Oxidation of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids by Acetobacter and Serratia strains

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110 g 2-furoic acid l(-1) from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2' biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86-91% molar yields.     

Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp.

Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K₂CrO₄ ml^–1 on nutrient agar, 25mgml^–1 in nutrient broth, and up to 10mgml^–1 in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing. Uptake of chromate was greater in living cells than in heat-killed on dried cells. CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000gml^–1 after 24h while CrT-13 reduced 41%, 14% and 9%. Other heavy metals at low concentrations did not affect these reductions. At 150 and 300gml^–1 in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13.Revisions requested 17 May 2004; Revisions received 2 July 2004     

Simultaneous and selective production of levan and poly(γ-glutamic acid) by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis(natto) Takahashi, used to prepare the fermented soybean product natto, was grown in a basal medium containing 5% (w/w) sucrose and 1.5% (w/w) l-glutamate and produced 58% (w/w) poly(-glutamic acid) and 42% (w/w) levan simultaneously. After 21 h, 40–50 mg levan ml^-1had been produced in medium containing 20% (w/w) sucrose but without l-glutamate. In medium containing l-glutamic acid but without sucrose, mainly poly(-glutamic acid) was produced. Revisions requested 28 August 2004/14 October 2004; Revisions received 11 October 2004/22 November 2004     

A novel psychrotrophic fungus, <Emphasis Type="Italic">Mortierella minutissima</Emphasis>, for <Emphasis Type="BoldSmallCaps">D</Emphasis>-limonene biotransformation

Of 98 strains of moulds, isolated from arctic soils, Mortierella minutissima 01, grew the best on agar plates with limonene vapor. Perillyl alcohol and perillic acid were the main products of limonene biotransformation. Maximal yield of perillyl alcohol (125mgl^–1) occurred in medium containing 0.8% substrate, at 15°C, pH 6 and after 4–5 d. Revisions requested 27 October 2004; Revisions received 27 November 2004     

Survival of <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> during heating,drying and storage in the dried state when growth has occurred in the presence of sucrose or monosodium glutamate

Spray-dried cells of Lactobacillus sakei CTC 494 survived ca. 60% longer in the spray dried state when cells were grown in the presence of 20g sucrose l^–1or 12.5g monosodium glutamate l^–1. No significant differences were observed in viability during storage in the freeze dried state with the addition of these compounds to the growth medium, nor in survival during a heat treatment (55°C). Both sucrose and glutamate in the growth medium suppressed intracellular accumulation of total amino acids and changed the overall pattern of the individual amino acids. Glutamate in the growth medium enhanced intracellular glutamate by ca. 38%. Revisions requested 4 November 2004; Revisions received 13 December 2004     

Enhancement of photorespiration in immobilized <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Immobilization of Chlamydomonas reinhardtii in alginate increases its photorespiration rate. In the immobilized cells, the photorespiratory enzyme, phosphoglycolate phosphatase, was 75% higher than in freely suspended cells. Thus, the immobilized cells produced glycolate at twice the rate than in freely suspended cells when treated with aminooxyacetate (a transaminase inhibitor). With immobilized cells in a batch reactor, 270mol glycolatemg^–1 Chl was produced after 12h.Revisions requested 27 October 2004; Revisions received 13 December 2004     

Expression of <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA2 phytase in four yeast systems

To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ A), and one constitutive system: P. pastoris (pGAPZA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml^–1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml^–1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower K_mfor sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.Revisions requested 18 November 2004; Revisions received 7 January 2005     

Isolation and characterization of a fructosyl-amine oxidase from an <Emphasis Type="Italic">Arthrobacter </Emphasis>sp.

An Arthrobacter sp. was isolated that, when induced by fructosyl-valine, expressed a fructosyl-amine oxidase (FAOD) that was specific for -glycated amino acids. The N-terminal amino acid sequence of the purified oxidase was determined and used to design oligonucleotides to amplify the gene by inverse PCR. Expression of the gene in Escherichia coli produced 0.23 units FAOD per mg protein, over 30-fold greater than native expression levels, with properties almost indistinguishable from the native enzyme. The presence of FAOD was confirmed in other Arthrobacter ssp.Revisions requested 8 September 2004; Revisions received 4 November 2004     

Continuous production of neo-fructooligosaccharides by immobilization of whole cells of <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

To produce neo-fructooligosaccharides (neo-FOSs) in a 500ml continuous packed-bed reactor using whole cell immobilization of Penicillium citrinum KCCM 11663, the optimum reaction conditions were 50 °C, pH 6 with 600 g sucrose l^-1 being fed as substrate at 1.3 ml min^-1 . Under these conditions, the maximum neo-FOSs production was 49 g l^-1. In a packed-bed reactor, continuous production of neo-FOSs was possible for 50 d indicating a potential for industrial production. Revisions requested 6 September 2004/14 October 2004; Revisions received 7 October 2004/29 November 2004     

Enhancement of poly-β-hydroxybutyrate accumulation in <Emphasis Type="Italic">Nostoc muscorum</Emphasis> under mixotrophy,chemoheterotrophy and limitations of gas-exchange

Nostoc muscorum, a heterocystous cyanobacterium, produced poly--hydroxybutyrate ( PHB) up to 8% (w/w) dry cells when grown photoautotrophically but 35% when grown mixotrophically with 0.4% (w/v) glucose and acetate after 21 d. Gas-exchange limitations under mixotrophy and chemoheterotrophy with 0.4% (w/v) acetate enhanced the accumulation up to 40–43% (w/w) dry cells, the value almost 5-fold higher with respect to photoautotrophic condition.Revisions requested/10 September 2004; Revisions received 9 September 2004/11 November 2004     

Increased number of Arginine-based salt bridges contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100

Thermotolerant acetic acid bacteria (AAB), Acetobacter tropicalis SKU1100, can grow above 40 °C. To investigate the basis of its thermotolerance, we compared the genome of A. tropicalis SKU1100 with that of mesophilic AAB strain Acetobacter pasteurianus IFO3283-01. The comparative genomic study showed that amino acid substitutions from large to small residue and Lys to Arg occur in many orthologous genes. Furthermore, comparative modeling study was carried out with the orthologous proteins between SKU1100 and IFO3283-01 strains, indicating that the number of Arg-based salt bridges increased in protein models. Since it has been reported that Arg-based salt bridges are important factor for thermo-stability of protein structure, our results strongly suggest that the increased number of Arg-based salt bridges may contributes to the thermotolerance of A. tropicalis SKU1100 (the thermo-stability of proteins in A. tropicalis SKU1100).     

Ethanol production from cellulosic materials by genetically engineered <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

To confer the ability to ferment cello-oligosaccharides on the ethanol-producing bacterium, Zymomonas mobilis, the -glucosidase gene from EmRuminococcus albus, tagged at its N-terminal with the 53-amino acid Tat signal peptide from the periplasmic enzyme glucose–fructose oxidoreductase from Z. mobilis, was introduced into the strain. The tag enabled 61% of the -glucosidase activity to be transported through the cytoplasmic membrane of the recombinant strain which then produced 0.49 g ethanol/g cellobiose. Revisions requested 9 November 2004; Revisions received 10 December 2004; Accepted 13 December 2004     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9gg^-1 dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.Revisions requested; Revisions received 6 September 2004     

Changes of primary sequence and secondary structure proximal to the 5′ end of the stop codon substantially increases the expression of the variable region of an antibody in <Emphasis Type="Italic">E. coli</Emphasis>

The sequence context at the 5 end of the stop codon may influence the efficiency of termination and translation. To increase the expression of a designed variable region of an antibody (named as VH5) against tumor necrosis factor (TNF), two nucleotides (TC) at 25 and 26 nucleotides (nt) upstream of termination codon were substituted with AG, respectively. The free energy of 70 nt (arbitrarily defined from the 32 nt upstream of termination codon to 38 nt downstream) was changed from –13.5 kcal mol^-1 to –17.3 kcal mol^-1. The expression level was increased from 1 ± 0.3% to 10 ± 1.2% of total cellular protein. Although the precise mechanism of this phenomenon remains to be elucidated, this report provides an alternative means to increase the expression of a foreign gene in E. coli. Revisions requested 18 October 2004; Revisions received 26 November 2004     

Structural characterization of an acidic exoheteropolysaccharide produced by the nitrogen-fixing bacterium Burkholderia tropica

An acidic exopolysaccharide (EPS) produced by the diazotrophic bacterium Burkholderia tropica, strain Ppe8, was isolated from the culture supernatant of bacteria grown in a synthetic liquid medium containing mannitol and glutamate. Monosaccharide composition showed Rha, Glc and GlcA in a 2.0:2.0:1.0 molar ratio, respectively. Further structural characterization was performed by a combination of NMR, mass spectrometry and chemical methods. Partial acid hydrolysis of EPS provided a mixture of acidic oligosaccharides that were characterized by ESI-MS, giving rise to ions with m/z 193 (GlcA-H)⁻, 339 (GlcA,Rha-H)⁻, 501 (GlcA,Rha,Glc-H)⁻, 647 (GlcA,Rha₂,Glc,-H)⁻, 809 (GlcA,Rha₂,Glc₂,-H)⁻ and 851 (GlcA,Rha₂,Glc₂,OAc-H)⁻. Carboxyreduced EPS (EPS-CR) had Glc and Rha in a 3:2 ratio, present as d- and l-enantiomers, respectively. Methylation and NMR analysis of EPS and EPS-CR showed a main chain containing 2,4-di-O-Rhap, 3-O-Rhap and 4-O-Glcp. A GlcA side chain unit was found in the acidic EPS, substituting O-4 of α-l-Rhap units. This was observed as a non-reducing end unit of glucopyranose in the EPS-CR. Acetyl esters occured at O-2 of β-l-Rhap units. From the combined results herein, we determined the structure of the exocellular polysaccharide produced by B. tropica, Ppe8, as being a pentasaccharide repeating unit as shown:

Full-size image (8K)

     

Dehalogenation of chlorinated aromatic compounds using a hybrid bioinorganic catalyst on cells of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis>

A novel bioinorganic catalyst was obtained via reduction of Pd(II) to Pd⁰ on to the surface of cells of Desulfovibrio desulfuricans at the expense of H₂. Palladised biomass, supplied with formate or H₂ as an electron donor, catalysed the dehalogenation of 2-chlorophenol and polychlorinated biphenyls. In the example of 2,3,4,5-tetrachlorobiphenyl, the bioinorganic catalyst promoted a rate of chloride release of 9.33 ± 0.17 nmol min^–1 mg ^–1and only ~5% of this value was obtained using chemically reduced or commercially available Pd ⁰. In the case of 2,2,4,4,6,6-hexachlorobiphenyl the rate was more than four orders of magnitude faster than the degradation reported using a sulfidogenic culture. Negligible chloride release occurred from any of the chloroaromatic compounds using biomass alone, or from palladised biomass challenged with hexane carrier solvent only. Analysis of the spent solution showed that in addition to catalysis of reductive dehalogenation the new material was able to remove very effectively the organic residua, with neither any PCB nor any breakdown products identifiable by GC/MS.Revisions requested 8 September 2004; Revisions received 21 October 2004;     

Establishment and dispersal of the biological control weevil Rhinoncomimus latipes on mile-a-minute weed, Persicaria perfoliata

Mile-a-minute weed, Persicaria perfoliata (L.) H. Gross (Polygonaceae), is an annual vine from Asia that has invaded the eastern US where it can form dense monocultures and outcompete other vegetation in a variety of habitats. The host-specific Asian weevil Rhinoncomimus latipes Korotyaev (Coleoptera: Curculionidae) was first released in the US in 2004 as part of a classical biological control program. The weevil was intensively monitored in three release arrays over 4 years, and field cages at each site were used to determine the number of generations produced. The weevil established at all three sites and produced three to four generations before entering a reproductive diapause in late summer. Weevils dispersed at an average rate of 1.5–2.9 m wk⁻¹ through the 50 m diameter arrays, which had fairly contiguous mile-a-minute cover. Weevils dispersing in the broader, more variable landscape located both large monocultures and small isolated patches of mile-a-minute 600–760 m from the release within 14 months. Weevil density ranged from fewer than 10 to nearly 200 weevils m⁻² mile-a-minute weed. Mile-a-minute cover decreased at the site with the highest weevil density. The production of P. perfoliata seed clusters decreased with increasing weevil populations at two sites, and seedling production declined over time at two sites by 75% and 87%. The ability of the weevil to establish, produce multiple generations per season, disperse to new patches, and likelihood of having an impact on plants in the field suggests that R. latipes has the potential to be a successful biological control agent.     

An improved method for the enzymatic transformation of nucleosides into 5′-monophosphates

An improved method to transform nucleosides into 5-monophosphates using nucleoside phosphotransferase from Erwinia herbicola is reported. The method is based on the shift in the equilibrium state of the reaction to the formation of desired product due to its precipitation by Zn²⁺. Under optimal conditions, the extent of nucleoside transformations into nucleoside-5-monophosphates were 41–91% (mol).Revisions requested 22 September 2004; Revisions received 11 October 2004     

Conidia production by <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (for the biocontrol of a diamondback moth) during solid-state fermentation in a packed-bed bioreactor

Conidia of Beauveria bassiana CS-1, which have the potential for the control of the diamondback moth (Plutella xylostella), were produced by solid-state fermentation (SSF) using a packed-bed bioreactor with rice straw and wheat bran. As the packing density and the bed height were increased, the production of conidia decreased. In a packed-bed bioreactor under no aeration and no addition of polypropylene (PP) foam (control), the total average of conidia was 4.9 × 10⁸ g^-1. The production of conidia was affected more by the addition of PP foam as an inert support than forced aeration and was approx. 23 times higher than that of the control. The total average of conidia produced by B. bassiana was 1.1–1.2 × 10¹⁰ g^-1 . Revisions requested 6 September 2004/2 November 2004; Revisions received 1 November 2004/8 December 2004     

Pre-germinated conidia of Coniothyrium minitans enhances the foliar biological control of Sclerotinia sclerotiorum

The relatively slow germination rate of Coniothyrium minitans limits its control efficiency against Sclerotinia sclerotiorum. Pre-germinated conidia of C. minitans enhanced its efficiency significantly: in foliar experiments with oilseed rape, hyphal extension of S. sclerotiorum was inhibited by 68%, while formation of sclerotia was completely inhibited when pre-germinated conidia were applied.Revisions requested 27 July 2004; Revisions received 7 September