Tanja Narancic

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.

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.

Metabolic versatility of Gram-positive microbial isolates from contaminated river sediments

Gram-positive bacteria from river sediments affected by the proximity of a petrochemical industrial site were isolated and characterized with respect to their ability to degrade a wide range of aromatic compounds. In this study we identified metabolically diverse Gram-positive bacteria capable of growth on wide range aromatic compounds in the presence of heavy metals and with the ability to accumulate biopolymers. Thirty-four isolates that were able to use 9 or more common aromatic pollutants, such as benzene, biphenyl, naphthalene etc. as a sole source of carbon and energy included members of Bacillus, Arthrobacter, Rhodococcus, Gordonia, Streptomyces, and Staphylococcus genus. Rhodococcus sp. TN105, Gordonia sp. TN103 and Arthrobacter sp. TN221 were identified as novel strains. Nine isolates were able to grow in the presence of one or more metals (mercury, cadmium, nickel) at high concentration (100mM). Seven isolates could degrade 15 different aromatic compounds and could grow in the presence of one or more heavy metals. Two of these isolates were resistant to multiple antibiotics including erythromycin and nalidixic acid. One third of isolates could accumulate at least one biopolymer. Twelve isolates (mainly Bacillus sp. and Arthrobacter sp.) accumulated polyphosphate, 3 Bacillus sp. accumulated polyhydroxybutyrate, while 4 isolates could accumulate exopolysaccharides.

Medium-chain-length polyhydroxyalkanoate production by newly isolated Pseudomonas sp. TN301 from a wide range of polyaromatic and monoaromatic hydrocarbons.

The aim of this study was to convert numerous polyaromatic and monoaromatic hydrocarbons into biodegradable polymer medium‐chain‐length polyhydroxyalkanoate (mcl‐PHA).

Microbial biotechnology addressing the plastic waste disaster

Oceans are a major source of biodiversity, they provide livelihood, and regulate the global ecosystem by absorbing heat and CO 2. However, they are highly polluted with plastic waste. We are discussing here microbial biotechnology advances with the view to improve the start and the end of life of biodegradable polymers, which could contribute to the sustainable use of marine and coastal ecosystems (UN Sustainability development goal 14).

Identification and characterization of an acyl-CoA dehydrogenase from Pseudomonas putida KT2440 that shows preference towards medium to long chain length fatty acids

Diverse and elaborate pathways for nutrient utilization, as well as mechanisms to combat unfavourable nutrient conditions make Pseudomonas putida KT2440 a versatile micro-organism able to occupy a range of ecological niches. The fatty acid degradation pathway of P. putida is complex and correlated with biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) biosynthesis. Little is known about the second step of fatty acid degradation (β-oxidation) in this strain. In silico analysis of its genome sequence revealed 21 putative acyl-CoA dehydrogenases (ACADs), four of which were functionally characterized through mutagenesis studies. Four mutants with insertionally inactivated ACADs (PP_1893, PP_2039, PP_2048 and PP_2437) grew and accumulated mcl-PHA on a range of fatty acids as the sole source of carbon and energy. Their ability to grow and accumulate biopolymer was differentially negatively affected on various fatty acids, in comparison to the wild-type strain. Inactive PP_2437 exhibited a pattern of reduced growth and PHA accumulation when fatty acids with lengths of 10 to 14 carbon chains were used as substrates. Recombinant expression and biochemical characterization of the purified protein allowed functional annotation in P. putida KT2440 as an ACAD showing clear preference for dodecanoyl-CoA ester as a substrate and optimum activity at 30 °C and pH 6.5-7.

Biodegradable Plastic Blends Create New Possibilities for End-of-Life Management of Plastics but They Are Not a Panacea for Plastic Pollution

Plastic waste pollution is a global environmental problem which could be addressed by biodegradable plastics. The latter are blended together to achieve commercially functional properties, but the environmental fate of these blends is unknown. We have tested neat polymers, polylactic acid (PLA), polyhydroxybutyrate, polyhydroxyoctanoate, poly(butylene succinate), thermoplastic starch, polycaprolactone (PCL), and blends thereof for biodegradation across seven managed and unmanaged environments. PLA is one of the worlds best-selling biodegradable plastics, but it is not home compostable. We show here that PLA when blended with PCL becomes home compostable. We also demonstrate that the majority of the tested bioplastics and their blends degrade by thermophilic anaerobic digestion with high biogas output, but degradation times are 3-6 times longer than the retention times in commercial plants. While some polymers and their blends showed good biodegradation in soil and water, the majority of polymers and their blends tested in this study failed to achieve ISO and ASTM biodegradation standards, and some failed to show any biodegradation. Thus, biodegradable plastic blends need careful postconsumer management, and further design to allow more rapid biodegradation in multiple environments is needed as their release into the environment can cause plastic pollution.

Design of a tailor-made platform for syngas bioconversion into polyhydroxybutyrate

Biodegradable polymers such as polyhydroxybutyrate (PHB) are part of the emerging portfolio of renewable materials, which are addressing the issue of plastic waste. Syngas, as a cheap, renewable and sustainable resource that can be obtained from biomass or waste, is viewed as an excellent feedstock for different bioprocesses, including syngas to PHB bioconversion. However, due to the hazardous nature of syngas, it is of utmost importance to consider safety aspects of the process. This recently developed tailor‐made platform for safe syngas fermentation and PHB production addresses safety aspects and demonstrates the importance of robust online and in‐line analytical tools allowing for monitoring and controlling of this bioprocess.