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A novel bacterial tyrosinase for biomaterials and biocatalysis
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
medium using sucrose as a sole carbon source under pH-stat feeding control for high level PHB production. In semi-optimized conditions, the wild-type strain was capable of producing 30 g L−1 PHB at a productivity of 1.1 ± 0.1 g L−1 h−1 . In order to improve sucrose utilization, the sucrose operon repressor gene was knocked out. The engineered strain produced 46 g L−1 PHB at a productivity of 1.8 ± 0.1 g L−1 h−1 , a 50% increase in yield over the wild type. To further improve PHB production, we have engineered central carbon metabolism to increase carbon flux to PHB precursors. Specifically, we have knocked out pgi and overexpressed eda, fbaA, and tpiA. Preliminary data shows that tpiA overexpression on its own can increase PHB production by 50%, while the other genes have insignificant effect on PHB production on their own. We are currently evaluating overexpression of the gene targets in the pgi knock-out strain. doi:10.1016/j.jbiotec.2010.08.189 [I.12] Potential applications of microbial biomass and PHA elastomer from glycerol to obtain biodegradable and compostable films Rosa Palmeri 1,∗ , Manuela Fragalà 1 , Stefano La Porta 2 , Antonino Felice Catara 1 1
Parco Scientifico e Tecnologico della Sicilia, Italy Università di Messina, Italy Keywords: Biopolymer; Glycerol; Paper; Organic waste 2
Due to the great concern about the long lasting time for the degradation of plastic materials different approaches are under evaluation to find friendly solutions to the problem and assure a sustainable growth. The use of 100% biodegradable filmsappears as a challenging alternative for specific applications, as organic agriculture and composting. Among them is the coating of different supports with different blends of oils, polymers and natural products to improve their performances. Based on successful conversion of fatty acids in PHA by Pseudomonas corrugata CFBP5454, we have improved the quality of the polymer by approaching the metabolic pathway of fatty acids through the conversion of glycerol with P mediterranea CFBP5447 strain. A high biomass fermentation pHstat fed-batch type was settled in order to obtain an unbalance of carbon (glycerol)/ammonia source. At the end of fermentation, the accumulation of intracellular PHA was detected with Nile-red and centrifuged to spin and wash the broth. The biomass was freezedried and later acetone extracted to recover the elastomer. Different solutions of biomass or PHA were sprayed or calendered on paper and fiber sheets and tested for their potential applications, as mulching films or moist organic waste bags. Results show that both coatings reduces the water and oil absorption improving the resistance and maintaining a good air permeation as compared with polyethylene films and untreated paper. Biomass coated paper sheets were unbroken up to 96 hours whereas the control was broken within five minutes. Biomass coated paper and fiber bags of different size appear very promising to manage moist organic wastes. doi:10.1016/j.jbiotec.2010.08.190
S73
[I.13] A novel bacterial tyrosinase for biomaterials and biocatalysis M. Fairhead ∗ , L. Thoeny-Meyer Swiss Federal Laboratories for Materials Testing and Research, Switzerland Keywords: Tyrosinase; Melanin; Immobilized enzymes; Protein engineering Tyrosinase (EC 1.14.18) is a type 3 copper oxidase enzyme that converts tyrosine and DOPA into dopaquinone, which is a precursor in melanin biosynthesis (Claus and Decker, 2006). Tyrosinases have many potential biotechnological applications, one of them being the ability to catalyze protein–protein or protein–polysaccharide cross-linking (Halaouli et al., 2006). We report on the successful expression of a tyrosinase-like gene from the aquatic bacterium Verrucomicrobium spinosum in Escherichia coli (RCTEBT, 2010). The gene codes for a 57 kDa protein (full length unprocessed form) which bioinformatic analysis suggests has a TAT signal peptide, the two copper binding motifs typical of the tyrosinase protein family and a C-terminal extension similar to that found in the pro-form of plant and fungal polyphenol oxidases. The protein was purified in a cytoplasmic form to homogeneity and shown to contain 1.72 copper atoms per polypeptide, in agreement with a binuclear copper site. Both mono-and diphenol oxidase activites were present as shown by spectrophotometric assays for tyrosine hydroxylation. We expressed various mutants of the recombinant enzyme in E. coli and found that removal of the C-terminal extension by genetic engineering or limited trypsin digest of the pro-form results in a more active enzyme, i.e. 30–100 fold increase in monophenolase and diphenolase activities. In addition, cross-linking experiments demonstrate that this enzyme may be used for the formation of cross-linked enzyme aggregates. The protein can also be stamped onto surfaces allowing the formation of thin melanin films in-situ.
References Claus, H., Decker, H., 2006. Bacterial tyrosinases. Syst Appl Microbiol. 29, 3–14. Halaouli, S., Asther, M., Sigoillot, J.C., Hamdi, M., Lomascolo, A., 2006. Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. J Appl Microbiol. 100, 219–232. Role of the C-terminal extension in a bacterial tyrosinase. (2010) Michael Fairhead and Linda Thöny-Meyer. FEBS J. In press.
doi:10.1016/j.jbiotec.2010.08.191
medium using sucrose as a sole carbon source under pH-stat feeding control for high level PHB production. In semi-optimized conditions, the wild-type strain was capable of producing 30 g L−1 PHB at a productivity of 1.1 ± 0.1 g L−1 h−1 . In order to improve sucrose utilization, the sucrose operon repressor gene was knocked out. The engineered strain produced 46 g L−1 PHB at a productivity of 1.8 ± 0.1 g L−1 h−1 , a 50% increase in yield over the wild type. To further improve PHB production, we have engineered central carbon metabolism to increase carbon flux to PHB precursors. Specifically, we have knocked out pgi and overexpressed eda, fbaA, and tpiA. Preliminary data shows that tpiA overexpression on its own can increase PHB production by 50%, while the other genes have insignificant effect on PHB production on their own. We are currently evaluating overexpression of the gene targets in the pgi knock-out strain. doi:10.1016/j.jbiotec.2010.08.189 [I.12] Potential applications of microbial biomass and PHA elastomer from glycerol to obtain biodegradable and compostable films Rosa Palmeri 1,∗ , Manuela Fragalà 1 , Stefano La Porta 2 , Antonino Felice Catara 1 1
Parco Scientifico e Tecnologico della Sicilia, Italy Università di Messina, Italy Keywords: Biopolymer; Glycerol; Paper; Organic waste 2
Due to the great concern about the long lasting time for the degradation of plastic materials different approaches are under evaluation to find friendly solutions to the problem and assure a sustainable growth. The use of 100% biodegradable filmsappears as a challenging alternative for specific applications, as organic agriculture and composting. Among them is the coating of different supports with different blends of oils, polymers and natural products to improve their performances. Based on successful conversion of fatty acids in PHA by Pseudomonas corrugata CFBP5454, we have improved the quality of the polymer by approaching the metabolic pathway of fatty acids through the conversion of glycerol with P mediterranea CFBP5447 strain. A high biomass fermentation pHstat fed-batch type was settled in order to obtain an unbalance of carbon (glycerol)/ammonia source. At the end of fermentation, the accumulation of intracellular PHA was detected with Nile-red and centrifuged to spin and wash the broth. The biomass was freezedried and later acetone extracted to recover the elastomer. Different solutions of biomass or PHA were sprayed or calendered on paper and fiber sheets and tested for their potential applications, as mulching films or moist organic waste bags. Results show that both coatings reduces the water and oil absorption improving the resistance and maintaining a good air permeation as compared with polyethylene films and untreated paper. Biomass coated paper sheets were unbroken up to 96 hours whereas the control was broken within five minutes. Biomass coated paper and fiber bags of different size appear very promising to manage moist organic wastes. doi:10.1016/j.jbiotec.2010.08.190
S73
[I.13] A novel bacterial tyrosinase for biomaterials and biocatalysis M. Fairhead ∗ , L. Thoeny-Meyer Swiss Federal Laboratories for Materials Testing and Research, Switzerland Keywords: Tyrosinase; Melanin; Immobilized enzymes; Protein engineering Tyrosinase (EC 1.14.18) is a type 3 copper oxidase enzyme that converts tyrosine and DOPA into dopaquinone, which is a precursor in melanin biosynthesis (Claus and Decker, 2006). Tyrosinases have many potential biotechnological applications, one of them being the ability to catalyze protein–protein or protein–polysaccharide cross-linking (Halaouli et al., 2006). We report on the successful expression of a tyrosinase-like gene from the aquatic bacterium Verrucomicrobium spinosum in Escherichia coli (RCTEBT, 2010). The gene codes for a 57 kDa protein (full length unprocessed form) which bioinformatic analysis suggests has a TAT signal peptide, the two copper binding motifs typical of the tyrosinase protein family and a C-terminal extension similar to that found in the pro-form of plant and fungal polyphenol oxidases. The protein was purified in a cytoplasmic form to homogeneity and shown to contain 1.72 copper atoms per polypeptide, in agreement with a binuclear copper site. Both mono-and diphenol oxidase activites were present as shown by spectrophotometric assays for tyrosine hydroxylation. We expressed various mutants of the recombinant enzyme in E. coli and found that removal of the C-terminal extension by genetic engineering or limited trypsin digest of the pro-form results in a more active enzyme, i.e. 30–100 fold increase in monophenolase and diphenolase activities. In addition, cross-linking experiments demonstrate that this enzyme may be used for the formation of cross-linked enzyme aggregates. The protein can also be stamped onto surfaces allowing the formation of thin melanin films in-situ.
References Claus, H., Decker, H., 2006. Bacterial tyrosinases. Syst Appl Microbiol. 29, 3–14. Halaouli, S., Asther, M., Sigoillot, J.C., Hamdi, M., Lomascolo, A., 2006. Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. J Appl Microbiol. 100, 219–232. Role of the C-terminal extension in a bacterial tyrosinase. (2010) Michael Fairhead and Linda Thöny-Meyer. FEBS J. In press.
doi:10.1016/j.jbiotec.2010.08.191