Gatersleben, Germany
Gatersleben, Germany

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Jac P.,Friedrich - Schiller University of Jena | Jac P.,Charles University | Elschner T.,Friedrich - Schiller University of Jena | Reiter C.,Bene PharmaChem GmbH & Co. KG | And 6 more authors.
Cellulose | Year: 2014

Hemicelluloses such as xylans play an increasing role as renewable raw materials for technological applications. The complex and variable composition of hemicelluloses requires powerful analytical techniques in order to assess their composition. In the present study, the neutral fraction of hydrothermally isolated xylan from beech wood was characterized by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) upon derivatization with 8-aminopyrene-1,3,6-trisulfonic acid. Reproducible separation of the xylo-oligosaccharides was achieved using a polyvinyl alcohol coated capillary and a 25 mM sodium acetate buffer, pH 4.75, as background electrolyte at an applied voltage of −30 kV. Intermediate precision expressed as relative standard deviation was below 2.0 % for migration times and below 10 % for relative peak areas except for the oligomers present at very low concentrations only. At the same time, derivatization conditions proved to be robust as well. Samples obtained by fractionation of the xylan were subsequently characterized by CE-LIF. In addition, capillary electrophoresis with mass spectrometry detection indicated the presence of small amounts of xylo-oligosaccharides containing additional sugar moieties such as 4-O-methylglucuronic acid. Moreover, minor components containing acetyl groups could be detected. The presence of these impurities was confirmed by nuclear magnetic resonance analysis of the fractions. In conclusion, although none of the techniques applied here gave a complete picture of the composition of the investigated xylan or its fractions, the combination provided insight into the complexity of the sample. © 2014, Springer Science+Business Media Dordrecht.


Kasprzak J.,Leibniz Institute of Plant Genetics and Crop Plant Research | Rauter M.,Orgentis Chemicals GmbH | Denter S.,ARTES Biotechnology GmbH | Becker K.,Orgentis Chemicals GmbH | And 6 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2016

Yeast cell catalysts carrying a recombinant Carboxydothermus hydrogenoformans alcohol dehydrogenase (ChADH) gene were used to synthesise ethyl (R)-mandelate. Transgenic Arxula adeninivorans and Hansenula polymorpha strains were constructed to produce recombinant ChADH at high concentrations. Biochemical parameters such as pH and temperature optima, thermostability and substrate specificity were determined for the enzyme synthesized in Arxula adeninivorans. The recombinant enzyme combined with a substrate-coupled cofactor regeneration system and permeabilized Arxula adeninivorans and Hansenula polymorpha cell catalysts co-expressing ChADH and Bacillus megaterium glucose dehydrogenase (BmGDH) for enzyme-coupled cofactor regeneration, were used to synthesize ethyl (R)-mandelate. Comparison of purified recombinant ChADH/BmGDH and both of the yeast cell based catalysts ability to synthesize ethyl (R)-mandelate demonstrated that Hansenula polymorpha cell catalysts were able to produce the highest yield (Hansenula polymorpha 6.07 mmol l−1 h−1) while Arxula adeninivorans produced approximately half this amount (3.07 mmol l−1 h−1). The maximum conversion achieved was 98% with a high enantiomeric excess (>98%). © 2016 Elsevier B.V.


Rauter M.,Orgentis Chemicals GmbH | Kasprzak J.,Leibniz Institute of Plant Genetics and Crop Plant Research | Denter S.,ARTES Biotechnology GmbH | Becker K.,Orgentis Chemicals GmbH | And 4 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2014

An Arxula adeninivorans strain co-expressing ADH of Rhodococcus ruber and GDH of Bacillus megaterium, coding for (S)-specific alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) respectively, was used for the synthesis of enantiomerically pure 1-(S)-phenylethanol as permeabilized yeast cells (PC), permeabilized immobilized cells (PIC) or immobilized crude extract (IE). Permeabilization was achieved with Triton X-100 and resulted in cells that had the same activity as crude extract. Calcium alginate immobilization allowed the entrapment of enzymes and PIC, which resulted in a gain of 60 and 82% activity respectively compared to crude extract. All enzyme preparations were used in successive reaction cycles. PC and IE lost activity after 10 reactions whereas PIC were active with over 80% activity for 28 cycles. After 5 PIC catalyzed reactions, 94.8% chemically pure 1-(S)-phenylethanol with ee of ≥99% was isolated, which is a conversion of 79.6% of the substrate, acetophenone. Furthermore the reaction medium containing BIS-TRIS buffer could be regenerated by the addition of Ca(OH)2, which precipitated calcium gluconate and restored full buffering capacity. Stability is a key factor for the cost-effective use of an enzyme system in industry and these results indicate that A. adeninivorans PIC will be useful for the synthesis enantiomerically pure alcohols. © 2014 Elsevier B.V.


Rauter M.,Orgentis Chemicals GmbH | Prokoph A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Kasprzak J.,Leibniz Institute of Plant Genetics and Crop Plant Research | Becker K.,Orgentis Chemicals GmbH | And 4 more authors.
Applied Microbiology and Biotechnology | Year: 2015

The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200 U l-1 ADH and 370 U l-1 G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700 U l-1 ADH and 170 U l-1 GDH activity. However, the crude extract of both strains reduced 40 mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99 % in 60 min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH–LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP+, makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74 g l−1 day−1 or 406 g kg−1 dry cell weight (dcw) day−1 isolated 1-(R)-phenylethanol with an ee of 100 % and a TTN of 206 mol product per mole NADP+. In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols. © 2014, Springer-Verlag Berlin Heidelberg.


Rauter M.,Orgentis Chemicals GmbH | Schwarz M.,Leibniz Institute of Plant Genetics and Crop Plant Research | Becker K.,Orgentis Chemicals GmbH | Baronian K.,University of Canterbury | And 3 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2013

The LAC4 gene of Kluyveromyces lactis encoding for β-galactosidase was overexpressed in the yeast Arxula adeninivorans to produce the enzyme, which can be used for the synthesis of β-d-galactosides. These compounds play a major role as precursors for the synthesis of glycolipids and glycoproteins in medicine or for the production of tensides. The Xplor®2 transformation/expression platform was used because it enabled stable integration of the gene in the Arxula genome and the production of high levels of the enzyme. The recombinant β-galactosidase, fused with C-terminal His-tag region (Lac4-6hp), was purified by precipitation with ammonium sulphate and FPLC using hydroxylapatite. The enzyme exhibited optimal activity at 37 to 40 C, pH 6.5 in 50 mM sodium acetate buffer. Activity was measured by the formation of p-nitrophenol at 405 nm from the hydrolyzed chromogenic substrate, p-nitrophenyl-β-d-gal. Biochemical characterization included the calculation of KM and apparent kcat values of the enzyme. The formation of benzyl β-d-gal by 0.1 U enzyme from A. adeninivorans with transgalactosylation was six times higher than that for the prokaryotic enzyme from E. coli. Moreover, the partially purified enzyme was used for the selective hydrolysis of allyl β-d-gal in a mixture of allyl β- and allyl α-d-gal, with 4 g l-1 being hydrolysed within one day by 1 U ml-1. Thus, the recombinant β-galactosidase produced in A. adeninivorans is of potential interest for the enzymatic synthesis of benzyl β-d-gal and other galactosides as well as the selective hydrolysis of anomeric mixtures and could be used to replace difficult chemical procedures. © 2013 Elsevier B.V. All rights reserved.


Rauter M.,Orgentis Chemicals GmbH | Kasprzak J.,Leibniz Institute of Plant Genetics and Crop Plant Research | Becker K.,Orgentis Chemicals GmbH | Riechen J.,Jackering Muhlen und Nahrmittelwerke GmbH | And 9 more authors.
Microbial Cell Factories | Year: 2016

Background: The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation. Results: In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs' (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000 U/L and 500 U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400 U/L and 200 U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and ∆aadh2 mutant cultures had the effect of accelerating 1-butanol consumption. Conclusions: Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation. © 2016 The Author(s).


Kasprzak J.,Leibniz Institute of Plant Genetics and Crop Plant Research | Bischoff F.,Leibniz Institute of Plant Genetics and Crop Plant Research | Rauter M.,Orgentis Chemicals GmbH | Becker K.,Orgentis Chemicals GmbH | And 5 more authors.
Biochemical Engineering Journal | Year: 2016

The ReADH gene of Rhodococcus erythropolis DSM 43297 encoding alcohol dehydrogenase (ReADH) was expressed in the yeast Arxula adeninivorans and Hansenula polymorpha to determine which host accumulated the highest concentration of recombinant alcohol dehydrogenase for the production of enantiometrically pure alcohols. The ReADH gene was fused with a His-tag encoding sequence at its 3'-end and expressed in both yeast species. The recombinant ReADH-6H preparations exhibited small host-strain dependent differences in their pH and temperature optima, thermo-stability and substrate specificity. The recombinant enzymes in combination with a substrate-coupled cofactor regeneration system were used to synthesize 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate. Permeabilized A. adeninivorans whole cell catalysts co-expressing R. erythropolis alcohol dehydrogenase and Bacillus megaterium glucose dehydrogenase, for enzyme-coupled cofactor regeneration, were also used for the synthesis reactions. © 2015 Elsevier B.V..


Rauter M.,Orgentis Chemicals GmbH | Kasprzak J.,Leibniz Institute of Plant Genetics and Crop Plant Research | Becker K.,Orgentis Chemicals GmbH | Baronian K.,University of Canterbury | And 3 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2014

The RrADH gene of Rhodococcus ruber coding for (S)-specific alcohol dehydrogenase (RrADH) was overexpressed in the yeast Arxula adeninivorans and used for the synthesis of enantiomerically pure alcohols. The substrates acetophenone, 2,5-hexandione and 2-nonanon used for this synthesis, were reduced by RrADH to produce S-configured alcohols. Regeneration of the cofactor, NADH, was required for the reaction and this was provided by using isopropanol as a second ADH substrate to reduce NAD+ or by cloning the glucose dehydrogenase gene from Bacillus megaterium (BmGDH) into the yeast which regenerated NADH by oxidizing glucose. Expressing both RrADH and BmGDH in the yeast provided a strain that could synthesize 1-(S)-phenylethanol from acetophenone with NADH being regenerated by the oxidation of glucose. Both bioreduction systems led to the synthesis of pure (S) enantiomer of 1-phenylethanol, but only the enzyme coupled approach reduced 40 mM acetophenone completely in 150 min. 75 mg of 98% pure product could be isolated from 20 ml. In conclusion the synthesis potential of the RrADH expressed in A. adeninivorans is very promising for 1-(S)-phenylethanol synthesis. © 2014 Elsevier B.V.


PubMed | University of Canterbury, University of Greifswald, Orgentis Chemicals GmbH, Jackering Muhlen und Nahrmittelwerke GmbH and Leibniz Institute of Plant Genetics and Crop Plant Research
Type: Journal Article | Journal: Microbial cell factories | Year: 2016

The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation.In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000U/L and 500U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400U/L and 200U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and aadh2 mutant cultures had the effect of accelerating 1-butanol consumption.Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation.


PubMed | University of Canterbury, University of Greifswald, Orgentis Chemicals GmbH, Jackering Muhlen und Nahrmittelwerke GmbH and Leibniz Institute of Plant Genetics and Crop Plant Research
Type: Journal Article | Journal: FEMS yeast research | Year: 2016

In this study, alcohol dehydrogenase 1 from Arxula adeninivorans (Aadh1p) was identified and characterized. Aadh1p showed activity with short and medium chain length primary alcohols in the forward reaction and their aldehydes in the reverse reaction. Aadh1p has 64% identity with Saccharomyces cerevisiae Adh1p, is localized in the cytoplasm and uses NAD(+) as cofactor. Gene expression analysis showed a low level increase in AADH1 gene expression with ethanol, pyruvate or xylose as the carbon source. Deletion of the AADH1 gene affects growth of the cells with 1-butanol, ethanol and glucose as the carbon source, and a strain which overexpressed the AADH1 gene metabolized 1-butanol more rapidly. An ADH activity assay indicated that Aadh1p is a major enzyme for the synthesis of ethanol and the degradation of 1-butanol in A. adeninivorans.

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