; K. Iwasaki; H. Nishihara; Y. Takamura; O. Yagi (pp. 731-736).
We developed a method based on real-time PCR for the specific and rapid enumeration of a trichloroethylene-degrading methanotroph, Methylocystis sp. M, with the aim of monitoring the strain in groundwater. A primer set designed from the nucleotide sequence of the mmoC gene of a soluble methane monooxygenase (sMMO) gene cluster from Methylocystis sp. M was specific to amplify the DNA region from the strain and no PCR products were amplified with the sMMO gene clusters from six other methanotroph strains. The real-time PCR reliably quantified Methylocystis sp. M over at least five orders of magnitude (5×106 to 5×102 cells/PCR tube, or 2×108 to 2×104 cells/ml). Five cells of Methylocystis sp. M per PCR tube (2×102 cells/ml) were detectable when the cells were suspended in distilled water. The concomitant presence of other methanotrophs in samples did not affect the reliability of enumeration; and recovery of the cells with a membrane filter enabled us to quantify cells of the strain in groundwater. This quantification procedure was completed within 3 h, including preparation time of environmental samples. We conclude that real-time PCR using the mmoC primer set can be used practically to analyze the behavior of Methylocystis sp. M at bioremediation sites.
Isolation and characterization of a moderate thermophile, Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization by K. J. Kayser; L. Cleveland; H.-S. Park; J.-H. Kwak; A. Kolhatkar; J. J. Kilbane II (pp. 737-746).
An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30–52°C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50°C M. phlei GTIS10 culture was determined to be 1.1±0.07 µmol 2-HBP min–1 (g dry cell)–1. M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolis IGTS8 reach the highest rate of DBT metabolism is near 30°C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50°C, and activity is detectable at temperatures as high as 57°C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.