Jasmina Nikodinovic-Runic

Carbon-rich wastes as feedstocks for biodegradable polymer (polyhydroxyalkanoate) production using bacteria.

Research into the production of biodegradable polymers has been driven by vision for the most part from changes in policy, in Europe and America. These policies have their origins in the Brundtland Report of 1987, which provides a platform for a more sustainable society. Biodegradable polymers are part of the emerging portfolio of renewable raw materials seeking to deliver environmental, social, and economic benefits. Polyhydroxyalkanoates (PHAs) are naturally-occurring biodegradable-polyesters accumulated by bacteria usually in response to inorganic nutrient limitation in the presence of excess carbon. Most of the early research into PHA accumulation and technology development for industrial-scale production was undertaken using virgin starting materials. For example, polyhydroxybutyrate and copolymers such as polyhydroxybutyrate-co-valerate are produced today at industrial scale from corn-derived glucose. However, in recent years, research has been undertaken to convert domestic and industrial wastes to PHA. These wastes in todays context are residuals seen by a growing body of stakeholders as platform resources for a biobased society. In the present review, we consider residuals from food, plastic, forest and lignocellulosic, and biodiesel manufacturing (glycerol). Thus, this review seeks to gain perspective of opportunities from literature reporting the production of PHA from carbon-rich residuals as feedstocks. A discussion on approaches and context for PHA production with reference to pure- and mixed-culture technologies is provided. Literature reports advocate results of the promise of waste conversion to PHA. However, the vast majority of studies on waste to PHA is at laboratory scale. The questions of surmounting the technical and political hurdles to industrialization are generally left unanswered. There are a limited number of studies that have progressed into fermentors and a dearth of pilot-scale demonstration. A number of fermentation studies show that biomass and PHA productivity can be increased, and sometimes dramatically, in a fermentor. The relevant application-specific properties of the polymers from the wastes studied and the effect of altered-waste composition on polymer properties are generally not well reported and would greatly benefit the progress of the research as high productivity is of limited value without the context of requisite case-specific polymer properties. The proposed use of a waste residual is advantageous from a life cycle viewpoint as it removes the direct or indirect effect of PHA production on land usage and food production. However, the question, of how economic drivers will promote or hinder advancements to demonstration scale, when wastes generally become understood as resources for a biobased society, hangs today in the balance due to a lack of shared vision and the legacy of mistakes made with first generation bioproducts.

Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions.

Pseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating medium-chain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogen-limited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth).

Properties and applications of undecylprodigiosin and other bacterial prodigiosins

The growing demand to fulfill the needs of present-day medicine in terms of novel effective molecules has lead to reexamining some of the old and known bacterial secondary metabolites. Bacterial prodigiosins (prodiginines) have a long history of being re markable multipurpose compounds, best examined for their anticancer and antimalarial activities. Production of prodigiosin in the most common producer strain Serratia marcescens has been described in great detail. However, few reports have discussed the ecophysiological roles of these molecules in the producing strains, as well as their antibiotic and UV-protective properties. This review describes recent advances in the production process, biosynthesis, properties, and applications of bacterial prodigiosins. Special emphasis is put on undecylprodigiosin which has generally been a less studied member of the prodigiosin family. In addition, it has been suggested that proteins involved in undecylprodigiosin synthesis, RedG and RedH, could be a useful addition to the biocatalytic toolbox being able to mediate regio- and stereoselective oxidative cyclization. Judging by the number of recent references (216 for the 2007–2013 period), it has become clear that undecylprodigiosin and other bacterial prodigiosins still hold surprises in terms of valuable properties and applicative potential to medical and other industrial fields and that they still deserve continuing research curiosity.

In vitro evolution of styrene monooxygenase from Pseudomonas putida CA-3 for improved epoxide synthesis

The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, (S)-styrene oxide, was formed from styrene while (1S,2R)-indene oxide was the predominant enantiomer (e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively.

The anti-cancer activity of a cationic anti-microbial peptide derived from monomers of polyhydroxyalkanoate

The biodegradable polymer medium chain length polyhydroxyalkanoate (mclPHA), produced by Pseudomonas putida CA-3, was depolymerised and the predominant monomer (R)-3-hydroxydecanoic acid (R10) purified. R10 was conjugated to a d-peptide DP18 and its derivatives. All peptides conjugated with R10 exhibited greater anti-cancer activity compared to the unconjugated peptides. Unconjugated and conjugated peptides were cytocidal for cancer cells. Conjugation of R10 to peptides was essential for enhanced anti-proliferation activity, as unconjugated mixes did not result in enhancement of anti-cancer activity. The conjugation of R10 resulted in more rapid uptake of peptides into HeLa and MiaPaCa cells compared to unconjugated peptide. Both unconjugated and R10 conjugated peptides localized to the mitochondria of HeLa and MiaPaCa cells and induced apoptosis. Peptide conjugated with a terminally hydroxylated decanoic acid (ω-hydroxydecanoic acid) exhibited 3.3 and 6.3 fold higher IC(50) values compared to R10 conjugated peptide indicating a role for the position of the hydroxyl moiety in enhancement of anti-cancer activity. Conjugation of decanoic acid (C10) to peptides resulted in similar or higher IC(50) values compared to R10 conjugates but C10 conjugates did not exhibit any cancer selectivity. Combination studies showed that R10DP18L exhibited synergy with cisplatin, gemcitabine, and taxotere with IC(50) values in the nanomolar range.

Recent developments in biocatalysis beyond the laboratory

Recent developments in biocatalysis, where implementation beyond the laboratory has been demonstrated, are explored: the use of transglutaminases to modify foods, reduce allergenicity and produce advanced materials, lipases for biodiesel production, and transaminases for biochemical production. The availability and application of enzymes at pilot and larger scale opens up possibilities for further improvements of biocatalyst-based processes and the development of new processes. Enzyme production, stability, activity, re-use, and product retrieval are common challenges for biocatalytic processes. We explore recent advances in biocatalysis within the process chain, such as protein engineering, enzyme expression, and biocatalyst immobilization, in the context of these challenges.

Toxic essential oils. Part III: Identification and biological activity of new allylmethoxyphenyl esters from a Chamomile species (Anthemis segetalis Ten.)

To determine the exact structure of previously tentatively identified minor essential-oil constituents of a Chamomile species (Antemis segetalis Ten. (Asteraceae)), we have synthesized a small combinatorial library of 54 regioisomeric allylmethoxyphenyl pentanoates and 2-pentenoates (49 completely new compounds). GC-MS in combination with 1D- and 2D-NMR analyses of the library compounds provided unambiguous data that led to a straightforward identification of the mentioned A. segetalis constituents as eugenyl angelate, 2-methylbutanoate and 3-methylbutanoate (0.21, 0.22, and 0.13 mg/100 g of fresh plant material, respectively). To assess the safety and potential beneficial pharmacological uses of these naturally occurring esters and several other library compounds (these were tested to provide relevant data for a SAR (structure-activity relationship) analysis), we have studied the effect of these compounds in several models of toxicity (acute toxicity against Artemia salina, cytotoxicity against two cell lines (fibroblast and melanoma)), as well as their acetylcholinesterase inhibitory and antibacterial activities. Anthemis segetalis constituents showed low to moderate activity in all tests. The obtained results suggest that the intake of these compounds in naturally available amounts, on their own, would probably not represent a risk to human health but the possible adverse interactions with the plant matrix should not be neglected.

Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches

The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2‐fold improvement in kcat, 5.2‐fold lower Km and 16‐fold improvement in catalytic efficiency for D‐tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased kcat for D‐tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher kcat and Km value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D‐tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9‐fold) improvement in kcat and a 2.4‐fold increase in Km compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the Km up to 2.6‐fold for D‐tyrosine but one variant 145_V153A increased the Km 2.4‐fold compared to the WT. Homology based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an α‐helix providing one of the conserved histidine residues of the active site. The kcat and Km values for L‐tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1‐fold high catalytic efficiency compared to the WT which is a 7.6‐fold lower improvement compared to D‐tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D‐tyrosine:D‐DOPA and a 1.4‐fold higher L‐tyrosine:L‐DOPA activity ratio compared to the WT. Biotechnol. Bioeng. 2013; 110: 1849–1857.

Highly efficient Michael-type addition of acetaldehyde to β-nitrostyrenes by whole resting cells of Escherichia coli expressing 4-oxalocrotonate tautomerase.

A novel whole cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) was developed and shown to be an effective biocatalyst for the asymmetric Michael addition of acetaldehyde to β-nitrostyrenes. Optimal ratio of substrates (2mM β-nitrostyrenes and 20mM acetaldehyde) and biocatalyst of 5 g of cell dry weight of biocatalyst per liter was determined. Through further bioprocess improvement by sequential addition of substrate 10mM nitrostyrene biotransformation was achieved within 150 min. Excellent enantioselectivity (>99% ee) and product yields of up to 60% were obtained with β-nitrostyrene substrate. The biotransformation product, 4-nitro-3-phenyl-butanal, was isolated from aqueous media and further transformed into the corresponding amino alcohol. The biocatalyst exhibited lower reaction rates with p-Cl-, o-Cl- and p-F-β-nitrostyrenes with product yields of 38%, 51%, 31% and ee values of 84%, 88% and 94% respectively. The importance of the terminal proline of 4-OT was confirmed by two proline enriched variants and homology modeling.

Synthesis and Evaluation of Series of Diazine-Bridged Dinuclear Platinum(II) Complexes through in Vitro Toxicity and Molecular Modeling: Correlation between Structure and Activity of Pt(II) Complexes

Polynuclear Pt(II) complexes are a novel class of promising anticancer agents with potential clinical significance. A series of pyrazine (pz) bridged dinuclear Pt(II) complexes with general formulas {[Pt(L)Cl]2(μ-pz)}(2+) (L, ethylenediamine, en; (±)-1,2-propylenediamine, 1,2-pn; isobutylenediamine, ibn; trans-(±)-1,2-diaminocyclohexane, dach; 1,3-propylenediamine, 1,3-pd; 2,2-dimethyl-1,3-propylenediamine, 2,2-diMe-1,3-pd) and one pyridazine (pydz) bridged {[Pt(en)Cl]2(μ-pydz)}(2+) complex were prepared. The anticancer potential of these complexes were determined through in vitro cytotoxicity assay in human fibroblasts (MRC5) and two carcinoma cell lines (A375 and HCT116), interaction with double stranded DNA through in vitro assay, and molecular docking study. All complexes inhibited cell proliferation with inhibitory concentrations in the 0.5-120 μM range. While {[Pt(1,3-pd)Cl]2(μ-pz)}(2+) showed improved activity and {[Pt(en)Cl]2(μ-pydz)}(2+) showed comparable activity to that of clinically relevant cisplatin, {[Pt(en)Cl]2(μ-pydz)}(2+) was less toxic in an assay with zebrafish (Danio rerio) embryos, causing no adverse developmental effects. The in vitro cytotoxicity of all diazine-bridged dinuclear Pt(II) complexes is discussed in correlation to their structural characteristics.