Search

Summary: The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl‐PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l−1 in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l−1 in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l−1 containing 65% PHA at the end of a 25‐h incubation. [ABSTRACT FROM AUTHOR]

Kanamycin A is an aminoglycoside antibiotic isolated from Streptomyces kanamyceticus and used against a wide spectrum of bacteria, including Mycobacterium tuberculosis. Biosynthesis of kanamycin involves an oxidative deamination step catalyzed by kanamycin B dioxygenase (KanJ), thereby the C2' position of kanamycin B is transformed into a keto group upon release of ammonia. Here, we present for the first time, structural models of KanJ with several ligands, which along with the results of ITC binding assays and HPLC activity tests explain substrate specificity of the enzyme. The large size of the binding pocket suggests that KanJ can accept a broad range of substrates, which was confirmed by activity tests. Specificity of the enzyme with respect to its substrate is determined by the hydrogen bond interactions between the methylamino group of the antibiotic and highly conserved Asp134 and Cys150 as well as between hydroxyl groups of the substrate and Asn120 and Gln80. Upon antibiotic binding, the C terminus loop is significantly rearranged and Gln80 and Asn120, which are directly involved in substrate recognition, change their conformations. Based on reaction energy profiles obtained by density functional theory (DFT) simulations, we propose a mechanism of ketone formation involving the reactive FeIV = O and proceeding either via OH rebound, which yields a hemiaminal intermediate or by abstraction of two hydrogen atoms, which leads to an imine species. At acidic pH, the latter involves a lower barrier than the OH rebound, whereas at basic pH, the barrier leading to an imine vanishes completely. Databases: Structural data are available in PDB database under the accession numbers: 6S0R, 6S0T, 6S0U, 6S0W, 6S0V, 6S0S. Diffraction images are available at the Integrated Resource for Reproducibility in Macromolecular Crystallography at http://proteindiffraction.org under DOIs: 10.18430/m36s0t, 10.18430/m36s0u, 10.18430/m36s0r, 10.18430/m36s0s, 10.18430/m36s0v, 10.18430/m36s0w. A data set collection of computational results is available in the Mendeley Data database under DOI: 10.17632/sbyzssjmp3.1 and in the ioChem‐BD database under DOI: 10.19061/iochem‐bd‐4‐18. [ABSTRACT FROM AUTHOR]

ABSTRACT High Cell Density (HCD) cultivation of bacteria is essential for the majority of industrial processes to achieve high volumetric productivity (g L−1 h−1) of a bioproduct of interest. This study developed a fed batch bioprocess using glucose as sole carbon and energy source for the HCD of the well described biocatalyst Pseudomonas putida KT2440 without the supply of oxygen enriched air. Growth kinetics data from batch fermentations were used for building a bioprocess model and designing feeding strategies. An exponential followed by linearly increasing feeding strategy of glucose was found to be effective in maintaining biomass productivity while also delaying the onset of dissolved oxygen (supplied via compressed air) limitation. A final cell dry weight (CDW) of 102 g L−1 was achieved in 33 h with a biomass productivity of 3.1 g L−1 h−1 which are the highest ever reported values for P. putida strains using glucose without the supply of pure oxygen or oxygen enriched air. The usefulness of the biomass as a biocatalyst was demonstrated through the production of the biodegradable polymer polyhydroxyalkanoate (PHA). When nonanoic acid (NA) was supplied to the glucose grown cells of P. putida KT2440, it accumulated 32% of CDW as PHA in 11 h (2.85 g L−1 h−1) resulting in a total of 0.56 kg of PHA in 18 L with a yield of 0.56 g PHA g NA−1. Biotechnol. Bioeng. 2015;112: 725-733. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The primary enzyme involved in polyphosphate (poly P) synthesis, poly P kinase ( ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate ( PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative ( H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in poly P levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, poly P kinase ( PPK) was undetectable, but up-regulation of the polyp-associated proteins poly P adenosine triphosphate ( ATP)/nicotinamide adenine dinucleotide ( NAD) kinase ( PpnK), a putative poly P adenosine monophosphate ( AMP) phosphotransferase ( PP_1752), and exopolyphosphatase was observed. Δ ppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δ ppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting poly P accumulation. [ABSTRACT FROM AUTHOR]

Biodegradable and biocompatible novel materials of natural origin are gaining more and more attention in recent years. These so called biopolymers, characterized by their biointegrity and biocompatibility, find completely new and promising applications in biomedical sciences. The presented work focuses on the medium chain length elastomeric polyhydroxyalkanoate biopolymer—polyhydroxyoctanoate (PHO). This biopolymer is fully biodegradable without formation of harmful byproducts.We investigated PHO's physical properties with nanoindentation technique and scratch testing to determine Young's modulus and friction coefficient. Further, the work focused on the impact of PHO, used as growth substrate, on the physiology and morphology of mouse embryonic fibroblast cells (MEF 3T3). Application of fluorescent staining protocols and advanced microscopic techniques allowed to study the morphological changes in the cytoskeletons of cells grown on PHO and also gave an insight into their migration strategies on the polymer surface. We found that PHO exhibits no cellular cytotoxicity, similarly to a glass substrate. MEF cells spread better on glass surface than on each tested PHO substrate though there was almost no difference between PHO substrates cast from different solvents. However, a detailed analysis of actin and microtubule cytoskeletal architecture reveals changes in the density of actin and microtubular networks. Migration of MEF cells on PHO substrates was slower than on the glass substrate. To elucidate the molecular mechanisms of observed changes in cytoskeletal architecture and migration parameters can be of special interest for future medical application of PHO polymer. [ABSTRACT FROM AUTHOR]

Insight into the topographic and mechanical properties of biomaterials allows for efficient selection of a material for a specific application. Here, atomic force microscopy (AFM) and force spectroscopy were exploited to reveal the topographic and mechanical characteristics of charcoal‐purified, solvent‐cast polyhydroxyoctanoate (PHO) film. The root mean square surface roughness of a PHO surface derived from ethyl acetate, acetone, or chloroform solution was 13.2, 11.5, or 30.9 nm, respectively, for 100 μm2 AFM images. The distribution of the local Young's modulus had a maximum of 25.4, 14.1, and 12.6 MPa for PHO films obtained from ethyl acetate, acetone, and chloroform solution, respectively. The positron annihilation spectroscopy measurements allowed us to determine the free volume in the polymer film structure (9.38%). Moreover, a number of additional techniques (X‐ray diffraction, thermogravimetric analysis, differential scanning calorimetry, gel permeation chromatography, NMR, infrared spectroscopy, and polarized light microscopy) were used to reveal PHO features. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47192. [ABSTRACT FROM AUTHOR]