Degradation kinetics of medium chain length Polyhydroxyalkanoate degrading enzyme: a quartz crystal microbalance study.

This study investigates the enzymatic degradation processes of different classes of polyhydroxyalkanoates (PHAs), a group of biopolymers naturally synthesized by various microorganisms. Medium chain length PHAs (mcl-PHAs) are distinguished biopolymers due to their biodegradability and diverse material properties. Using quartz crystal microbalance measurements as a valuable tool for accurate real-time monitoring of the enzymatic degradation process, the research provides detailed kinetic data, describing the interaction between enzymes and substrates during the enzymatic degradation process. Thin films of poly-3-hydroxybutyrate (PHB) and polyhydroxyoctanoate copolymer (PHO), containing molar fractions of about 84% 3-hydroxyoctanoate and 16% 3-hydroxyhexanoate, were exposed to scl-depolymerases from Pseudomonas lemoignei LMG 2207 and recombinant mcl-depolymerase produced in Escherichia coli DH5α harboring the plasmid pMAD8, respectively. Analyses based on a heterogeneous kinetic model for the polymer degradation indicated a six-fold stronger adsorption equilibrium constant of mcl-depolymerase to PHO. Conversely, the degradation rate constant was approximately twice as high for scl-depolymerases acting on PHB. Finally, the study highlights the differences in enzyme-substrate interactions and degradation mechanisms between the investigated scl- and mcl-PHAs.

Defense Priming in Nicotiana tabacum Accelerates and Amplifies 'New' C/N Fluxes in Key Amino Acid Biosynthetic Pathways.

In the struggle to survive herbivory by leaf-feeding insects, plants employ multiple strategies to defend themselves. One mechanism by which plants increase resistance is by intensifying their responsiveness in the production of certain defense agents to create a rapid response. Known as defense priming, this action can accelerate and amplify responses of metabolic pathways, providing plants with long-lasting resistance, especially when faced with waves of attack. In the work presented, short-lived radiotracers of carbon administered as 11CO2 and nitrogen administered as 13NH3 were applied in Nicotiana tabacum, to examine the temporal changes in 'new' C/N utilization in the biosynthesis of key amino acids (AAs). Responses were induced by using topical application of the defense hormone jasmonic acid (JA). After a single treatment, metabolic partitioning of recently fixed carbon (designated 'new' carbon and reflected as 11C) increased through the shikimate pathway, giving rise to tyrosine, phenylalanine and tryptophan. Amplification in 'new' carbon fluxes preceded changes in the endogenous (12C) pools of these AAs. Testing after serial JA treatments revealed that fluxes of 'new' carbon were accelerated, amplified and sustained over time at this higher rate, suggesting a priming effect. Similar results were observed with recently assimilated nitrogen (designated 'new' nitrogen reflected as 13N) with its partitioning into serine, glycine and glutamine, which play important roles supporting the shikimate pathway and downstream secondary metabolism. Finally, X-ray fluorescence imaging revealed that levels of the element Mn, an important co-factor for enzyme regulation in the shikimate pathway, increased within JA treated tissues, suggesting a link between plant metal ion regulation and C/N metabolic priming.

Influence of Unusual Co-substrates on the Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates Produced in Multistage Chemostat.

A two-stage chemostat cultivation was used to investigate the biosynthesis of functionalized medium-chain-length polyhydroxyalkanoate (mcl-PHA) in the ß-oxidation weakened strain of Pseudomonas putida KTQQ20. Chemostats were linked in sequence and allowed separation of biomass production in the first stage from the PHA synthesis in the second stage. Four parallel reactors in the second stage provided identical growth conditions and ensured that the only variable was the ratio of decanoic acid (C10) to an unusual PHA monomer precursor, such as 10-undecenoic acid (C11:1) or phenylvaleric acid (PhVA). Obtained PHA content was in the range of 10 to 25 wt%. When different ratios of C10 and C11:1 were fed to P. putida, the produced PHA had a slightly higher molar ratio in favor of C11:1-based 3-hydroxy-10-undecenoate. However, in case of PhVA a significantly lower incorporation of 3-hydroxy-5-phenylvalerate over 3-hydroxydecanoate took place when compared to the ratio of their precursors in the feed medium. A result that is explained by a less efficient uptake of PhVA compared to C10 and a 24% lower yield of polymer from the aromatic fatty acid ( y P H A - M P h V A = 0.25). In addition, PHA isolated from cultivations with PhVA resulted in the number average molecular weight M n ¯ two times lower than the PHA produced from C10 alone. Detection of products from PhVA metabolism in the culture supernatant showed that uptaken PhVA was not entirely converted into PHA, thus explaining the difference in the yield polymer from substrate. It was concluded that PhVA or its related metabolites increased the chain transfer rate during PHA biosynthesis in P. putida KTQQ20, resulting in a reduction of the polymer molecular weight.

Microplastics - from Anthropogenic to Natural.

In the context of the ongoing discussion of accumulating plastic debris in the environment, this article summarizes recent reports on the topic of microplastic pollution. Moreover, it gives an overview of the results from the activities of the research group for Biotechnology and Sustainable Chemistry of the Institute of Life Technologies at the HES-SO ValaisWallis concerning the fabrication of biodegradable microstructured plastics and microplastics from renewable resources. Polyhydroxyalkanoate biopolyesters are proposed as one suitable alternative in specific applications to reduce the use of persistent plastics. Standard techniques are described that have been successfully applied to produce porous materials, fibers and particles in the micro- and nanometer range.

Tailored biosynthesis of polyhydroxyalkanoates in chemostat cultures.

Polyhydroxyalkanoates (PHAs) are accumulated intracellularly by many bacteria and serve as a carbon and energy storage compound. PHAs are polyesters of high molecular weight and can be isolated by solvent extraction and precipitation in antisolvents. The material properties of PHAs are of great interest due to the inherent biodegradability and excellent biocompatibility. To date, more than 150 different PHA monomers have been described in literature and it has been found that the monomeric unit composition significantly influences the physico-chemical properties of PHAs. The monomer composition may be controlled to some extent by the choice of the PHA production strain but also by the cultivation conditions and the carbon substrate/PHA precursor supply. In previous studies, it has been shown that the most reproducible production method of PHA is the chemostat cultivation of suitable bacteria under multiple nutrient limited growth conditions. This chapter is dedicated to provide step-by step instructions to produce PHAs in a chemostat culture and specifically describes how the composition of PHA copolymers can be tailored during biosynthesis, as well as a set of analytical tools and methods to characterize PHAs.

Branched Polymers via ROMP of Termimers.

Today's olefin metathesis catalysts show high reactivity, selectivity, and functional group tolerance and allow the design of new syntheses of precisely functionalized polymers. Here the synthesis of a new end-capping reagent is investigated allowing the introduction of a highly reactive activated ester end-group at the polymer chain end as well as its prefunctionalization to directly introduce functional moieties. The versatility of this new end-capping reagent is demonstrated by utilizing it to synthesize a so-called termimer (a monomer with termination capabilities). Copolymerization of a norbornene derivative with the termimer leads to hyperbranched ring-opening metathesis polymerization polymers as proven by gel permeation chromatography and MALDI-ToF-(matrix-assisted laser desorption/ionization time of flight) mass spectrometry.

Polyethylenimine is a strong inhibitor of human papillomavirus and cytomegalovirus infection.

Polyethylenimines are cationic polymers with potential as delivery vectors in gene therapy and with proven antimicrobial activity. However, the antiviral activity of polyethylenimines has not been addressed in detail thus far. We have studied the inhibitory effects of a linear 25-kDa polyethylenimine on infections with human papillomaviruses and human cytomegaloviruses. Preincubation of cells with polyethylenimine blocked primary attachment of both viruses to cells, resulting in a significant reduction of infection. In addition, the dissemination of human cytomegalovirus in culture cells was efficiently reduced by recurrent administration of polyethylenimine. Polyethylenimine concentrations required for inhibition of human papillomavirus and cytomegalovirus did not cause any cytotoxic effects. Polyethylenimines and their derivatives may thus be attractive molecules for the development of antiviral microbicides.

Partitioning of new carbon as ¹¹C in Nicotiana tabacum reveals insight into methyl jasmonate induced changes in metabolism.

We examined the timeline by which methyl jasmonate (MeJA) reprograms new carbon partitioning into key metabolite pools. The radioactive isotope ¹¹C (t(¹/2) 20.4 min), administered to intact leaves of Nicotiana tabacum L. (cv Samsun) as ¹¹CO(2) gas enabled us to measure changes in new carbon partitioning into soluble sugar and amino acid pools of [¹¹C]photosynthate. A 500 µM MeJA treatment resulted in a decrease in the [¹¹C]soluble sugar pool and an increase in the [¹¹C]amino acid pool after 4 h. This pattern was more pronounced 15 h after treatment. We also examined the timeline for ¹¹C-partitioning into aromatic amino acid metabolites of the shikimate pathway. [¹¹C]Tyrosine, [C¹¹C]phenylalanine and [¹¹C]tryptophan were elevated 1.5-fold, 12-fold and 12-fold, respectively, relative to controls, 4 h after MeJA treatment, while endogeneous pools were unchanged. This suggests that only new carbon is utilized during early stages of defense induction. By 15 h, [C¹¹C]tyrosine and [¹¹C]phenylalanine returned to baseline while [¹¹C]tryptophan was elevated 30-fold, suggesting that MeJA exerts selective control over the shikimate pathway. Finally, we measured trans-cinnamic acid levels as a gauge of downstream phenolic metabolism. Levels were unchanged 4 h after MeJA treatment relative to controls, but were increased 2-fold by 15 h, indicating a lag in response of secondary metabolism.

Use of gaseous 13NH3 administered to intact leaves of Nicotiana tabacum to study changes in nitrogen utilization during defence induction.

Nitrogen-13 (t(1/2) 9.97 m), a radioactive isotope of nitrogen, offers unique opportunities to explore plant nitrogen utilization over short time periods. Here we describe a method for administering (13)N as gaseous (13)NH(3) to intact leaves of Nicotiana tabacum L. (cv Samsun), and measuring the labelled amino acids using radio high-performance liquid chromatography (HPLC) on tissue extract. We used this method to study the effects of defence induction on plant nitrogen utilization by applying treatments of methyl jasmonate (MeJA), a potent defence elicitor. MeJA caused a significant increase relative to controls in key [(13)N]amino acids, including serine, glycine and alanine by 4 h post-treatment, yet had no effect on (13)NH(3) incorporation, a process that is primarily under the control of the glutamine synthatase/glutamate synthase pathway (GS/GOGAT) in cellular photorespiration. We suggest that the reconfiguration of nitrogen metabolism may reflect induction of non-photorespiratory sources of nitrogen to better serve the plant's defences.