Publications by authors named "Michael Breuer"

42 Publications

Physical and Chemical Traits of Grape Varieties Influence Preferences and Performance.

Front Plant Sci 2021 21;12:664636. Epub 2021 Apr 21.

Department of Chemical Ecology, Bielefeld University, Bielefeld, Germany.

The cuticle-covered surface forms the interface between plant parts, including fruits, and their environment. The physical and chemical properties of fruit surfaces profoundly influence plant-frugivore interactions by shaping the susceptibility and suitability of the host for the attacker. Grapevine (, Vitaceae) serves as one of the various host plants of the spotted wing drosophila, Matsumura (Diptera: Drosophilidae), which is invasive in several parts of the world and can cause major crop losses. The susceptibility of wine towards this pest species differs widely among varieties. The objective of our study was to identify physical and chemical traits of the berry surface that may explain the differences in susceptibility of five grape varieties to . Both preferences of adult and offspring performance on intact dewaxed (epicuticular wax layer mechanically removed) grape berries were investigated in dual-choice assays. Moreover, the morphology and chemical composition of cuticular waxes and cutin of the different varieties were analyzed. Bioassays revealed that the epicuticular wax layer of most tested grape varieties influenced the preference behavior of adult flies; even less susceptible varieties became more susceptible after removal of these waxes. In contrast, neither offspring performance nor berry skin firmness were affected by the epicuticular wax layer. The wax morphology and the composition of both epi- and intracuticular waxes differed pronouncedly, especially between more and less susceptible varieties, while cutin was dominated by ω-OH-9/10-epoxy-C18 acid and the amount was comparable among varieties within sampling time. Our results highlight the underestimated role of the epicuticular surface and cuticle integrity in grape susceptibility to
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http://dx.doi.org/10.3389/fpls.2021.664636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8098983PMC
April 2021

Effects of Variety and Grape Berry Condition of on Preference Behavior and Performance of .

Insects 2019 Nov 30;10(12). Epub 2019 Nov 30.

Department of Chemical Ecology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.

is an invasive fruit pest and represents a potential economic threat to viticulture. After first observations of in Europe in 2008, research mainly focused on the evaluation of the host range and infestation risk for fruit and berry crops. However, the risk assessment of in viticulture has only recently started. Understanding the factors influencing preferences of for host species and varieties as well as offspring performance is essential to improve management strategies. We investigated the field infestation of different grape varieties across Baden-Wuerttemberg, southwestern Germany, between 2015 and 2018. Moreover, we performed dual-choice assays in the laboratory to investigate whether adults show preferences for certain varieties and whether offspring performance differs between varieties. Furthermore, we studied the impact of grape damage on choice behavior. Field monitoring revealed that show preferences for red varieties, whereas almost no oviposition occurred in white varieties. The results of dual-choice assays confirmed that preference and performance are influenced by grape variety and that flies preferred damaged over intact "Pinot Noir", "Pinot Blanc", and "Müller-Thurgau" berries. Overall, these findings may have important implications for winegrowers regarding cultivated varieties, grape health, and insecticide reduction.
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http://dx.doi.org/10.3390/insects10120432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955987PMC
November 2019

Enzymatic network for production of ether amines from alcohols.

Biotechnol Bioeng 2016 09 9;113(9):1853-61. Epub 2016 Mar 9.

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, Groningen, 9747AG, The Netherlands.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.25954DOI Listing
September 2016

Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from 'Aromatoleum aromaticum' EbN1.

J Mol Microbiol Biotechnol 2015 22;25(5):327-39. Epub 2015 Oct 22.

Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg, Oldenburg, Germany.

The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium 'Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the 'classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD(+)-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe(187)), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr(93)) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93].
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http://dx.doi.org/10.1159/000439113DOI Listing
July 2016

Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

Appl Microbiol Biotechnol 2016 Feb 17;100(3):1265-1273. Epub 2015 Oct 17.

Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.
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http://dx.doi.org/10.1007/s00253-015-7041-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717159PMC
February 2016

Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades.

Science 2015 Sep;349(6255):1525-9

School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, Manchester M1 7DN, UK.

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product.
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http://dx.doi.org/10.1126/science.aac9283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4883652PMC
September 2015

Enzymatic enantioselective decarboxylative protonation of heteroaryl malonates.

Chemistry 2015 Apr 12;21(17):6557-63. Epub 2015 Mar 12.

School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7ND (UK).

The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase.
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http://dx.doi.org/10.1002/chem.201406014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517146PMC
April 2015

Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure.

J Biotechnol 2014 Dec 26;191:86-92. Epub 2014 Aug 26.

Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany(1). Electronic address:

In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.
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http://dx.doi.org/10.1016/j.jbiotec.2014.08.022DOI Listing
December 2014

Influence of environment and climate on occurrence of the cixiid planthopper Hyalesthes obsoletus, the vector of the grapevine disease 'bois noir'.

Bull Entomol Res 2013 Dec 29;103(6):621-33. Epub 2013 Apr 29.

State Institute of Viticulture and Oenology, Freiburg, Germany.

Species distribution models (SDMs), which are well established in many fields of biological research, are still uncommon in the agricultural risk analysis of pest insects. To exemplify the use of SDMs, we investigated the influence of environmental factors on the occurrence of Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae). The planthopper is the only known vector of the grapevine yellows disease 'bois noir'. The study was conducted in 145 locations in the Baden region of southwest Germany. The planthopper was surveyed on host plant patches, consisting of stinging nettle and/or bindweeds. We used a stratified modelling framework where (1) species presence-absence data were related to an extensive environmental dataset using logistic regressions; and (2) different types of average models were developed based on an information theoretic method. The results show that the incidence of H. obsoletus is associated to above- as well as below-ground environmental factors, particularly to the amount of fine soil and average annual precipitation. This result was consistent across all average models. The relative importance of other environmental variables was dependent upon the average model under consideration and thus may vary according to their intended use, either the explanation of habitat requirements or the prediction and mapping of occurrence risks. The study showed that SDMs offer a quantification of species' habitat requirements and thus, could represent a valuable tool for pest management purposes. By providing examples of current issues of grapevine pests in viticulture, we discuss the use of SDMs in agricultural risk analysis and highlight their advantages and caveats.
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http://dx.doi.org/10.1017/S0007485313000163DOI Listing
December 2013

Biocatalytic and structural properties of a highly engineered halohydrin dehalogenase.

Chembiochem 2013 May 12;14(7):870-81. Epub 2013 Apr 12.

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Two highly engineered halohydrin dehalogenase variants were characterized in terms of their performance in dehalogenation and epoxide cyanolysis reactions. Both enzyme variants outperformed the wild-type enzyme in the cyanolysis of ethyl (S)-3,4-epoxybutyrate, a conversion yielding ethyl (R)-4-cyano-3-hydroxybutyrate, an important chiral building block for statin synthesis. One of the enzyme variants, HheC2360, displayed catalytic rates for this cyanolysis reaction enhanced up to tenfold. Furthermore, the enantioselectivity of this variant was the opposite of that of the wild-type enzyme, both for dehalogenation and for cyanolysis reactions. The 37-fold mutant HheC2360 showed an increase in thermal stability of 8 °C relative to the wild-type enzyme. Crystal structures of this enzyme were elucidated with chloride and ethyl (S)-3,4-epoxybutyrate or with ethyl (R)-4-cyano-3-hydroxybutyrate bound in the active site. The observed increase in temperature stability was explained in terms of a substantial increase in buried surface area relative to the wild-type HheC, together with enhanced interfacial interactions between the subunits that form the tetramer. The structures also revealed that the substrate binding pocket was modified both by substitutions and by backbone movements in loops surrounding the active site. The observed changes in the mutant structures are partly governed by coupled mutations, some of which are necessary to remove steric clashes or to allow backbone movements to occur. The importance of interactions between substitutions suggests that efficient directed evolution strategies should allow for compensating and synergistic mutations during library design.
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http://dx.doi.org/10.1002/cbic.201300005DOI Listing
May 2013

Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

Appl Microbiol Biotechnol 2013 Feb 12;97(4):1571-80. Epub 2012 Apr 12.

Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany.

Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.
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http://dx.doi.org/10.1007/s00253-012-4008-1DOI Listing
February 2013

Activation-independent cyclization of monoterpenoids.

Appl Environ Microbiol 2012 Feb 9;78(4):1055-62. Epub 2011 Dec 9.

Institute of Microbiology, University of Stuttgart, Stuttgart, Germany.

The biosynthesis of cyclic monoterpenes (C(10)) generally requires the cyclization of an activated linear precursor (geranyldiphosphate) by specific terpene cyclases. Cyclic triterpenes (C(30)), on the other hand, originate from the linear precursor squalene by the action of squalene-hopene cyclases (SHCs) or oxidosqualene cyclases (OSCs). Here, we report a novel terpene cyclase from Zymomonas mobilis (ZMO1548-Shc) with the unique capability to cyclize citronellal to isopulegol. To our knowledge, ZMO1548-Shc is the first biocatalyst with diphosphate-independent monoterpenoid cyclase activity. A combinatorial approach using site-directed mutagenesis and modeling of the active site with a bound substrate revealed that the cyclization of citronellal proceeds via a different mechanism than that of the cyclization of squalene.
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http://dx.doi.org/10.1128/AEM.07059-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273027PMC
February 2012

The case for risk-based premiums in public health insurance.

Health Econ Policy Law 2006 Apr;1(Pt 2):171-88

Socioeconomic Institute of the University of Zurich, Switzerland.

Uniform, risk-independent insurance premiums are accepted as part of 'managed competition' in health care. However, they are not compatible with optimality of health insurance contracts in the presence of both ex ante and ex post moral hazard. They have adverse effects on insurer behaviour even if risk adjustment is taken into account. Risk-based premiums combined with means-tested, tax-financed transfers are advocated as an alternative.
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http://dx.doi.org/10.1017/S1744133105001064DOI Listing
April 2006

Improved (R)-phenylacetylcarbinol production with Candida utilis pyruvate decarboxylase at decreased organic to aqueous phase volume ratios.

Biotechnol Lett 2008 Feb 18;30(2):281-6. Epub 2007 Sep 18.

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

The effect of decreasing the organic (octanol) to aqueous phase volume ratio was evaluated in a two-phase enzymatic process for (R)-phenylacetylcarbinol (PAC) production. Decreasing the ratio from 1:1 to 0.43:1 at 4 degrees C increased PAC in the organic phase from 112 g/l to 183 g/l with a 10% improvement in overall productivity. Interestingly, the rate of enzyme (pyruvate decarboxylase) activity loss was unaffected by the reduced phase ratio over the reaction period (48 h). At 20 degrees C and 0.43:1 phase ratio the organic phase PAC concentration increased to 212 g/l and the overall productivity increased by 30% although the PAC yield (based on pyruvate) declined by about 10% due to greater byproduct acetoin formation at the higher temperature. Product recovery in such a system is facilitated both by the higher PAC concentration and the reduced organic phase volume.
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http://dx.doi.org/10.1007/s10529-007-9525-0DOI Listing
February 2008

Hexadecane and Tween 80 stimulate lipase production in Burkholderia glumae by different mechanisms.

Appl Environ Microbiol 2007 Jun 27;73(12):3838-44. Epub 2007 Apr 27.

Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Burkholderia glumae strain PG1 produces a lipase of biotechnological relevance. Lipase production by this strain and its derivative LU8093, which was obtained through classical strain improvement, was investigated under different conditions. When 10% hexadecane was included in the growth medium, lipolytic activity in both strains could be increased approximately 7-fold after 24 h of growth. Hexadecane also stimulated lipase production in a strain containing the lipase gene fused to the tac promoter, indicating that hexadecane did not affect lipase gene expression at the transcriptional level, which was confirmed using lipA-gfp reporter constructs. Instead, hexadecane appeared to enhance lipase secretion, since the amounts of lipase in the culture supernatant increased in the presence of hexadecane, with a concomitant decrease in the cells, even when protein synthesis was inhibited with chloramphenicol. In the presence of olive oil as a carbon source, nonionic detergents, such as Tween 80, increased extracellular lipase activity twofold. Like hexadecane, Tween 80 appeared to stimulate lipase secretion, although in a more disruptive manner, since other, normally nonsecreted proteins were found in the culture supernatant. Additionally, like olive oil, Tween 80 was found to induce lipase gene expression in strain PG1 in medium containing sucrose as a carbon source but not in glucose-containing medium, suggesting that lipase gene expression is prone to catabolite repression. In contrast, lipase production in the lipase-overproducing strain LU8093 was independent of the presence of an inducer and was not inhibited by glucose. In conclusion, hexadecane and Tween 80 enhance lipase production in B. glumae, and they act via different mechanisms.
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http://dx.doi.org/10.1128/AEM.00097-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932709PMC
June 2007

Yeast pyruvate decarboxylases: variation in biocatalytic characteristics for (R)-phenylacetylcarbinol production.

FEMS Yeast Res 2007 Jan;7(1):33-9

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.

Based on previous studies, Candida utilis pyruvate decarboxylase (PDC) proved to be a stable and high productivity enzyme for the production (R)-phenylacetylcarbinol (PAC), a pharmaceutical precursor. However, a portion of the substrate pyruvate was lost to by-product formation. To identify a source of PDC which might overcome this problem, strains of four yeasts -- C. utilis, Candida tropicalis, Saccharomyces cerevisiae and Kluyveromyces marxianus -- were investigated for their PDC biocatalytic properties. Biotransformations were conducted with benzaldehyde and pyruvate as substrates and three experimental systems were employed (in the order of increasing benzaldehyde concentrations): (I) aqueous (soluble benzaldehyde), (II) aqueous/benzaldehyde emulsion, and (III) aqueous/octanol-benzaldehyde emulsion. Although C. utilis PDC resulted in the highest concentrations of PAC and was the most stable enzyme, C. tropicalis PDC was associated with the lowest acetoin formation. For example, in system (III) the ratio of PAC over acetoin was 35 g g(-1) for C. tropicalis PDC and 9.2 g g(-1) for C. utilis PDC. The study thereby opens up the potential to design a PDC with both high productivity and high yield characteristics.
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http://dx.doi.org/10.1111/j.1567-1364.2006.00138.xDOI Listing
January 2007

Improving prediction of aerosol deposition in an idealized mouth using large-Eddy simulation.

J Aerosol Med 2006 ;19(3):290-300

Department of Mechanical and Aerospace Engineering, Carleton University, Ottawa, ON, Canada.

Monodisperse aerosol deposition in an idealized mouth geometry with a relatively small inlet diameter (D (in) = 3.0 mm) was studied numerically using a standard Large Eddy Simulation (LES). A steady inhalation flow rate of Q = 32.2 L/min was used. Thousands of particles (2.5, 3.7, and 5.0 microm in diameter and rho (f) = 912.0 kg/m(3) density) were released separately in the computational domain and aerosol deposition was determined. The total aerosol deposition results in this idealized mouth were in relatively good agreement when compared with measured data obtained in separate experiments, showing considerable improvement over the standard RANS/EIM (Reynolds Averaged Navier-Stokes/Eddy Interaction Model) approach.
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http://dx.doi.org/10.1089/jam.2006.19.290DOI Listing
January 2007

Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning.

J Biotechnol 2007 Jan 12;127(4):575-92. Epub 2006 Aug 12.

Fraunhofer Institute for Interfacial Engineering and Biotechnology, Institute for Interfacial Engineering, University of Stuttgart, Nobelstrasse 12, D-70569 Stuttgart, Germany.

Metagenome cloning has become a powerful tool to exploit the biocatalytic potential of microbial communities for the discovery of novel biocatalysts. In a novel variant of direct expression cloning, metagenomic DNA was isolated from compost by a modified direct lysis method, purified by size exclusion chromatography and cloned into an expression vector allowing bidirectional transcription. Transformation of Escherichia coli DH5alpha resulted in a metagenomic expression library with an average insert size of 3.2 kb. To estimate the functional diversity of the constructed library, it was screened by different approaches based on functional heterologous expression. A large number of active clones were identified, including lipolytic enzymes, amylases, phosphatases and dioxygenases. Molecular analysis of one important class of industrial biocatalysts, the lipolytic enzymes, confirmed the novelty and dissimilarity of all recovered genes, which exhibited only limited similarity to known enzymes. Equally, the novelty of another three genes encoding phosphatase or dioxygenase activity, respectively, was shown. These results demonstrate the suitability of this direct cloning approach, which comprised a dual-orientation expression vector and a simple one-step DNA purification method, for the efficient discovery of numerous active novel clones. By this means it provides an efficient way for the rapid generation of large libraries of hitherto unknown enzyme candidates which could be screened for different specific target reactions.
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http://dx.doi.org/10.1016/j.jbiotec.2006.07.036DOI Listing
January 2007

A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.

Comb Chem High Throughput Screen 2006 May;9(4):289-93

BASF Aktiengesellschaft, Fine Chemicals and Biocatalysis Research, GVF/E - A030, D-67056 Ludwigshafen, Germany.

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.
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http://dx.doi.org/10.2174/138620706776843228DOI Listing
May 2006

Crystal structure and enzyme kinetics of the (S)-specific 1-phenylethanol dehydrogenase of the denitrifying bacterium strain EbN1.

Biochemistry 2006 Jan;45(1):82-93

BASF AG, Physical Chemistry and Informatics, 67056 Ludwigshafen, Germany.

(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.
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http://dx.doi.org/10.1021/bi051596bDOI Listing
January 2006

Degradation profile of [His7]-corazonin in the hemolymph of the desert locust Schistocerca gregaria.

Peptides 2006 Mar 23;27(3):539-48. Epub 2005 Nov 23.

Laboratory of Developmental Physiology, Genomics and Proteomics, Zoological Institute, K.U. Leuven, B-3000 Leuven, Belgium.

Degradation of the neuropeptide [His7]-corazonin, a key hormone in phase transition in locusts was studied using [3H][His7]-corazonin, RP-HPLC and mass spectrometry. After 4h incubation, 50 and 75% of [His7]-corazonin could still be found in hemolymph of gregarious and solitarious Schistocerca gregaria, respectively. Under in vivo conditions the half-life was 30 min. These results are in contrast to many other neuropeptides that usually have half lives of a few minutes. The peptide is cleaved first by an endopeptidase, either just before or after the Tyr residue at position 5. Next, the C-terminal degradation fragments are further degraded by a dipeptidyl-peptidase, whereas the N-terminal fragments are further broken down one amino acid at a time. In addition, [Dopa5][His7]-corazonin was detected. Upon synthesis, this unexpected molecular modification turned out to be biologically active in bringing about cuticular melanization.
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http://dx.doi.org/10.1016/j.peptides.2005.08.021DOI Listing
March 2006

Enzymatic (R)-phenylacetylcarbinol production in a benzaldehyde emulsion system with Candida utilis cells.

Appl Microbiol Biotechnol 2006 Mar 13;70(2):170-5. Epub 2005 Sep 13.

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.

Recent progress in enzymatic (R)-phenylacetylcarbinol (PAC) production has established the need for low cost and efficient biocatalyst preparation. Pyruvate decarboxylase (PDC) added in the form of Candida utilis cells showed higher stability towards benzaldehyde and temperature in comparison with partially purified preparations. In the presence of 50 mM benzaldehyde and at 4 degrees C, a half-life of 228 h was estimated for PDC added as C. utilis cells, in comparison with 24 h for the partially purified preparation. Increasing the temperature from 4 to 21 degrees C for PAC production with C. utilis cells resulted in similar final PAC levels of 39 and 43 g l(-1) (258 and 289 mM), respectively, from initial 300 mM benzaldehyde and 364 mM pyruvate. The overall volumetric productivity was enhanced 2.8-fold, which reflected the 60% shorter reaction time at the higher temperature. Enantiomeric excess values of 98 and 94% for R-PAC were obtained at 4 and 21 degrees C, respectively, and benzyl alcohol (a potential by-product from benzaldehyde) was not formed.
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http://dx.doi.org/10.1007/s00253-005-0063-1DOI Listing
March 2006

pH shift enhancement of Candida utilis pyruvate decarboxylase production.

Biotechnol Bioeng 2005 Oct;92(2):183-8

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.

Pyruvate decarboxylase (PDC) catalyses the synthesis of asymmetric carbinols, e.g., chiral precursors for pharmaceuticals such as ephedrine and pseudoephedrine. The production of PDC by Candida utilis in a minimal medium was improved by manipulating the pH during fermentation in a 5 L bioreactor. At an aeration rate of 0.1 vvm with a stirrer speed of 300 rpm at constant pH 6, a specific PDC activity of 141 U/g dry cell weight (DCW) was achieved (average of two fermentations +/-13%). By allowing the yeast to acidify the growth medium from pH 6 to 2.9, the final specific PDC activity increased by a factor of 2.7 to 385 U/g DCW (average from 4 fermentations +/-16%). The effect of this pH drift on PDC production was confirmed by another experiment with a manual shift of pH from 6 to 3 by addition of 5 M sulfuric acid. The final PDC activity was 392 U/g DCW (average from two fermentations +/-5%). However, experiments with constant pH of 6, 5, 4, or 3 resulted in average specific activities of only 102 to 141 U/g DCW, suggesting that a transitional pH change rather than the absolute pH value was responsible for the increased specific PDC activity.
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http://dx.doi.org/10.1002/bit.20588DOI Listing
October 2005

Cells of Candida utilis for in vitro (R)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor.

Biotechnol Lett 2005 Apr;27(8):575-81

School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Sydney, Australia.

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 degrees C, a screen of several 1-alcohols (C4-C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l-1 (PDC activity 2.5 U ml-1), PAC levels of 103 g l-1 in the octanol phase and 12.8 g l-1 in the aqueous phase were produced in 15 h at 21 degrees C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 degrees C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U-1 h-1) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l-1 h-1) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g-1).
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http://dx.doi.org/10.1007/s10529-005-3252-1DOI Listing
April 2005

(R)-phenylacetylcarbinol production in aqueous/organic two-phase systems using partially purified pyruvate decarboxylase from Candida utilis.

Biotechnol Bioeng 2005 Jul;91(2):190-8

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

Aqueous/organic two-phase systems have been evaluated for enhanced production of (R)-phenylacetylcarbinol (PAC) from pyruvate and benzaldehyde using partially purified pyruvate decarboxylase (PDC) from Candida utilis. In a solvent screen, octanol was identified as the most suitable solvent for PAC production in the two-phase system in comparison to butanol, pentanol, nonanol, hexane, heptane, octane, nonane, dodecane, methylcyclohexane, methyl tert butyl ether, and toluene. The high partitioning coefficient of the toxic substrate benzaldehyde in octanol allowed delivery of large amounts of benzaldehyde into the aqueous phase at a concentration less than 50 mM. PDC catalyzed the biotransformation of benzaldehyde and pyruvate to PAC in the aqueous phase, and continuous extraction of PAC and byproducts acetoin and acetaldehyde into the octanol phase further minimized enzyme inactivation, and inhibition due to acetaldehyde. For the rapidly stirred two-phase system with a 1:1 phase ratio and 8.5 U/mL carboligase activity, 937 mM (141 g/L) PAC was produced in the octanol phase in 49 h with an additional 127 mM (19 g/L) in the aqueous phase. Similar concentrations of PAC could be produced in the slowly stirred phase separated system at this enzyme level, although at a much slower rate. However at lower enzyme concentration very high specific PAC production (128 mg PAC/U carboligase at 0.9 U/mL) was achieved in the phase separated system, while still reaching final PAC levels of 102 g/L in octanol and 13 g/L in the aqueous phase. By comparison with previously published data by our group for a benzaldehyde emulsion system without octanol (50 g/L PAC, 6 mg PAC/U carboligase), significantly higher PAC concentrations and specific PAC production can be achieved in an octanol/aqueous two-phase system.
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http://dx.doi.org/10.1002/bit.20513DOI Listing
July 2005

Insecticidal activity of the pyrimidine nucleoside analogue (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU).

Pest Manag Sci 2005 Aug;61(8):737-41

Zoological Institute, Katholieke Universiteit Leuven, Naamsestraat 59, B-3000 Leuven, Belgium.

The insecticidal activity of the antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was assessed in in vivo assays against the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera, Noctuidae). BVDU, mixed into an artificial diet, caused a variety of effects, depending on the concentration used. Compared with controls, food intake was lower, larval growth was retarded and larval development was prolonged. The treated larvae formed smaller pupae and the hatching moths often showed morphogenetic defects. A higher mortality could be found in larval and pupal stages and was generally caused by moult disruption. A choice assay showed that BVDU has very slight feeding-deterrent properties, which only partly explain the toxic effects. The agent most probably acts through its cytostatic activity that has been described previously using cell lines of different insect species.
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http://dx.doi.org/10.1002/ps.1053DOI Listing
August 2005

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde.

J Biotechnol 2005 Jan;115(1):91-9

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.
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http://dx.doi.org/10.1016/j.jbiotec.2004.08.002DOI Listing
January 2005

Transgenic expression in tobacco of a poly-proctolin construct leading to production of the bioactive peptide.

Biotechnol Lett 2004 Sep;26(18):1413-20

Dipartimento di Scienze del Suolo, della Pianta e dell'Ambiente, Università degli Studi di Napoli Federico II, Portici, Italy.

Short peptides can be expressed in plants using synthetic genes encoding multiple copies of the peptide spaced by dibasic endoproteolytic cleavage sites. A synthetic gene encoding an array of repeated copies of proctolin, a very well characterized insect myotropic peptide, spaced by Arg residues, was synthesized and expressed in tobacco plants. The successful production of bioactive proctolin from the precursor in transgenic plants was demonstrated by immunoblot, HPLC, mass spectrometry and a bioassay based on the contraction of isolated cockroach hindgut. These results suggest that in planta, as in animals and yeasts, endopeptidases of the serine proteases family may be involved in precursor processing.
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http://dx.doi.org/10.1023/B:BILE.0000045644.52747.6aDOI Listing
September 2004

The NMDA receptor antagonist MK-801 inhibits vitellogenesis in the flesh fly Neobellieria bullata and in the desert locust Schistocerca gregaria.

J Insect Physiol 2004 Oct;50(10):927-34

Zoological Institute, Katholieke Universiteit Leuven, Naamsestraat 59, 3000 Leuven, Belgium.

We found that in the flesh fly Neobellieria bullata, vitellogenesis can be inhibited in a dose-dependent way by two injections of 60 microg MK-801/g body mass. In the desert locust Schistocerca gregaria, vitellogenesis can also be fully inhibited but only by repeated injections of 200-400 microg/g body mass. In this species, the inhibition can be overruled by coapplication of juvenile hormone. Vitellogenin bands remained visible in electropherograms of hemolymph of MK-801-treated female locusts, but vitellogenin did not accumulate as might be expected when only its uptake by the oocytes, and not its synthesis by the fat body, would be affected. Whether MK-801 acts by inhibiting juvenile hormone synthesis by the corpora allata remains to be investigated.
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http://dx.doi.org/10.1016/j.jinsphys.2004.07.003DOI Listing
October 2004

Kinetic analysis and modelling of enzymatic (R)-phenylacetylcarbinol batch biotransformation process.

J Biotechnol 2004 Jul;111(2):179-89

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

Initial rate and biotransformation studies were applied to refine and validate a mathematical model for enzymatic (R)-phenylacetylcarbinol (PAC) production from pyruvate and benzaldehyde using Candida utilis pyruvate decarboxylase (PDC). The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U ml-1 carboligase. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde, and acetoin. These values were determined from data for three batch biotransformations performed over a range of initial concentrations (viz. 50-150 mM benzaldehyde, 60-180 mM pyruvate, 1.1-3.4 U ml-1 enzyme activity). The finalized model was then used to predict a batch biotransformation profile at 120/100 mM initial pyruvate/benzaldehyde (initial enzyme activity 3.0 U ml-1). The simulated kinetics gave acceptable fitting (R2 = 0.9963) to the time courses of these latter experimental data for substrates pyruvate and benzaldehyde, product PAC, by-products acetaldehyde and acetoin, as well as enzyme activity level.
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http://dx.doi.org/10.1016/j.jbiotec.2004.04.001DOI Listing
July 2004