ASA Spezialenzyme GmbH

Wolfenbüttel, Germany

ASA Spezialenzyme GmbH

Wolfenbüttel, Germany

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Tonin F.,University of Insubria | Melis R.,University of Insubria | Cordes A.,ASA Spezialenzyme GmbH | Sanchez-Amat A.,University of Murcia | And 4 more authors.
New Biotechnology | Year: 2016

Laccases from different sources are employed in a number of biotechnological processes, each characterized by specific reaction constraints and thus requiring an enzyme with suitable properties. In order to avoid the bias generated by different assay methodologies, in this work we investigated the main properties of ten laccases from fungi and bacteria under identical conditions. As a general rule, the optimal activity was apparent at pH 3-and was lost at pH ≥ 7.0 (all laccases were stable at pH ≥ 7.0); enzymes active at neutral pH values were also identified. For all tested laccases, activity increased with temperature up to 80°C and stability was good at 25°C. Interestingly, laccases insensitive to high salt concentration were identified, this favoring their use in treating waste waters. Indeed, bacterial laccases retained a significant activity in the presence of DMSO (up to 40% final concentration) and of surfactants, suggesting that they can be applied in lignin degradation processes requiring solvents. The available laccases are versatile and satisfy requirements related to different processes. Notably, the recombinant laccase from Bacillus licheniformis favorably compares with the tested enzymes, indicating that it is well suited for different biotechnological applications. © 2016 Elsevier B.V.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: KBBE.2013.3.4-01 | Award Amount: 9.95M | Year: 2013

The Valor-Plus supports the realisation of sustainable, economically viable closed loop integrated biorefineries through the development of new knowledge, (bio-)technologies and products that enable valorisation of key biorefinery by-products. The project comprises five key areas: Pre-treatment and fractionation: development of a novel methodology for the controlled and selective breakdown, release and fractionation of the lignocellulose biomass to minimally degraded cellulose, hemicellulose and lignin fractions that are suitable for further downstream refinement and processing to value product streams Hemicellulose Valorisation: engineering of new enzymes and microorganisms for the controlled hydrolysis and transformation of hemicellulose to high value oligomers and bulk fermentation product streams (butanol, ethanol and single cell proteins for animal feed) Lignin Valorisation: utilisation of combined chemo-enzymatic and chemo-microbial processes for the controlled depolymerisation and transformation of standardised lignin feedstocks to discrete families of platform and intermediate macromolecular and monomer chemicals; and their subsequent transformation to value product streams (fuels, platform chemicals, monomers for synthesis of resins and functional additives) Glycerol Valorisation: engineering of new microorganisms that are the suitable for the fermentation of crude glycerol to higher value product streams (lipids, alcohols and organic acids) Demonstration of the technological and economic potential for integration and scale-up within existing and future biorefinery value chains: including: demonstration of component technologies, focused biodiesel refinery case study, roadmaps for technology and product stream integration, and a full life cycle assessment The project assembles an industrially focused pan-European consortium spanning the complete biorefinery value chain, including 9 SMEs, 1 large enterprise, 2 research centres and 3 universities


PubMed | University of Murcia, University of Insubria, ASA Spezialenzyme GmbH and Polytechnic of Milan
Type: Comparative Study | Journal: New biotechnology | Year: 2016

Laccases from different sources are employed in a number of biotechnological processes, each characterized by specific reaction constraints and thus requiring an enzyme with suitable properties. In order to avoid the bias generated by different assay methodologies, in this work we investigated the main properties of ten laccases from fungi and bacteria under identical conditions. As a general rule, the optimal activity was apparent at pH 3-4 and was lost at pH7.0 (all laccases were stable at pH7.0); enzymes active at neutral pH values were also identified. For all tested laccases, activity increased with temperature up to 80C and stability was good at 25C. Interestingly, laccases insensitive to high salt concentration were identified, this favoring their use in treating waste waters. Indeed, bacterial laccases retained a significant activity in the presence of DMSO (up to 40% final concentration) and of surfactants, suggesting that they can be applied in lignin degradation processes requiring solvents. The available laccases are versatile and satisfy requirements related to different processes. Notably, the recombinant laccase from Bacillus licheniformis favorably compares with the tested enzymes, indicating that it is well suited for different biotechnological applications.


Jankowska D.A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Faulwasser K.,Leibniz Institute of Plant Genetics and Crop Plant Research | Trautwein-Schult A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Cordes A.,ASA Spezialenzyme GmbH | And 5 more authors.
Journal of Applied Microbiology | Year: 2013

Aims: Construction of a transgenic Arxula adeninivorans strain that produces a high concentration of adenine deaminase and investigation into the application of the enzyme in the production of food with low purine content. Methods and Results: The A. adeninivorans AADA gene, encoding adenine deaminase, was expressed in this yeast under the control of the strong inducible nitrite reductase promoter using the Xplor®2 transformation/expression platform. The recombinant enzyme was biochemically characterized and was found to have a pH range of 5·5-7·5 and temperature range of 34-46°C with medium thermostability. A beef broth was treated with the purified enzyme resulting in the concentration of adenine decreasing from 70·4 to 0·4 mg l-1. Conclusions: It was shown that the production of adenine deaminase by A. adeninivorans can be increased and that the recombinant adenine deaminase can be used to lower the adenine content in the food. Significance and Impact of the Study: Adenine deaminase is one component of an enzymatic system that can reduce the production of uric acid from food constituents. This study gives details on the expression, characterization and application of the enzyme and thus provides evidence that supports the further development of the system. © 2013 The Society for Applied Microbiology.


Trautwein-Schult A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Jankowska D.,Leibniz Institute of Plant Genetics and Crop Plant Research | Cordes A.,ASA Spezialenzyme GmbH | Hoferichter P.,ASA Spezialenzyme GmbH | And 7 more authors.
Journal of Molecular Microbiology and Biotechnology | Year: 2013

Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food. © 2013 S. Karger AG, Basel.


Jankowska D.A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Trautwein-Schult A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Cordes A.,ASA Spezialenzyme GmbH | Hoferichter P.,ASA Spezialenzyme GmbH | And 4 more authors.
Journal of Applied Microbiology | Year: 2013

Aims: Isolation and characterization of xanthine oxidoreductase and its application in the production of food with low purine content. Methods and Results: The A. adeninivorans xanthine oxidoreductase is an inducible enzyme. The best inducers were identified by enzyme activity tests and real-time PCR and used to produce large amounts of the protein. Xanthine oxidoreductase was partially purified and biochemically characterized, showing pH and temperature optimum of 8·5 and 43°C, respectively. The enzyme decreased xanthine and hypoxanthine concentrations in yeast extract and was active simultaneously with other purine-degrading enzymes so that all of the substrates for uric acid production were reduced in a single step. Conclusions: It was shown that induced A. adeninivorans can produce sufficient amount of xanthine dehydrogenase and that the enzyme is able to reduce xanthine and hypoxanthine content in food, and when used in conjunction with other enzymes of the pathway, uric acid concentration is significantly reduced. Significance and Impact of the Study: Reduction in dietary purines is recommended to people suffering from hyperuricemia. Elimination of most purine-rich foods may affect balanced nutrition. Food with lowered purine concentration will assist in controlling the disease. This study is a continuation of previous studies that characterized and overexpressed other enzymes of the purine degradation pathway. © 2013 The Society for Applied Microbiology.


Bischoff F.,Leibniz Institute of Plant Genetics and Crop Plant Research | Litwinska K.,Leibniz Institute of Plant Genetics and Crop Plant Research | Cordes A.,ASA Spezialenzyme GmbH | Baronian K.,University of Canterbury | And 3 more authors.
Applied and Environmental Microbiology | Year: 2015

The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6×His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6×His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml-1 could be achieved even with a nonoptimized cultivation procedure. © 2015, American Society for Microbiology.


Trautwein-Schult A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Jankowska D.,Leibniz Institute of Plant Genetics and Crop Plant Research | Cordes A.,ASA Spezialenzyme GmbH | Hoferichter P.,ASA Spezialenzyme GmbH | And 6 more authors.
Journal of Molecular Microbiology and Biotechnology | Year: 2014

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods. © 2014 S. Karger AG, Basel.


Jankowska D.A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Trautwein-Schult A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Cordes A.,ASA Spezialenzyme GmbH | Bode R.,University of Greifswald | And 2 more authors.
Bioengineered Bugs | Year: 2015

The purine degradation pathway in humans ends with uric acid, which has low water solubility. When the production of uric acid is increased either by elevated purine intake or by impaired kidney function, uric acid will accumulate in the blood (hyperuricemia). This increases the risk of gout, a disease described in humans for at least 1000 years. Many lower organisms, such as the yeast Arxula adeninivorans, possess the enzyme, urate oxidase that converts uric acid to 5-hydroxyisourate, thus preventing uric acid accumulation. We have examined the complete purine degradation pathway in A. adeninivorans and analyzed enzymes involved. Recombinant adenine deaminase, guanine deaminase, urate oxidase and endogenous xanthine oxidoreductase have been investigated as potential additives to degrade purines in the food. Here, we review the current model of the purine degradation pathway of A. adeninivorans and present an overview of proposed enzyme system with perspectives for its further development. © 2015 Taylor & Francis Group, LLC.


PubMed | University of Canterbury, University of Greifswald, ASA Spezialenzyme GmbH and Leibniz Institute of Plant Genetics and Crop Plant Research
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2015

The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6 His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6 His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml(-1) could be achieved even with a nonoptimized cultivation procedure.

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