Production of Lactobacillus kefir cells for asymmetric synthesis of a 3,5-dihydroxycarboxylate

An efficient fedbatch process for the production of Lactobacillus kefir DSM 20587 cells was developed. An improvement in space time yield of 270% (3.7 g_DCW l^–1 day^–1) and in final enzyme activity of 440% (9.1 U/ml) was achieved on a 150 l scale by controlling the oxygen transfer rate to 7–9 mmol l^–1 h^–1. The cells exhibited good and highly stereoselective reducing activities against tert-butyl 6-chloro-3,5-dioxohexanoate. tert-Butyl (3R,5S)-6-chloro-dihydroxyhexanoate, a chiral building block for HMG-CoA reductase inhibitor synthesis, was produced with 47.5% yield and >99% ee at C₃3 and C₅5 in a simple batch biotransformation process.     

Preparation of enantiomerically pure (R)-(1-hydroxyethyl)dimethyl(phenyl)silane using resting cells of Saccharomyces cerevisiae (DHW S-3) as biocatalyst

The prochiral sila-ketone acetyldimethyl-(phenyl)silane (1) was reduced enantioselectively into (R)-(1-hydroxyethyl)dimethyl(phenyl)silane [(R)-2] using resting cells of the commercially available yeast Saccharomyces cerevisiae (DHW S-3) as the biocatalyst. The bioconversion was performed on a 2.0-g scale in a 5-1 bioreactor. Starting with a substrate (1) concentration of 0.4 g·1^–1, the highest production rate measured for this bioconversion was about 45–55 mol (R)-2·1^–1·min^–1. After an incubation time of 1 h, all substrate in the medium had been converted, either biocatalytically reduced to (R)-2 or (probably chemically) converted into dimethyl(phenyl)silanol (Me₂PhSiOH). After extraction of the cell-free medium with ethyl acetate/dichloromethane and subsequent purification of the extract by Kugelrohr distillation and chromatography on silica gel (medium-pressure liquid chromatography), 800 mg (yield 40%) of the bioconversion product (R)-2 was isolated. As shown by HPLC studies (cellulose triacetate as the chiral stationary phase) and ¹H-nuclear magnetic resonance experiments (after derivatization of the bioconversion product with a chiral auxiliary agent), compound (R)-2 was almost enantiomerically pure (> 99% enantiomeric excess).This article is dedicated to Prof. Dr. Fritz Wagner on the occasion of his 65th birthday     

Role of trehalose synthesis pathways in salt tolerance mechanism of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> f. sp. <Emphasis Type="Italic">denitrificans</Emphasis> IL106

Two strains of trichloroethylene (TCE)-degrading bacteria were isolated from soils at polluted and unpolluted sites. The isolates, strains TE26^T and K6, showed co-substrate-independent TCE-degrading activity. TCE degradation was accelerated by preincubation with tetrachloroethylene, cis-dichloroethylene (DCE) and 1,1-DCE. TCE-degrading activities of strains TE26^T and K6 were 0.23, 0.24 mol min^–1 g^–1 dry cells, respectively. 16S rDNA sequences of strains TE26^T and K6 were almost identical (99.7% similarity), and most closely related to Ralstonia basilensis (ATCC17697^T) (98.5% similarity). From the results of DNA–DNA hybridizations, strain TE26^T was genetically coherent to strain K6 (94 and 88% hybridization), and exhibited lower relatedness to R. basilensis (DSM11853^T) (44% and 15%). In addition, because of the differences in chemotaxonomic properties, strain TE26^T and strain K6 appear to be distinct from all established species of the Ralstonia group. Based on these results and the proposal of transferring R. basilensis and related species to Wautersia gen. nov., we propose that these strains should be assigned to the genus Wautersia as Wautersia numadzuensis sp. nov.     

Enantioselective resolution of 3-phenylthio-2-propanol with Humicola lanuginosa lipase

The acetylation of 3-phenylthio-2-propanol (168 mg) was performed with vinyl acetate (1 ml) using different lipases from 15°C to 51°C. As a result, the (R)-enantiomer was selectively acetylated and the (S)-enantiomer was non-reactive in all the cases. An appropriate choice of conditions can be made to isolate both (R)-alcohol (ee 99%, 36 h, conversion 46%, sub/enz: 1/2) and (S)-alcohol (ee 93%, 38 h, conversion 46%, THF, sub/enz: 1 l^–1) using Humicola lanuginosalipase (Lipolase). Increasing the amount of enzyme increased the ee.     

The Structure of Uncommon Lipid A from the Marine Bacterium <Emphasis Type="Italic">Marinomonas communis</Emphasis> ATCC 27118<Superscript>T</Superscript>

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118^T with 1% AcOH. Using chemical analysis and 1D and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be β-1′,6-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-{(R)-3-hydroxydecanoyloxy)]decanoic acid and (R)-3-hydroxydecanoic acid at the C2, C2′ and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M .communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 404–413.Original Russian Text Copyright © 2005 by Vorob’eva, A. Dmitrenok, P. Dmitrenok, Isakov, Krasikova, Solov’eva.The article was translated by the authors.     

Response surface optimization for the continuous glucose isomerization process

Production of fructose via a continuous glucose isomerization process was optimized using response surface methodology. Glucose isomerization was performed using immobilized glucose isomerase in a flow-through tubular reactor. Process factors eg pH (7.0–7.8), temperature (50–60°C), flow rate (5–17 ml min^–1) and glucose content (30–50% w/w) of the feedstock solution were simultaneously tested according to a central composite experimental design. Measured responses such as % isomerization, and fructose yield (gh^–1) has an excellent correlation with tested factors. The highest desirability,D, (geometric mean of % isomerization and fructose yield) was obtained when the feedstock (56–60°C) had 34–36% glucose, a pH of 7.4–7.8 and was pumped at 15 ml min^–1.     

DNA-DNA homology studies among strains of Arthrobacter and Brevibacterium

Sixteen named strains of Arthrobacter and two strains of Brevibacterium were investigated by nucleic acid hybridisation. The Arthrobacter strains show homology values ranging between 11 and 55% to the type strain A. globiformis DSM 20124 (ATCC 8010), indicating only a low to moderate relationship. Two strains of A. globiformis, DSM 20124 and DSM 20125, exhibit only poor relationship to one another (30%). Among all the Arthrobacter strains the homology data range between 10 to 70% demonstrating separate status of almost all species. Only A. polychromogenes DSM 20136 was found to be a subspecies of A. oxydans DSM 20119. The type strain of A. citreus, DSM 20133 shows a remarkable lack of homology to four other strains of A. citreus, deposited as ATCC 15170, ATCC 17775, ATCC 21040 and ATCC 21348 (11–13%) which themselves can be separated into two groups according to the homology data (24–31%). Each of the two strains of Brevibacterium share high genetic relatedness with one of these A. citreus groups (71 and 73%, respectively). According to the DNA-DNA homology data, most of the species of Arthrobacter can actually be ranged taxonomically as species.Abbreviation DSM German Collection of Microorganisms, Menzinger Strasse 67, D-8000 Munich 19, FRG - ATCC American Type Culture Collection, Rockville, Maryland, U.S.A. - CCM Czechoslovak Collection of Microorganisms, J. E. Purkyne University, Tr. Obracu miru 10, Brno, CSSR - NCIB National Collection of Industrial Bacteria Aberdeen, Scotland     

Transformation of arachidonic acid to 19-hydroxy- and 20-hydroxy-eicosatetraenoic acids using Candida bombicola

Candida bombicola (ATCC 22214) and C. apicola (ATCC 96134), grown on glucose (100 g l^–1) and arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid; AA), 1.25 g l^–1, synthesized sophorolipid up to 0.93 g l^–1. Acid hydrolysis of sophorolipid yielded 19-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (19-HETE) and 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) which were identified by TLC and GC-MS; the ratio of synthesis was 73:27, respectively. Conversion of AA by immobilized Candida bombicola, suspended in beads of 2% (w/v) calcium alginate for 96 h, gave an 83% conversion of 1 g AA l^–1 to 19- and 20-HETE. There was no significant loss in the efficiency of the immobilized cells after ten uses.     

An easy access to optically pure (R)-malic acid via enantioselective hydrolysis of diethyl malate byRhizopus lipase

Summary Racemic diethyl malate was enantioselectively hydrolyzed by crudeRhizopus lipase (Saiken) to leave optically pure (99% ee) (R)-(+)- malate in c.a. 20% recovery. The combination of dipropyl malate and lipase AY (Amano) also gave (R)-enantiomer with a high ee of 97% and about 20% recovery in a short reaction time.     

Effect of inoculum level on xylitol production from rice straw hemicellulose hydrolysate byCandida guilliermondii

The effect of inoculum level on xylitol production byCandida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L^–1 to 2.41 g L^–1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g^–1) and volumetric productivity (0.56 g L^–1 h^–1) were reached with an inoculum level of 0.9 g L^–1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeastC. guilliermondii with efficiency values as high as 77% of the theoretical maximum.     

Enantioselective reduction of acetyldimethylphenylsilane by Trigonopsis variabilis (DSM 70714)

Summary Growing and resting cells of the yeast Trigonopsis variabilis (DSM 70714) can be used for the enantioselective reduction of the organosilicon compound acetyldimethylphenylsilane (1) to give optically active (R)-(1-hydroxyethyl)dimethylphenylsilane [(R)-2] in good yields. The enantiomeric purity of the isolated product was determined to be 62–86% ee depending on the substrate concentration used. Both substrate and product caused an inhibition of the reaction at concentrations higher than 0.35 and 0.5 g/l, respectively. Besides, higher substrate and product concentrations led to increased formation of the by-product 1,1,3,3-tetramethyl-1,3-diphenyldisiloxane. Considering the limiting substrate and product concentrations, it was possible to use the same biomass at least 5 times without significant loss of enzyme activity. 3-Methyl-3-phenyl-2-butanone (5) and acetyldimethylphenylgermane (7), which represent carbon and germanium analogues of 1, were also found to be accepted as substrates by Trigonopsis variabilis (DSM 70714). The reduction rates of the silicon (1) and germanium compound (7) were much higher than the transformation rate of the corresponding carbon analogue 5.     

Biocatalysts for production of chiral epoxides

Summary A number of bacterial strains, representing a range of genera, were isolated in pure culture with ethene or propene as the sole source of carbon and energy. The organisms included Aerococcus, Alcaligenes, Micrococcus and Staphylococcus spp. and a variety of Gram-negative, Gram-positive and Gram-variable mesophilic rods/ coccobacilli not yet identified. This suggests that the ability to utilize gaseous olefins is more widespread in nature than previously recognised. All 18 organisms tested stereospecifically formed R-1,2-epoxypropane (enantiomeric excess, ee=90–96%), R-1,2-epoxybutane (ee=90–98%) and trans-(2R,3R)-epoxybutane (ee=64–88%) from the corresponding olefins. In addition to Micrococcus sp. M90C, the substrate specificities of six other organisms were studied. The pattern of reactivity for the group of four ethene (M26, M90C, M93A, M186)- and two propene (M142, M156)-utilizers differed from that found with peracids, whereas the chemical reactivity of the substrate appeared to affect enzymatic epoxidations in Staphylococcus sp. M97B. Offprint requests to: M. Mahmoudian     

Alcoholic fermentation of glucose and xylose by Pichia stipitis,Candida shehatae,Saccharomyces cerevisiae and Zymomonas mobilis: oxygen requirement as a key factor

Summary To investigate simultaneous alcoholic fermentation of glucose and xylose derived from lignocellulosic material by separate or co-culture processes, the effect of oxygen transfer rate (OTR) on the fermentation of 50 g/l xylose by Pichia stipitis NRRL Y 7124 and Candida shehatae ATCC 22984, and the fermentation of 50 g/l glucose by Saccharomyces cerevisiae CBS 1200 and Zymomonas mobilis ATCC 10988 was carried out in batch cultures. The kinetic parameters of the xylose-fermenting yeasts were greatly dependent on the OTR. The optimum OTR values were found to be 3.9 and 1.75 mmol·1^–1·h^–1 for C. shehatae and P. stipitis, respectively. By contrast the fermentative parameters of S. cerevisiae were poorly affected by the OTR range tested (0.0–3.5 mmol·l^–1·h^–1) Under these conditions the ethanol yields ranged from 0.41 g·g^–1 to 0.45 g·g^–1 and the specific ethanol productivity was around 0.70 g·g^–1·h^–1. Z. mobilis gave the highest fermentative performance under strictly anaerobic conditions (medium continually flushed with nitrogen): under these conditions, the ethanol yield was 0.43 g·g^–1 and the average specific ethanol productivity was 2.3 g·g^–1·h^–1. Process considerations in relation to the effect of OTR on the fermentative performance of the tested strains are discussed. Offprint requests to: J. P. Delgenes     

Implications of element deficits for zooplankton growth

Daily measurements of mass and phosphorus (P) were made for Daphnia magna feeding on a high concentration (1 mg C l^–1) of P-sufficient (molar C:P = 70–80, `P+') or P-deficient (C:P = 1000, `P–') green algae (Scenedesmus obliquus or Ankistrodesmus falcatus). Animals feeding on P+ algae for 12 d showed a sharp decline in growth during the last juvenile instar (mean ± SE from 0.57 ± 0.02 to 0.25 ± 0.03 d^–1) and a modest decline in P-content at the first adult instar (from 1.55 ± 0.01 to 1.46 ± 0.02% mass). Animals feeding on P– algae showed declines in P-content to as low as 0.84% after 5 d, with the sharpest decline in the first day. Growth of animals feeding on P– algae declined sharply over time and showed a linear relationship with the grazer's P-content. Growth during the first day of exposure to P– algae was relatively high (mean 0.39 ± 0.03 d^–1) but approached zero as P-content declined to <0.90% mass. For animals feeding on P+ algae, the ontogenetic decline in P-content resulted in only a small difference between growth calculated for mass and growth calculated for Daphnia P. In contrast, for animals feeding for 4 d on P– algae, growth calculated for mass was 2–5 times higher than growth based on P. Thus, this study shows that short-term growth assays may substantially underestimate the negative impact of P-deficient resources, unless the decline in the grazer's P-content is taken into account. In this situation, measurement of growth in terms of nutrient content (e.g. grazer's P-content) should provide a better indication of the potential for sustained growth than calculations based on change in mass. This study also supports the `integration of growth' hypothesis (Sterner & Schwalbach, 2001), in which animals undergoing diel migration between water layers with P-deficient and P-sufficient resources experience a reduced growth penalty.     

Differentiation during asexual reproduction and regeneration in a microturbellarian

Affordable biological technology for the reclamation of wastes and water of the waste streams from intensive livestock units is important in a country short of water. This study tested the concept of reclamation of waste by Streptocephalus macrourus (Crustacea: Anostraca) from the effluent of a high rate algal pond processing livestock wastes. S. macrourus showed a growth efficiency of 39% to 74% when fed optimal rations and cultured at densities between 10 and 400 1^–1. The maximum daily growth rates (0.15–0.21) approximate the growth rates of cladoceran or rotifer cultures managed for maximal biomass production. S. macrourus' ability to withstand crowding enabled the production from S. macrourus cultures (up to 91.8 mg dry mass l^–1 d^–1, or 1241 mg wet mass l^–1 d^–1) to exceed production recorded from cladoceran or rotifer cultures. Temperature influenced growth rate, with the highest growth rate occurring at 24 °C. The dilution rate of continuously fed cultures influenced growth rate, with the optimum dilution rate tested being 10 ml organism ^–1 d^–1. Mass mortality occurred when organisms were held at a density of 4000 l^–1. S. macrourus is able to convert algae grown on livestock waste efficiently into anostracan biomass, and is able to give a very high daily production.     

Kinetic resolution of ketoprofen ester catalyzed by lipase from a mutant of CBS 5791

A biotransformation process was developed for the production of (S)-ketoprofen by enantioseletive hydrolysis of racemic ketoprofen ester using the mutant Trichosporon laibacchii strain CBS 5791. A satisfactory result was obtained, in which the E was 82.5, with an ee of 0.94 and a conversion of 0.47 under the optimum hydrolysis conditions [E is enantiomeric ratio, E=ln[1–X(1+ee)]/ln[1–X(1–ee)]; ee is enantiomeric excess, ee=(C_S–C_R)/(C_S+C_R): temperature of hydrolysis was 23°C]. The medium used in biotransformation was a mixture of growth broth and biotransformation broth at a ratio of 1:9, the concentration of Tween 80 was 15 g/l, the time of hydrolysis, 72 h. These results are promising for further scale-up. Tween 80 significantly improved lipase enantioselectivity and activity at the optimum concentration.     

Asymmetric Hydrolysis of (R,S)-5-Acetoxymethyl-3-tert-butyl-oxazolidin-2-one with Enzymes and Microorganisms†

Enzymes and microorganisms were screened for the asymmetric hydrolysis of (R, S)-5-acetoxymethyl-3-tert-butyl-oxazolidin-2-one 1. Lipases from Pseudomonas aeruginosa and Alcaligenes species, and microorganisms which belong to Enterobacter species or Klebsiella species were found to hydrolyze 1 enantioselectively to give (R)-5-hydroxymethyl-3-tert-butyl-oxazolidin-2-one (R)-2 and (S)-l. (S)-2, one of the typical intermediates for preparing optically active β-blocking agents (β-blockers), was obtained with high enantiomeric excess (91~98% e.e.) from (S)-1.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Phytoplankton communities in channel catfish ponds

Seasonal changes in the quantity and quality of phytoplankton were studied in six channel catfish culture ponds. Chlorophyll a concentrations were generally highest in the summer (averaging >200 g 1^–1) but the highest individual chlorophyll a value recorded (910 g 1^–1) occurred in the winter during a bloom of Dictyosphaerium pulchellum. On the average, green algae (Chlorophyta) and euglenoids (Euglenophyta) represented relatively constant proportions of the phytoplankton community seasonally (about 35 and 10%, respectively). In the summer and fall, blue-green algae (Cyanophyta) became abundant. Diatoms were relatively abundant at all times and constituted the majority of the community in the winter and spring.     

Evaluation of plastic composite-supports for enhanced ethanol production in biofilm reactors

Biofilms are a natural form of cell immobilization that result from microbial attachment to solid supports. Biofilm reactors with polypropylene composite-supports containing up to 25% (w/w) of various agricultural materials (corn hulls, cellulose, oat hulls, soybean hulls or starch) and nutrients (soybean flour or zein) were used for ethanol production. Pure cultures ofZymomonas mobilis, ATCC 31821 orSaccharomyces cerevisiae ATCC 24859 and mixed cultures with either of these ethanol-producing microorganisms and the biofilm-formingStreptomyces viridosporus T7A ATCC 39115 were evaluated. An ethanol productivity of 374g L^–1 h^–1 (44% yield) was obtained on polypropylene composite-supports of soybean hull-zein-polypropylene by usingZ. mobilis, whereas mixed-culture fermentations withS. viridosporus resulted in ethanol productivity of 147.5 g L^–1 h^–1 when polypropylene composite-supports of corn starch-soybean flour were used. WithS. cerevisiae, maximum productivity of 40 g L^–1 h^–1 (47% yield) was obtained on polypropylene composite-supports of soybean hull-soybean flour, whereas mixed-culture fermentation withS. viridosporus resulted in ethanol productivity of 190g L^–1 h^–1 (35% yield) when polypropylene composite-supports of oat hull-polypropylene were used. The maximum productivities obtained without supports (suspension culture) were 124 g L^–1 h^–1 and 5 g L^–1 h^–1 withZ. mobilis andS. cerevisiae, respectively. Therefore, forZ. mobilis andS. cerevisiae, ethanol productivities in biofilm fermentations were three- and eight-fold higher than suspension culture fermentations, respectively. Biofilm formation on the chips was detected by weight change and Gram staining of the support material at the end of the fermentation. The ethanol production rate and concentrations were consistently greater in biofilm reactors than in suspension cultures.This is Journal Paper No. J-16356 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253