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Gatersleben, Germany

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. Source

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. Source

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.. Source

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. Source

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. Source

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