Enzyme stability: Process engineering requirements

Transcription

1 Enzyme stability: Process engineering i requirements Ulrika Törnvall, Mathias Nordblad, Pär Tufvesson and John M Woodley Center for Process Engineering and Technology Department of Chemical and Biochemical Engineering Technical University of Denmark Kgs Lyngby Denmark

2 Outline Introduction to the process perspective Enzyme operational stability Process development strategy Stabilization methods The search for the deactivation mechanism Measurement of operational stability Case study: Chemo-enzymatic epoxidation Take home messages 2 DTU Chemical Engineering

3 Demands on the enzyme Thermal stability Chemical stability - Solvents -ph - Substrates t - Products Mechanical stab. - Stirring - Pumping Interfaces 3 DTU Chemical Engineering

4 The cost-effective process $$$ Fermentation Formulation Enzyme characteristics Process DSP - Expression level (g/l) -Time - Carrier and/or - Crosslinker - Scale - Procedure - Activity - Substrate - Chemicals - Selectivity - Energy - Energy - Stability -Equipment -Equipment See also Tufvesson et al (2011) Org. Proc. Res Dev DTU Chemical Engineering

5 Productivity targets g product /g immob enzyme Fine echemicals cas Bulk chemicals 1000 Pharma Pharmaceuticals Enz. contribution $ / Kg Bulk chemicals Enz. contribution 0.05 $ / Kg g product /L 5 DTU Chemical Engineering

6 Process development strategy Economic constraints Process constraints Scrutinize market/literature for available enzymes (active and stable within operational window) Operational window (upper and lower limits for temperature, ph, etc) Does biocatalysis seem feasible? Best available candiate(-s) Experiments in small scale with reaction media engineering, based on theoretically determined conditions Process parameters 6 DTU Chemical Engineering

7 Process parameters influencing productivity Metric Equation Hurdle Maximum rate Product concentration Yield Enantiomeric excess Enzymatic efficiency Purity d[p] dt < 15 g.l -1.hr - max P [P] 100 g.l -1 Y P F ee final [P] [F] [P] final 0 final [P] final [RP] final 1 50% 99% 10gg - [P] [E final 1 ] 10 g.g dcw [P] final X 100 [P] [RP] [RM] [SM] 95% final final final final 1 7 DTU Chemical Engineering

8 Window for enzyme concentration 8 DTU Chemical Engineering

9 Process development strategy Economic constraints Process constraints Scrutinize market/literature for available enzymes (active and stable within operational window) Operational window (upper and lower limits for temperature, ph, etc) Does biocatalysis seem feasible? Best available candiate(-s) Experiments in small scale with reaction media engineering, based on theoretically determined conditions Process parameters Deactivation causes Are extra costs such as immobilization possible? Enzyme stabilization procedure started 9 DTU Chemical Engineering

10 Stabilization methods for operational stability Method Reported stabilization work (%) Immobilization 34 Exploring extremophiles, environmental samples or similar Chemical modification of enzyme or other stabilization during enzyme formulation Reaction media engineering 11 Process conditions (temp, ph) 8 Protein engineering 7 Computer aided simulation/modeling 3 Process design (substrate supply, reactor type) 10 DTU Chemical Engineering 2

11 Process development strategy Economic constraints Process constraints Scrutinize market/literature for available enzymes (active and stable within operational window) Operational window (upper and lower limits for temperature, ph, etc) Does biocatalysis seem feasible? Best available candiate(-s) Are extra costs such as immobilization possible? Experiments in small scale with reaction media engineering, based on theoretically determined conditions Immobilization - Carrier and/or - Crosslinking Protein engineering - Site-directed - Random Process parameters Deactivation causes Are extra costs such as immobilization possible? Enzyme production -Host Enzyme stabilization procedure started Explore extremophiles Search for a better - Procedure -Etc. enzyme candidate 11 DTU Chemical Engineering

12 Process development strategy Economic constraints Process constraints Scrutinize market/literature for available enzymes (active and stable within operational window) Operational window (upper and lower limits for temperature etc) Does biocatalysis seem feasible? Best available candiate(-s) Experiments in small scale with reaction media engineering, based on theoretically ti determined d conditions Process and economic model Process parameters Deactivation causes Are extra costs such as immobilization possible? Reactor configuration Enzyme stabillization procedure started 12 DTU Chemical Engineering

13 Enzyme stability considerations during process design and scale-up PBR Reactor type Concentration gradient No mechanical disruption CSTR No concentration gradient Mechanical disruption Scale-up No concentration or temperature gradients Concentration ti and temperature gradients 13 DTU Chemical Engineering

14 Process development strategy Economic constraints Process constraints Scrutinize market/literature for available enzymes (active and stable within operational window) Operational window (upper and lower limits for temperature, ph, etc) Does biocatalysis seem feasible? Best available candiate(-s) Experiments in small scale with reaction media engineering, based on theoretically ti determined d conditions Process and economic model Process parameters Deactivation causes Are extra costs such as immobilization possible? Reactor configuration Enzyme stabilization procedure started Experiments according to model, in small/lab scale Remaining deactivation causes Optimal conditions for maximal productivity 14 DTU Chemical Engineering

15 The search for the deactivation mechanism Pioneering work by Sadana, A. (1988) Biotech. Adv First order, single step Series deactivation k 1 E E d k 1 k 2 E E 1 E d Reversible deactivation k 1 E E d k DTU Chemical Engineering

16 The search for the deactivation mechanism Example: oxygen supply Gas supply Mixing Bubble size E O 2 Case 1 E O 2 Case 2 16 DTU Chemical Engineering

17 Measurement of operational stability Half-life life Repeated batches Apparent stability Yield (%) 100 Suggestions Process-like conditions Report productivities Continuously operated tank reactor Fast procedures (e.g. T-ramping) Batch nr 17 DTU Chemical Engineering

18 Stabilization work case study Chemo-enzymatic epoxidation O epoxide H 2 O 2 O R1 R2 Lipase R olefin OH O H 2 O R O peracid OH R1 R2 Törnvall et al. (2007) Enzyme Microb. Tech DTU Chemical Engineering

19 Epoxidation - Stabilization efforts Type of enzyme Immobilization procedure Temperature H 2 O 2 concentration ti and addition time Site-directed mutagenesis Fatty substrate 80-fold improvement still needed Orellana-Coca et al. (2005) Biocatal Biotransfor Reactor design Törnvall et al. (2009) Ind. Biotech Hagström et al. (2011) Biotechnol. Prog Törnvall et al. Unpublished data. 19 DTU Chemical Engineering

20 Deactivation mechanism Intensity, coun nts Oxidized residues detected Intact active site residues Disrupted disulphide bridges Loss of secondary structure Aggregation Törnvall et al. (2009) Rapid Commun. Mass Spectrom Törnvall et al. (2010) Biochimie Ellipticity (mdeg) A( Wavelength (nm) log( / s) m/z, amu DTU Chemical Engineering

21 Take home messages Report stabilization work as improved productivity, and include cost estimization Screening procedures should always include the stability aspect Avoid suboptimizations 21 DTU Chemical Engineering

22 Take home messages Process engineering g is essential for implementation of bioprocesses Process engineering tools are required to assist in designing the biocatalyst We are always open to collaboration and student/faculty exchange 22 DTU Chemical Engineering

23 Acknowledgment People at DTU: Prof. John Woodley Dr. Pär Tufvesson Dr. Mathias Nordblad Hemalata Ramesh Stuart Tindal UT acknowledges the support of BIONEXGEN, financed through the European Union 7th Framework Programme (Grant agreement no.: ) Contact: 23 DTU Chemical Engineering