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Optimisation of laccase immobilization on modified magnetic nanoparticles for biocatalytic reactions
Abstracts / New Biotechnology 33S (2016) S1–S213
O3-9
O3-11
Biotechnological process design for the production and purification of three recombinant thermophilic phosphotriesterases
High diversity of enzymes in plant-associated microbiomes
Restaino ∗ ,
Maria Giovanna Borzacchiello, Odile Francesca Scognamiglio Ilaria, Luigi Fedele, Chiara Schiraldi Second University of Naples, Italy Bacterial phosphotriesterases able to degrade organophosphate molecules, highly toxic compounds widely used as pesticides, are nowadays particularly interesting from scientific and industrial points of view because potentially employable as decontaminating agents in environmental bioremediation procedures. In this study a new biotechnological process was designed for the production and purification of three engineered, recombinant thermostable phosphotriesterases (PTE): SsoPox W263F and SsoPox C258L/I261F/W263A, whose wild type genes were originally isolated from Sulfolobus solfataricus, and SacPox isolated from Sulfolobusacidocaldarius. New induction strategies, alternative to IPTG ones, were exploited and optimization fermentation development studies allowed to reach high cell density cultures for all the three bacteria. Enzyme concentrations in the range from 2950 ± 200 to 6700 ± 150 U L−1 were obtained, according to the different strains, and the production processes resulted scalable from 2.5-L to150-L reactors. A new downstream purification scheme was designed coupling a step of thermal precipitation with an ultrafiltration/diafiltration membrane based purification protocol, after enzyme extraction from biomass by using a mechanical homogenizer. This procedure resulted robust enough to be applied to all three strains allowing to achieve a final enzyme recovery that ranged from 75.0 to 80.0 ± 4.5% and a purity grade from 77.0 to 83.0 ± 5.0%, according to the enzyme. http://dx.doi.org/10.1016/j.nbt.2016.06.776
S15
Christina A. Müller 1,∗ , Veronika Perz 2 , Stephanie N. Hollauf 1 , Christoph Provasnek 1 , Georg M. Gübitz 3 , Gabriele Berg 3 1
Graz University of Technology, Austria Austrian Centre of Industrial Biotechnology (acib GmbH), Austria 3 University of Natural Resources and Life Sciences, Vienna, Austria 2
Plants and their inhabiting microorganisms can be considered one of the richest sources for new bioactive compounds. With growing demand for novel bioactive compounds and biocatalysts it is important to investigate the unexplored biotechnological potential of the plant-associated microbiome. Mosses produce plenty of bioactive substances and the antimicrobial activity of their microbiome has been reported [1]. We investigated the metagenome of Sphagnum moss as an untapped resource for novel enzymes. Nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are commonly involved in the production of microbial bioactive secondary metabolites. Screening for NRPS and PKS genes in the moss metagenome revealed a high level of diversity and enabled identification of thirteen novel NRPS-related sequences that share similarities from 48% to 91% to annotated sequences, mainly from Proteobacteria, Actinobacteria, and Cyanobacteria [2]. Their putative role in the production of microbial metabolites such as siderophores, phytotoxins, and antibiotics is currently being evaluated. In a second screening approach we identified, isolated and expressed six novel esterase genes that display polyester degrading activity. Two of the esterases, which clustered into the family VIII of bacterial lipolytic enzymes (homology to class C ß lactamases) have been purified and characterized. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems for the discovery of biocatalyst and natural products.
O3-10 Optimisation of laccase immobilization on modified magnetic nanoparticles for biocatalytic reactions Ana
Xavier ∗ ,
Ana Tavares, Ana Daniel-da-Silva, Cristiano Fortes
University of Aveiro, Portugal Magnetic nanoparticles (MNPs) as supports for immobilised catalysts greatly improve their reutilization since recovery can be easily done by simple application of an external magnetic field. Laccase (EC 1.10.3.2) as an oxidative enzyme with a wide range of industrial applications requires new technologies for its immobilization in order to improve its catalytic activity with reduced costs. The conditions of laccase immobilization on magnetic nanoparticles were optimized by box-Benhken experimental design. At the optimal conditions, the highest recovery activity of immobilized laccase reached 36.3 U/L. Compared with free laccase, thermal, operational and storage stabilities of immobilized laccase were improved. The immobilized laccase obtained can retain above 75% of activity after 6 consecutive operations. Laccase was successfully bound on functionalized MNPs according to FTIR spectroscopy. MNPs can be used for immobilization of important enzymes at industrial level, as these nanomaterials can improve both enzymatic application properties and easy and fast recovery for reutilization. http://dx.doi.org/10.1016/j.nbt.2016.06.777
References [1] Opelt K, Berg C, Berg G. FEMS Microbiol Ecol 2007;61:38–53. [2] Müller CA, Oberauner-Wappis L, Peyman A, Amos GCA, Wellington EMH, Berg G. Appl Environ Microbiol 2015;81:5064–72.
http://dx.doi.org/10.1016/j.nbt.2016.06.778
O3-12 Carboxylic acid reductases and their use as well defined enzyme building blocks for the construction of in vitro cascade reactions William Finnigan University of Exeter, United Kingdom Biocatalysis is becoming increasingly attractive for the development of more efficient and cleaner chemical synthetic processes. The combination of multiple enzyme steps for cascade reactions allows for attractive one-pot processes with reduced operating costs. Whilst the use of whole-cells have a number of advantages for these reactions, the competing needs of the cell and limited transport across the cell membrane can result in a low final product concentration. In contrast, the use of isolated enzymes allows reactions to be easily controlled, with the use of stable enzymes such as those from thermophiles offering economically competitive processes.
O3-9
O3-11
Biotechnological process design for the production and purification of three recombinant thermophilic phosphotriesterases
High diversity of enzymes in plant-associated microbiomes
Restaino ∗ ,
Maria Giovanna Borzacchiello, Odile Francesca Scognamiglio Ilaria, Luigi Fedele, Chiara Schiraldi Second University of Naples, Italy Bacterial phosphotriesterases able to degrade organophosphate molecules, highly toxic compounds widely used as pesticides, are nowadays particularly interesting from scientific and industrial points of view because potentially employable as decontaminating agents in environmental bioremediation procedures. In this study a new biotechnological process was designed for the production and purification of three engineered, recombinant thermostable phosphotriesterases (PTE): SsoPox W263F and SsoPox C258L/I261F/W263A, whose wild type genes were originally isolated from Sulfolobus solfataricus, and SacPox isolated from Sulfolobusacidocaldarius. New induction strategies, alternative to IPTG ones, were exploited and optimization fermentation development studies allowed to reach high cell density cultures for all the three bacteria. Enzyme concentrations in the range from 2950 ± 200 to 6700 ± 150 U L−1 were obtained, according to the different strains, and the production processes resulted scalable from 2.5-L to150-L reactors. A new downstream purification scheme was designed coupling a step of thermal precipitation with an ultrafiltration/diafiltration membrane based purification protocol, after enzyme extraction from biomass by using a mechanical homogenizer. This procedure resulted robust enough to be applied to all three strains allowing to achieve a final enzyme recovery that ranged from 75.0 to 80.0 ± 4.5% and a purity grade from 77.0 to 83.0 ± 5.0%, according to the enzyme. http://dx.doi.org/10.1016/j.nbt.2016.06.776
S15
Christina A. Müller 1,∗ , Veronika Perz 2 , Stephanie N. Hollauf 1 , Christoph Provasnek 1 , Georg M. Gübitz 3 , Gabriele Berg 3 1
Graz University of Technology, Austria Austrian Centre of Industrial Biotechnology (acib GmbH), Austria 3 University of Natural Resources and Life Sciences, Vienna, Austria 2
Plants and their inhabiting microorganisms can be considered one of the richest sources for new bioactive compounds. With growing demand for novel bioactive compounds and biocatalysts it is important to investigate the unexplored biotechnological potential of the plant-associated microbiome. Mosses produce plenty of bioactive substances and the antimicrobial activity of their microbiome has been reported [1]. We investigated the metagenome of Sphagnum moss as an untapped resource for novel enzymes. Nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are commonly involved in the production of microbial bioactive secondary metabolites. Screening for NRPS and PKS genes in the moss metagenome revealed a high level of diversity and enabled identification of thirteen novel NRPS-related sequences that share similarities from 48% to 91% to annotated sequences, mainly from Proteobacteria, Actinobacteria, and Cyanobacteria [2]. Their putative role in the production of microbial metabolites such as siderophores, phytotoxins, and antibiotics is currently being evaluated. In a second screening approach we identified, isolated and expressed six novel esterase genes that display polyester degrading activity. Two of the esterases, which clustered into the family VIII of bacterial lipolytic enzymes (homology to class C ß lactamases) have been purified and characterized. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems for the discovery of biocatalyst and natural products.
O3-10 Optimisation of laccase immobilization on modified magnetic nanoparticles for biocatalytic reactions Ana
Xavier ∗ ,
Ana Tavares, Ana Daniel-da-Silva, Cristiano Fortes
University of Aveiro, Portugal Magnetic nanoparticles (MNPs) as supports for immobilised catalysts greatly improve their reutilization since recovery can be easily done by simple application of an external magnetic field. Laccase (EC 1.10.3.2) as an oxidative enzyme with a wide range of industrial applications requires new technologies for its immobilization in order to improve its catalytic activity with reduced costs. The conditions of laccase immobilization on magnetic nanoparticles were optimized by box-Benhken experimental design. At the optimal conditions, the highest recovery activity of immobilized laccase reached 36.3 U/L. Compared with free laccase, thermal, operational and storage stabilities of immobilized laccase were improved. The immobilized laccase obtained can retain above 75% of activity after 6 consecutive operations. Laccase was successfully bound on functionalized MNPs according to FTIR spectroscopy. MNPs can be used for immobilization of important enzymes at industrial level, as these nanomaterials can improve both enzymatic application properties and easy and fast recovery for reutilization. http://dx.doi.org/10.1016/j.nbt.2016.06.777
References [1] Opelt K, Berg C, Berg G. FEMS Microbiol Ecol 2007;61:38–53. [2] Müller CA, Oberauner-Wappis L, Peyman A, Amos GCA, Wellington EMH, Berg G. Appl Environ Microbiol 2015;81:5064–72.
http://dx.doi.org/10.1016/j.nbt.2016.06.778
O3-12 Carboxylic acid reductases and their use as well defined enzyme building blocks for the construction of in vitro cascade reactions William Finnigan University of Exeter, United Kingdom Biocatalysis is becoming increasingly attractive for the development of more efficient and cleaner chemical synthetic processes. The combination of multiple enzyme steps for cascade reactions allows for attractive one-pot processes with reduced operating costs. Whilst the use of whole-cells have a number of advantages for these reactions, the competing needs of the cell and limited transport across the cell membrane can result in a low final product concentration. In contrast, the use of isolated enzymes allows reactions to be easily controlled, with the use of stable enzymes such as those from thermophiles offering economically competitive processes.