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Langenfeld, Germany

Grant
Agency: Cordis | Branch: FP7 | Program: CP-TP | Phase: KBBE.2013.3.6-01 | Award Amount: 11.70M | Year: 2013

The Nano3Bio project convenes a consortium of world renowned experts from 8 EU universities, 1 large company, and 14 SME, to develop biotechnological production systems for nanoformulated chitosans. Chitosans, chitin-derived polysaccharides varying in their degree of polymerisation (DP), degree of acetylation (DA), and pattern of acetylation (PA), are among the most versatile and most promising biopolymers, with excellent physico-chemical and material properties, and a wide range of biological functionalities, but their economic potential is far from being exploited due to i) problems with reproducibility of biological activities as todays chitosans are rather poorly defined mixtures, and ii) the threat of allergen contamination from their typical animal origin. The Nano3Bio project will overcome these hurdles to market entry and penetration by producing in vitro and in vivo defined oligo- and polymers with controlled, tailor-made DP, DA, and PA. Genes for chitin synthases, chitin deacetylases, and transglycosylating chitinases/chitosanases will be mined from different (meta)genomic sources and heterologously expressed, the recombinant enzymes characterized and optimized by protein engineering through rational design and molecular evolution, e.g. targeting engineered glycosynthases. These enzymes and genes will be used for in vitro and in vivo biosynthesis in microbial and microalgal systems, focusing on bacteria and diatoms. The bioinspired chitosans will be formulated into biomineralised hydrogels, nanoparticles, nanoscaffolds, etc., to impart novel properties, including by surface nano-imprinting, and will be bench-marked against their conventional counterparts in a variety of cell based assays and routine industrial tests for e.g. cosmetics and pharma markets. The process will be accompanied by comprehensive life cycle assessments including thorough legal landscaping, and by dissemination activities targeted to the scientific community and the general public.


Schuttmann I.,Justus Liebig University | Bouws H.,Justus Liebig University | Szweda R.T.,Justus Liebig University | Suckow M.,ARTES Biotechnology GmbH | And 2 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2014

A β-carotene degrading versatile peroxidase (VP) was successfully induced in submerged cultures of the white-rot basidiomycete Pleurotus sapidus by use of residues of a biogas plant as a carbon and nitrogen source. After three chromatographic steps, the VP was isolated with an overall yield of 12% and a purification factor of 130. The purified enzyme showed a molecular mass of 38 kDa and an isoelectric point of 3.6. Highest affinity to β-carotene (Km = 50 ± 5 μM) was observed at 30 °C and pH 4.5. The purified VP was capable of degrading suspended lignin organosolv particles. N-terminal and internal peptide sequences were obtained from Edman degradation and mass spectrometric peptide sequencing. The VP encoding cDNA was identified by colony hybridization and amplified by PCR. Bioinformatic analyses revealed an open reading frame of 1083 bp and similarities of 90% to VPs from P. eryngii. The recombinant VP was produced successfully with an activity of 450 ± 20 mU mg-1 in cultures of H. polymorpha. © 2013 Elsevier B.V. Source


Kaur P.,University of Delhi | Singh B.,University of Delhi | Boer E.,Leibniz Institute of Plant Genetics and Crop Plant Research | Straube N.,Leibniz Institute of Plant Genetics and Crop Plant Research | And 3 more authors.
Journal of Biotechnology | Year: 2010

The Pichia anomala gene PPHY, which codes for a cell-bound phytase, was isolated from genomic DNA by PCR, using oligonucleotide sequences derived from the N-terminal region of the purified phytase protein (Pphyp) and a degenerate primer derived from conserved sequences of yeast and fungal phytases as primers. The gene harbours an ORF of 1389bp, encoding a 462-amino-acid protein. The deduced amino acid sequence has similarity, to a varied extent, with those of phosphatases from Pichia stipitis (62%), Candida dubliniensis (51%), Candida albicans (51%), Arxula adeninivorans (35%) and phytases from Debaryomyces castellii (50%) and Pichia fabianii (39%). The sequence contains the phytase consensus heptapeptide motif (-Arg-His-Gly-X-Arg-X-Pro-) as well as two phosphohistidine signature motifs found in histidine acid phosphatases. After transformation of PPHY into the yeasts Saccharomyces cerevisiae, A. adeninivorans and Hansenula polymorpha, the last species was selected as the most suitable for synthesis of recombinant Pphyp. The cell-bound enzyme activities produced by wild-type P. anomala and transgenic H. polymorpha strains bearing the PPHY gene placed under the control of the inducible H. polymorpha-derived FMD promoter were characterized. In both cases, a molecular mass of approximately 380kDa was determined for the native enzyme (corresponding to a hexamer); the pH and temperature optima for the activity were 4.0 and 60°C, respectively. The enzyme was active on phytic acid, p-nitrophenylphosphate, glucose-6-phosphate, ADP, sodium pyrophosphate, AMP, 1-naphthylphosphate and ATP. Based on the Km/Kcat and further biochemical parameters, the enzyme was classified as a cell-bound phytase with acid phosphatase activity and not as acid phosphatase, despite its strong similarity to the latter class of enzymes. The yeast biomass containing phytase has been demonstrated to be useful as a feed additive in poultry and aquaculture, and dephytinization of foods and feeds. © 2010 Elsevier B.V. Source


Eilert E.,ARTES Biotechnology GmbH | Rolf T.,ARTES Biotechnology GmbH | Heumaier A.,University of Hamburg | Hollenberg C.P.,ARTES Biotechnology GmbH | And 3 more authors.
Journal of Biotechnology | Year: 2013

The literature as well as databases are ambiguous about the exact start of human interleukin-6 (IL-6) - three possibilities for the initiation of the mature protein are described. These three variants of IL-6, different in the exact initiation of the mature protein (A28, P29, or V30), were expressed in Hansenula polymorpha using the Saccharomyces cerevisiae MFα prepro sequence instead of the homologous pre sequence. All three IL-6 variants were secreted but the processing by the Kex2 protease showed significant differences. V30-IL-6 showed correctly processed material but also a molecule species of higher molecular weight indicating incomplete processing of the MFα pro peptide. P29-IL-6 did not yield any correctly processed IL-6, instead only the unprocessed pro form was found in the culture supernatant. Only A28-IL-6 led to 100% correctly processed material. N-terminal sequencing of this material revealed a start at V30 - obviously the first two amino acids (Ala28-Pro29) have been removed by a so far unknown protease. Thus expression of both A28-IL-6 and V30-IL-6 as MFα prepro fusion proteins resulted in the very same mature V30-IL-6, however, the ratio of correctly processed molecules was significantly higher in the case of A28-IL-6.The expression of an MFα prepro-interferon α-2a (IFNα-2a) fusion protein in H. polymorpha leads to about 50% correctly processed molecules and 50% misprocessed forms which contain part of the pro peptide at the N-termini. The insertion of A28 and P29 of IL-6 between the pro peptide and the start of the mature IFNα-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments. © 2012 Elsevier B.V. Source


Grant
Agency: Cordis | Branch: FP7 | Program: CP-IP | Phase: KBBE-2007-3-2-07 | Award Amount: 8.23M | Year: 2009

The PolyModE project convenes an international, interdisciplinary, and intersectorial consortium to identify, characterise, and optimise novel polysaccharide modifying enzymes, and to develop robust fermentation strategies for their large-scale production, to exploit the potential of biopolymers for food, pharmaceutical, cosmetic, and technical applications. We have selected the six complex carbohydrates with the highest current market share or expected future market potential, namely alginate, carrageenan, chitosan, glycosaminoglycan, pectin, and xanthan gum. For each of these, the industrial partners have identified those enzymes which will answer to the most pressing needs or offer the most promising potential for improved production of polysaccharides with novel physico-chemical properties and biological functionalities. Primary targets will be alginate epimerases, carrageenan sulfatases, chitosan de-acetylases, glycosaminoglycan sulfatases, pectin de-acetylases, and xanthan gum de-acetylases. These enzymes together with secondary target enzymes, e.g. sequence specific lyases and hydrolases, will allow the generation and analysis of polymers and oligomers with novel, non-random patterns of modification. Two parallel approaches will be followed for each type of polysaccharide modifying enzyme, namely a knowledge-based genomic approach and a broad, un-biased metagenomic approach, e.g. using soil or sludge samples with a history of contact with the polysaccharide in question. A pipeline of three levels of fermentation systems will be established, ranging from lab-scale innovative expression systems with features shaped according to the specific characteristics of our target enzymes, through medium-scale, novel and unusual fermentation systems provided by a number of SME with highly specialised knowledge and expertise in developing and using such systems, to the established large-scale fermentation systems and facilities of market leaders in White Biotechnology.

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