Protein engineering – Tailor made enzymes
Selection of the right starting genes and enzymes
Starting from the needs of our customers in terms of targeted reaction and conditions of use, we screen and identify the suitable enzymes using literature study and sequence datamining in public databases, as well as in our exclusive collection of more than 1000 genomes sequenced from our proprietary microorganisms collection.
Screening of a diversity of enzymes from exclusive sequences collection
Once the most promising genes are selected and the HTS screening assay is set-up, we clone and express the genes. Expression can be performed in various expression hosts such as Escherichia coli and Yarrowia lipolytica (others on demand). We then test the activity of the produced enzymes.
Examples of past projects:
- Wahler D. , O. Boujard, F. Lefevre, J.-L. Reymond, Adrenaline profiling of lipases and esterases with 1,2-diol and carbohydrate acetates. Tetrahedron, 60, 703–710 2004
- Magnin, A., Pollet, E., Perrin, R., Ullmann, C., Persillon, C., Phalip, V., Avérous, Enzymatic recycling of thermoplastic polyurethanes: Synergistic effect of an esterase and an amidase and recovery of building blocks. Waste Management, 85, 141-150. 2019
- A diverse collection
- >1000 sequenced strains
- millions of exclusive sequences of enzymes
Selection and screening of a subset of exclusive sequences with the target activity.
Quick selection of a diverse subset of sequences presenting a putative activity on the target reaction :
- Enlarge the screening with a diversity of sequences
- Selection of a subset of enzymes to be tested: graphical representation of the identity matrix in order to illustrate the diversity of genes and select a representative subset of enzymes to be tested
- Screening on selected enzymes
Directed evolution using proprietary technologies
Our platform for molecular directed evolution includes both randomized gene shuffling ( L-ShufflingTM) and algorithm-based approaches ( L-ShufflingTM). It results in new proprietary enzymes with drastic enhanced performances, such as specific activity and stability under demanding industrial conditions.
Proteus directed evolution patented technologies allow drastic enzymatic activity improvements and thus competitive solutions for our customers applications.
Semi-rational methods (rational mutagenesis strategy) can also be used to generate further improvement, if a 3D structure of an enzyme with a sufficient sequence identity is available.
High Throughput screening
- UHPLC – UV, RI, ELSD and MS detectors
- GC – FID, GC – MS
- Micro GC with headspace
- Ionic Chromatography
- Development of chromogenic or fluorogenic substrates (Clips-OTM proprietary substrates)
CLIPS-O™ is chemistry suited for the discovery of novel enzymatic activities under a high throughput format. CLIPS-O™ is a versatile proprietary technology which enables the design of chromogenic or fluorogenic targets that closely mimic the structure and energy state of the targeted compounds.
Examples of past projects:
- Gonzalez-GarcÌa E., Grognux J., Wahler D., Reymond J.; Synthesis and Evaluation of Chromogenic and Fluorogenic Analogs of Glycerol for Enzyme Assays; Helvetica chemica acta; 86:2458-2470. 2003
- Badalassi F, Wahler D, Klein G, Crotti P, Reymond JL.
A Versatile Periodate-Coupled Fluorogenic Assay for Hydrolytic Enzymes Angew Chem Int Ed Engl. ; 39(22):4067- 4070. 2000
CONDITIONS of expression
Having an efficient expression system is key to a profitable biocatalytic process. For this purpose, we can test a variety of expression systems (including E. coli, Bacillus, Pseudomonas, Yarrowia lipolytica) and, for each of them, a panel of vectors and conditions allowing :
- intracellular, periplasmic or secreted expression
- inducible or constitutive expression
- Episomal or integrative vectors
Directed evolution technologies are also a way to improve expression and/or solubility of enzymes. High throughput assays for screening better expression and solubility are available.
Enzyme formulation improves stability in use and in storage. Immobilization allows enzyme recycling in case of batch process as well as for continuous processes ( including flow chemistry), Proteus selects for its customers the best support for immobilizing the target enzyme, depending on the process and regulatory constraints. A Design Of Experiments (DoE) approach is used to optimize the immobilization of the enzyme.