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  • ISSN: 2333-7117
    Special Issue entitled: Industrial Biotechnology-Made in Germany: The path from policies to sustainable energy, commodity and specialty products
    Thomas Brueck
    Professor
    Department of Chemistry
    Technical University of Munich, Germany
    Editorial
    Thomas Brück*
    Globally, Germany is the first nation dedicated to change its entire energy supply from fossil to renewable resources in the next decades. A clear political roadmap to accomplish this changeover fosters a climate for innovation and technology development leading to new sustainable energy and fuel solutions.
    Policy Opinion
    Müller W*
    Globally, Germany is the first nation dedicated to change its entire energy supply from fossil to renewable resources in the next decades. A clear political roadmap to accomplish this changeover fosters a climate for innovation and technology development leading to new sustainable energy and fuel solutions.
    Policy Opinion
    Haralabos Zorbas*, Sonja Völker and Katrin Härtling
    Abstract: The company Industrielle Biotechnologie Bayern Netzwerk GmbH (abbreviated: IBB Netzwerk GmbH) is a network organization in service of Industrial Biotechnology and sustainable economic growth. IBB Netzwerk GmbH manages the Network IBB. In this article, we clarify and define the relationship of Industrial Biotechnology to related areas and its relation to the goals of the bioeconomy. Second, we rationalize our commitment to Industrial Biotechnology by listing explicitly fields, for which Industrial Biotechnology is highly relevant. On this rationale, we then describe the company IBB Netzwerk GmbH and the Network IBB, their tasks, goals, mode of action and achievements so far and suggest explanations for some drawbacks of technology transfer. Finally, we attempt a bird's eye view on cluster benefits in general, on the cluster policy of Germany as well as on an important requirement for cluster maintenance.
    Short Communication
    Friedrich Streffer*
    Abstract: Great efforts are made to realize concepts for replacing oil and using renewable resources as starting material in biorefineries. Currently, biorefineries produce chemical base materials on an industrial scale from readily available sugar-or starch-containing plant components. However, thesefeedstocks only account for about 1% of the available plant biomass. The majority of available plant biomass, constitutes lignocellulose, which is currently inaccessible to conventional biorefineries and biogas processes.However, in future generating higher economic efficiency for biorefineries and biogas plants is important to ensure these operations can compete with the efficiency of oil refineries even in the absence of government subsidies. Further, it is desirable to increase the ecological efficiency of these operations in order to reduce the required agricultural land use and to improve the CO2 balance. All these claims could be achieved if hitherto waste products such as digestates, agricultural, food and municipal wastestreams could be used as feedstock. Physico-chemical and biotechnological pretreatment technologies, such as the LX process are being established, which would allow utilization of these feedstocks particularly for biogas plants. This review summarized the technical and economic framework to establish these enabling technologies with a particular focus on development of second generation biogas process.
    Review Article
    Andre Koltermann, Ulrich Kettling, Michael Kraus, Markus Rarbach, Christoph Reisinger, Michael Zavrel and Yvonne Söltl*
    Abstract: Cellulosic ethanol made from agricultural residues has been a scientific and commercial interest for decades however the development and commercial deployment of technologies have been limited. It constitutes an almost carbon neutral new energy source using an already existing renewable feedstock that doesn't compete with food or feed production and land use. The field of application is wide, from second generation biofuel to the chemical industry. A key controversial issue regarding technological developments aimed at the production of cellulosic ethanol is the commercial economic viability of the process. The challenges facing process development include optimization of the ethanol yield while lowering operational and capital costs such as the reduction in enzyme costs and energy efficiency improvements. Recent years have seen great success in the development and deployment of cellulosic ethanol technologies. Now policy makers are asked to facilitate the market entry of such innovative processes by setting a long-term stable framework. Clariant's sunliquid® technology overcomes the main challenges of competitive conversion of lignocellulosic feedstock into cellulosic sugars for fermentation to cellulosic ethanol. In July 2012 a demonstration plant with an annual output of 1000 tons of ethanol started operation. This is the last step on the way to commercializing a technology platform for second generation biofuels and biobased chemicals. The plant represents the complete production chain, including pretreatment, process-integrated production of feedstock and process specific enzymes, hydrolysis, simultaneous C5 and C6 fermentation and energy saving ethanol separation. The process itself is energy neutral, yielding cellulosic ethanol with about 95% of CO2 emission reductions.
    Review Article
    Bendig C#, Kraxenberger T# and Römer L*
    Abstract: The large-scale production of biopolymers has been an emerging branch of industry for decades. Besides mere substitution of oil-based polymers, biopolymers with innovative features are in the focus of research and industry. This review highlights modern high-impact biopolymers, their respective industrial production processes and relevant applications.
    The vast majority of prominent commercial biopolymers are either made from sugars (such as cellulose derivatives), acids (such as poly lactic acid) or proteins (such as silk). Some of the more simple biopolymers such as γ-polyglutamic acid and bacterial cellulose are mainly produced in their native microorganisms. A more challenging trend in biopolymer production is the switch from traditional extraction or conversion of natural products to recombinant/heterologous production techniques in microorganisms. This is analyzed in detail for collagen, hyaluronic acid and silk. Despite the complexity of these biopolymers in structure and production, all share important features such as biocompatibility, adjustable shapes and slow biodegradation. The combination of properties renders these polymers ideal materials for biomedical scaffolding, surgery and wound care as well as related pharmaceutical applications and drug delivery.
    Research Article
    Farah Qoura1, Dr. Thomas Brück2* and Garabed Antranikian3*
    Abstract: A new psychrophilic, strictly aerobic bacterium, strain 40-3, was isolated from seawater samples collected at Spitsbergen in the Arctic. The cells are gram negative, straight or curved rod shaped and non-spore forming (2-3 µm long and 0.4-0.6 µm wide). Colonies on agar medium are slightly orange, circular, smooth and convex. 40-3 strain grows optimally over the temperature range of 10-15 °C and a pH range of 7-8 in media containing 8 to 9 % NaCl (w/v). Growth occurs with α cyclo-dextrin, dextrin, tween 80, N-acetyl-D-glucosamine, α-D-glucose, maltose, sucrose, methyl pyruvate, D,L-lactitate, succiniate , bromo succinic acid, inosine, esculin ferric citrate, L-arabinose, potassium gluconate, malic acid and trisodium citrate. In the presence of glucose H2S was produced and nitrates are reduced to nitrites. The fatty acid methyl ester (FAME) are composed of 17.89 % straight chain saturated FAMEs, 14,85 % terminally branched saturated FAMEs and 17.73 % monounsaturated FAMEs. The DNA base ratio is 48 mol % G + C. Phylogenetic analysis reveals as close relationship to Shewanella putrefaciens with 99 % 16S rDNA composition identity and 50 % DNA-DNA similarity. The phylogenetic evidence, together with phenotypic characteristics, show that this psychrophilic strain constitute a novel species of the genus Shewanella. The name Shewanella arctica is proposed. Interestingly, when grown on glucose as a carbon source Shewanella arctica produced numerous industrially important enzyme systems including amylase, pullulanase, protease, ornithine decarboxylase, alkaline phospatase, esterase (C4), lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β- glucosaminidase. Due to the functional new physochrophilic Shewanella strainresource for the isolation of new enzyme system, which may operate at low reaction thereby increasing the energy efficiency of industrial processes.
    Research Article
    Saskia Blank1, Carola Schröder1, Georg Schirrmacher2, Christoph Reisinger2 and Garabed Antranikian1*
    Abstract: To discover new industrially relevant, thermoactive xylanases a gene library from Thermus brockianus was constructed. Function-based screening revealed a novel xylanase-encoding gene (xyn10) which was successfully expressed in E. coli BL21 (DE3). The resulting protein (38.7 kDa), a member of glycoside hydrolase family 10 was purified to homogeneity and biochemically characterized. Catalytic activity was detected up to 115 °C and highest activity was measured at 95 °C and pH 6.0. The protein was extremely thermostable and showed 80 % remaining activity after incubation at 50-70 °C for 24 h. HPLC analysis showed that Xyn10 hydrolyzes insoluble and soluble substrates, such as oat spelt xylan, xylan from beech- and birchwood forming xylobiose and xylose. Specific activity of the enzyme was 1119.5 U/mg for oat spelt xylan and 994.0 U/mg for beechwood xylan, respectively. The xylanase exhibited remarkable stability in the presence of various detergents and chaotropic agents, such as CHAPS, guanidine hydrochloride and urea.
    This is the first report of the heterologous production, purification and characterization of a xylanase from Thermus sp.
    Research Article
    Jochen Reiter1,2, Andre Pick1, Lars O Wiemann2, Doris Schieder2, Volker Sieber1,2*
    Abstract: The antioxidant glutathione (GSH) is an important reducing agent in cell physiology. Glutathione reductases (GR) of humans and higher organisms convert oxidized glutathione (GSSG) to two reduced GSH molecules under consumption of the co-factor NADPH. GSH acts as an antioxidant eliminating reactive oxygen species in the cell. We found a novel GR being able to accept both NADPH and much cheaper NADH for GSSG reduction. For the first time we produced it in E. coli and purified active GR from Allochromatium vinosum, determined its Km-values for NADH (0.026 mM) and NADPH (0.309 mM), as well as its temperature optimum (20 °C) and pH optimum (pH 8). Since numerous bio-diagnostic assays and enzymatic processes are dependent on GRs the possibility to use a cheaper co-substrate could help to overcome cost limitations in future.
    Research Article
    Robert Kourist1*, Frank Hollmann2 and Giang-Son Nguyen2
    Abstract: Lipases A and B from the psychrophilic basidiomyceteous yeast Pseudozyma antarctica (formerly known as Candida antarctica) belong to the most important industrial biocatalysts with numerous applications in the oleochemical, polymer, textile, biodiesel, and detergent industry.
    Both lipases have been intensively studied for decades. Nevertheless, several important achievements were made in the last few years.
    This highlight presents three recent trends that significantly widen the application of lipases. Firstly, improvements in enzyme formulation and reactor setups have improved the performance of lipase in solvent-free reactions systems, which have significantly broadened the scope of lipases for the environmentally friendly synthesis of cosmetic products. Secondly, combination with chemical reactions has a tremendous potential to widen the scope of lipases. For instance, metal-free racemization reactions proved to be a successful approach to increase the yield in the industrially established kinetic resolution of amines. Thirdly, the impressive process in the engineering of lipases shortened time horizons for catalyst development and led to series of novel biocatalysts with engineered selectivity. Successful examples include lipase variants with improved activity towards amines, increased substrate scope, increased or even inverted enantioselectivity and increased ability to discriminate cis and trans fatty acids.
    Review Article
    Kurzrock T* and Kress K
    Abstract: Imagine a future world with a significantly grown influence of biotechnology into all parts of society. Let us assume that the majority of products in a consumer's world, including food & beverage, furniture and clothes, plastics, buildings, streets and fuel are produced with the help of modern biotechnology. Meaning that at least parts of finished products are produced in an industrial environment with outcome from a biotechnological procedure.
    There might be many reasons for a biotechnological revolution like exhaustible raw materials, minimizing ecological pollution, the consumer's political will or just quality improvements.
    Whatever the final drivers are, a transformation like this will open new fields in laboratory work and create new requirements for laboratory devices.
    Without predicting the future world in details, it will be essential that biotechnological production processes are competitive to industrial productions. Main industrial challenges are 1st optimal economic parameter definitions in laboratory scale, 2nd a quick and reliable scale-up to minimize time to market and 3rd a maximal yield assurance in production. To overcome all these requirements for a successful biotechnological process development, a highly efficient screening tool, as the 2mag bioREACTOR, is urgently needed.
    Research Article
    Ibrahim Ahmed1,2*, HolgerHuebner1 and Rainer Buchholz1
    Abstract: Baculoviruses have a significant potential as biological pesticides. Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) could thus find an application to protect plants against the African Cotton Leafworm. For the in vitro production of SpliMNPV a cellular system has to be established. For this purpose three new continuous cell lines were established from the embryonic tissue of the cotton leaf worm S. littoralis.The three cell lines were designated Spli-C, Spli-S and Spli-B. They consisted mostly of spherical cells, but also contained spindle and giant cells. The population doubling time for the three cell lines Spli-C, Spli-S and Spli-B were 30.5, 31 and 44.5 hrs, respectively, at passage 19, while at passage 120 it decreased to 26, 27 and 32 hrs, respectively. RAPD and DAF DNA fingerprint confirmed that the cell lines originated from S. littoralis tissues. The lactate dehydrogenase (LDH) isozyme analysis demonstrated a distinguishable difference between the three new S. littoralis cell lines and the other insect cell lines which we use in our laboratory.All three new cell lines were susceptible to SpliMNPV and thus are suitable for virus multipication. Cells were immobilized using sodium cellulose sulfate (NaCS) and poly diallyl dimethyl ammoniumchloride (PDADMAC) capsules to protect cells from shear stress. This is caused during cultivation by agitation and gas sparging during supply of sufficient oxygen in order to reach high cell densities. The cell densities increased from 4-5x106 cells/ml in suspension cultureto1.3x107 cells/ml in capsules. Our results suggest that large-scale production of SpliMNPV as a biopesticide is possible with these cell lines.
    Research Article
    Sabine Mundt1*, Huong T Bui1, Michael Preisitsch1, Susann Kreitlow1, Ha TN Bui1, Hang TL Pham1, Elmi Zainuddin1, Tuyet TA Le1, Gerold Lukowski2 and Wolf-Dieter Jülich1
    Abstract: Due to the growing resistance of pathogenic bacteria and fungi against commercially available therapeutics, the search for new antimicrobial substances is of increasing importance. Based on the hypothesis that microorganisms living in an aquatic environment produce secondary metabolites as chemical weapons to survive in their daily fight against cohabitants of the biotope, a screening of 133 microalgae (121 cyanobacteria, 12 eukaryotic microalgae) was started. Biomass extracts and cultivation media were tested for activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa as well as the yeast Candida maltosa. Our data indicates that 56 cyanobacterial strains and 5 eukaryotic algae exhibited antimicrobial activity. Interestingly, 19 of the screened cyanobacteria inhibited the growth of MRSA. Further, screening experiments revealed activity against Helicobacter pylori as well as fish pathogenic bacteria and plant pathogenic fungi. Strains exhibiting significant antimicrobial activity were cultivated at 40L scale in order to conduct a bioassay-guided isolation and structure elucidation of the bioactive components. This procedure allowed identification of bioactive secondary metabolites encompassing the hydroxylated fatty acids coriolic and dimorphecolic acid, lyngbyazothrins, cyclic depsispeptides and carbamidocyclophanes, belonging to the class of polyketides, which are responsible for the observed antimicrobial activity.
    In addition to tests of purified bioactive compounds, whole biomass of selected microalgae was used to prepare microparticles by high pressure homogenization. Subsequent, in-vitro tests have shown that microparticles from biomass of the microalgal strain Bio 33, named Maresome®, inhibit dermal colonization of different MRSA strains. Since preliminary, clinical tests confirm the in-vitro data, the anti-pathogenic potential of microalgae might be utilized in form of a prophylactic skin care product to prevent nosocomial infections.
    Research Article
    Nicholas Schramek1, Claudia Huber1, Stefanie Schmidt1, Sabine E.-M. Dvorski1, Nihat Knispel1, Elena Ostrozhenkova1, Luis M. Peña-Rodríguez1,2, Rosa M. Cusidó3, Gesine Wischmann4 and Wolfgang Eisenreich1*
    Abstract: The biosynthesis of the triterpenoidginsenosides Rg1 and Rb1 was studied by 13CO2 pulse-chase experiments in six-year-old Panax ginseng growing in the field. A pulse period of 7 hours followed by a chase period of 8 days was shown to generate significant 13C-enrichments in both ginsenosides (Rg1> Rb1), as well as in free sugars and amino acids. More specifically, 13CO2-labeled Rg1 and Rb1 were characterized by specific NMR couplings due to13C2-units indicating the mevalonate origin of the triterpenes. 13C3-Labeled motifs in Rg1 or Rg1 that should be generated by the alternative methylerythritol phosphate pathway from a 13C3-triose phosphate precursor were apparently absent, whereas 13C3-isotopologues were detected in free sugars, amino acids and the sugar moieties of the ginsensosides from the same experiment. It can be concluded that ginsenosides are predominantly or entirely biosynthesized in P. ginseng via the mevalonate route, under the physiological conditions of the field experiment. The observed labeling patterns were also in perfect agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor entering the cyclization process with the dammarenyl intermediate. The higher enrichments of 13C2-isotopologuesinthe protopanaxatriol-type Rg1in comparison to the protopanaxadiol-type Rb1 indicated higher rates of Rg1biosynthesis during the pulse/chase experiment with the six-year-old plant of P. ginseng. In summary, the study reveals the nature and dynamics of the ginsenoside biosynthetic pathway as a welcome basis for future biotechnological means..
    Review Article
    Larisa Yurlova1, Andrea Buchfellner1,2 and KouroshZolghadr1*
    Abstract: Antibody-based reagents are indispensable for a broad range of biomedical sciences as well as for bioproduction. Here we highlight novel derivatives of heavy-chain antibodies and elaborate upon their beneficial application in proteomics, cell biological research and drug discovery. This special class of antibodies from Camelidae provides distinct advantages over conventional antibodies due to the naturally originating single-chain structure of their antigen-binding domains. At ChromoTek we have now developed new immunization and screening strategies to convert these advantages into superior tools for a broad range of applications.
    First, these extremely stable antigen-binding fragments of heavy chain antibodies (VHHs, single domain antibodies, sdABs) can be easily produced as recombinant proteins in bacteria with constant quality criteria. When coupled to solid matrices, the highly specific VHHs can function as Nano-Traps®, facilitating efficient affinity purification of proteins for proteomic analyses.
    Second, the tenfold smaller size of VHHs than that of the commonly used immunoglobulins offers a versatile substitute to traditional antibody staining as VHHs can also be fluorescently labeled. These Nano-Booster termed reagents are therefore especially useful for super-resolution microscopy and possess higher tissue penetrating ability.
    The third exciting application of VHHs as live cell biomarkers opens new possibilities for cell biological and pharma research. When VHHs are genetically fused to fluorescent proteins and expressed inside cells, these so-called Chromobodies® enable unique intracellular live cell antibody staining, unthinkable with conventional antibodies. These fluorescent nanoprobes are of special interest for high content analysis (HCA) in drug discovery, since they permit real-time analyses of the effects of drug candidates at endogenous targets. Here we show examples for Chromobodies® highlighting important cellular biomarkers.Beyond visualization, Chromobodies® can be designed to specifically modulate (e.g. inhibit) their intracellular targets.
    Further possible implementations of these extremely small, stable and soluble single-chain camelid antibody fragments vary from co-crystallization assistance and neutralization of toxins, to crop protection and eventual therapeutics.
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