ProteoNic BV

Leiden, Netherlands

ProteoNic BV

Leiden, Netherlands

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Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: KBBE.2013.3.3-04 | Award Amount: 9.11M | Year: 2013

OPTIBIOCAT is a 48 months project aimed at developing biocatalysts based on feruloyl esterases (FAEs) and glucuronoyl esterases (GEs) for production of phenolic fatty- and sugar- esters with antioxidant activity for cosmetic industry, expanding the number/type of industrial biotransformations. Selected FAEs and GEs available within the consortium will be improved for their thermo- and solvent- resistance and substrate specificity by site-directed mutagenesis and directed evolution. Novel enzymes will be discovered by mining for new genes from available genomes. An inventory of novel FAEs and GEs will be developed including 50 fungal and 500 bacterial esterases, 25 site-directed and 20 directed evolved mutants. Enzymatic performances will be optimized to enhance the yield (up to the theoretical yield of 100%) and productivity (up to 0.5-1 g/l/h) of reactions giving the main targeted antioxidants: butyl ferulate, p-coumarate, caffeate, sinapate and 5-O-(trans-feruloyl)-arabinofuranose (using FAEs), glucuronate and benzyl glucuronate (using GEs). FAEs and GEs will be also tested for production of other compounds with improved biological activity and properties of hydrophilicity/hydrophobicity for cosmetic applications. Cost-effective methods will be developed for production of the new biocatalysts, in the g/L scale, and for their technical application to produce antioxidants for cosmetic industry, up to 20L. Enzyme immobilization will increase their recyclability up to ten cycles. The ability of the developed catalysts to work in conditions miming the industrial ones with reduced use of solvents and lower temperature than the chemical routes will be demonstrated. The techno-economic viability and environmental friendliness will be assessed considering a full industrial scale scenario. OPTIBIOCAT involves a highly skilled and multidisciplinary partnership of 16 partners from 8 EU countries, and it is a strongly industry driven project through the participation of 8 SMEs and 1 large company.


Tamayo-Ramos J.A.,Wageningen University | Barends S.,ProteoNic BV | de Lange D.,ProteoNic BV | de Lange D.,Genencor | And 3 more authors.
Biotechnology and Bioengineering | Year: 2013

In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5′ untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5′UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5′UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5′UTR can lead to improvements in protein yields. Biotechnol. © 2012 Wiley Periodicals, Inc.

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