Biocatalysis allows to access to greener synthesis pathways
Enzymes involved in biocatalytic processes are not anymore limited to lipases and ketoreductases. Nowadays, sources of enzymes for process chemistry applications have expanded: Seqens offers an access to its catalog of enzymes covering all common biocatalysis mediated chemical transformations such as reduction, oxidation, chiral transformation, hydratation, hydrolysis, esterification.
Enzymes / Biocatalysts
We offer our customers
- Possibilities to identify biocatalysts among a unique biodiversity of microorganisms, with more than 4500 strains (bacteria, Archaea, Fungi, microalgae…) including over 4300 sequenced (meta)genomes. This collection is encompassing a large diversity of microorganisms from extreme environments (deep hydrothermal springs, salt marshes, volcanic environment, oil field…)
- More than 500 well characterized enzymes in collection, available on demand, covering 28 families of enzymes across main classes of enzymes (eg oxido-reductases, hydrolases, transferases, biomass treatment, cofactor recycling)
- State-of-the-art proprietary technologies for molecular engineering/assay development and screening
- Development and optimization of biocatalysis assays from enzyme selection to the industrial scale-up of greener chemical process
Seqens has developed a robust methodology for the development of biocatalytic processes, in two steps: a rapid proof of concept with selection and identification of the first hits. Then the optimization of enzymes as well as optimization of the biocatalytic process toward scale-up industrial development.
Proof of Concept (3 months)
The goal of the feasibility study is to determine the potential interest of the biocatalysis to perform the targeted chemical reaction. SEQENS team will prepare a proposal, typically within two weeks, based on this evaluation. Confidentiality agreement will be in place for this step.
SEQENS will select 50 new enzymes specifically for the target and will add several more from its own catalogue.
Enzymes selected in the previous step will be expressed using a microbial host system (bacteria or yeast). The analytical method will be transferred, if provided by the customer, or developed in order to prepare the screening assay.
A screening assay will be developed based on the analytical method and adapted to miniaturize the reaction in order to test a large number of enzymes and conditions.
Based on the results of the screening, the best enzymes and conditions (the “hits”) will be used as a starting point for the development of the biocatalytic process.
Optimization and scale-up
Conditions of screening assay previously developed will be used in order to refine the conditions to be used. Primary and secondary physicochemical parameters will be challenged for optimal ratio enzyme/substrate/co-factors.
When a non-natural chemical reaction is targeted, sometimes wild type enzymes have to be improved in order to reach performances compatible with industrialization. This is performed using enzyme engineering, in order to adapt the enzyme structure to its targeted non-natural substrate or conditions (temperature or salt resistance) via modifications of its amino acid sequence. Different methods can be used, such as semi-rational methods which allow to combine in silico modelling, in order to predict the amino acid positions or area to be mutated, with the screening of a large number of enzyme variants. Performing enzyme engineering allows to increase the performances of the enzyme and to generate intellectual property rights on the new variants generated.
If needed, the best reaction conditions can be defined using a Design of Experiments approach.
The biocatalytic reaction will be scaled up, as well as the fermentation process for the production of the enzyme for tailor-made enzymes (more details in the “Production of the biocatalysts: fermentation scale-up” section).
The biocatalytic process will then be transferred to the Seqens’Lab for further upscaling towards industrialization.
In silico protein analysis for protein engineering
These movements can affect the active site of the enzyme, which is the region where the substrate binds, and the chemical reaction takes place. By understanding the conformational dynamics of enzymes, the key amino acid residues can be identified, and targeted modifications can be applied specifically to favor interactions between residues and substrate having a positive impact on the activity of the enzyme.
There are several techniques in computing that have been used for enzyme evolution, each with its advantages and limitations. The choice of the best technique depends on the specific problem being addressed
In silico design of smart libraries
- Homology modeling of the proteins
- Identification of hotspots for saturation mutagenesis (HotSpot Wizard)
- Analysis of multiple-point mutation combined in active site cavity/tunnel (Carver/Carverdock)
- Combining docking, phylogenetic analyses & Rosetta design calculations, QM/MM modeling
- Provide better knowledge on catalytic cavity and substrate/co-factor and enzyme interactions)
- Prediction of distal active site mutations by conformationally driven Shortest Path Map (SPM)-based enzyme evolution approach
Production of the biocatalysts: fermentation scale-up
Seqens has internal fermentation capabilities up to 300L scale, which is compatible with some commercial applications, and has a network of external partners for the implementation of fermentation processes at a larger scale (from m3 scale).
- 1/ Strain selection, media optimization
- Lab-scale (5 – 200 ml) Benchtop bioreactors 1L
- Design of Experiments (DoE)
- Feed stock (Glucose, Glycerol…)
- Fermentation parameters optimization
- 6x 1L Applikon bioreactors
- 2/ Fementation process development
- Benchtop bioreactor 3L & Pilot plant 40L – 300L
- 2x 3L Applikon bioreactors
- 2x 40L Applikon bioreactors
- 300L Global Process Concept (GPC)
- Biomass separation by centrifugation
- Downstream process: cell disruption, membrane separation,…
- 3/ Tech Transfer, Scale-up
- Process book
- Biocatalyst manufacturing: at SEQENS or transfer to external production site
- Biocatalyst reaction: Integration within SEQENS plants or to customer site