L LOSCHMIDT , LABORATORIES PROTEIN ENGINEERING BIOTECHNOLOGY, ENZYME APPLICATIONS, PROTEIN ENGINEERING APPROACHES Radka Chaloupková Loschmidt Laboratories Department of Experimental Biology Masaryk University, Brno 1/64 Outline □ Biotechnology □ Enzymes in technologies □ Enzyme applications □ Enzyme advantages and disadvantages □ Common targets of protein engineering □ Enzymes with desired properties □ Protein engineering, strategies of protein engineering □ Examples of protein engineering Protein Engineering 2/64 Biotechnology □ any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use Protein Engineering 3/64 5 □ mostly proteins, RNA (ribozyme) □ catalysis of chemical reactions □ lowering of activation energy = increasing of reaction rate □ non toxic substances □ catalysis under mild conditions □ high efficiency, easy regulation □ high specificity (functional specificity, substrate specificity) □ requirement of cofactors (oxi do reductases, transferases) Protein Engineering 4/64 Enzymes in technologies □ to manufacture both bulk and high added-value products - food and animal feed - fine chemicals - pharmaceuticals □ to provide services - housework - industry - environmental technologies - analytical purposes - diagnostics food enzymes A detergent enzymes technical enzymes r Distribution of sales in Novozymes Protein Engineering 5/64 Detergent industry □ laundry and dishwashing detergents □ proteases, lipases, amylases, cellulases □ enzymes reduce the environmental load of detergents products - save energy and C02 emissions by enabling lower wash temperatures - have no negative environmental impact on sewage treatment process - do not present a risk to aquatic life Computer simulation: A laundry detergent enzyme (red) attacks the soil (yellow) ^« on a textile fiber (gray). Protein Engineering 6/64 Food processing □ improvement of bread quality (alpha-amylases) □ production of sugars from starch (amylases) □ fruit juice and wine manufacture (pectinases, cellulases, amylases) □ brewing industry (enzymes from barley) □ milk industry (chymosin, beta-galactosidases, lactases) □ meat tenderizers (papain, bromelain) Protein Engineering 7/64 Paper industry □ xylanases reduce bleach required for decolorizing □ cellulases smooth fibers and enhance water drainage □ amylases degrade starch to lower viscosity sugars □ lipases reduce pitch Protein Engineering 8/64 Textile industry □ cellulases are used in denim washing for a stone-washed look □ amylases are used for desizing of textile fibers □ catalases are used for bleach clean-up □ laccases are used as bleaching agents Protein Engineering 9/64 Biofuel industry □ bacterial and fungal cellulases break down cellulose into sugars that can be fermented Biomass is harvested and delivered to the biorefinery. Enzymes break down cellulose chains into sugars. Microbes ferment sugars into ethanol. Protein Engineering 10/64 Synthesis of chemicals and pharmacei □ kiloton-scale production of acrylamide (nitrile hydratase) □ synthesis of 6-APA - precursor of antibiotics (penicilin amidase) O /b—N O penicillin G OH + H20 COOH COOH 6-amino penicillanic acid □ synthesis of single enantiomers - precursors of drugs (lipases) dopa (/?)- causes granulocytopenia (5)- anti-parkinson Protein Engineering 11/64 Specialty enzymes □ clinical analytical applications - glucose biosensor (glucose oxidase) - alkaline phosphatase and peroxidase immunoassays □ flavor production - production of glutamates used in food flavouring (glutamases) □ personal care products - contact lens cleaning (lipase, proteinase) - in toothpaste to convert glucose to H202 (glucose oxidase) □ DNA technology - restriction enzymes (restriction endonuclease) - DNA-modifying enzymes (ligase) Protein Engineering 12/64 □ use of microorganisms or their enzymes to return the environment altered by contaminants to its original condition □ examples of biodegradation enzymes and pollutants - monooxygenases - alkane, steroids, fatty acid and aromatic compounds - dioxygenases - phenolic and aromatic compounds - peroxidases - lignin and other phenolic compounds - lipases - organic pollutants such as oil spill - cellulases - cellulosic substances - haloalkane dehalogenases - halogenated hydrocarbons - proteases - proteins Protein Engineering 13/64 Advantages of enzymes □ high catalytic efficiency □ high degree of selectivity □ compatibility of each other □ reusability □ sustainability - produced from biomass - easily biodegradable - non-toxic and non-flammable - less byproducts and wastes - operate at mild conditions Protein Engineering 14/64 Disadvantages of enzymes □ generally less stable □ insufficient activity □ insufficient selectivity □ cofactor requirement □ allergies Protein Engineering 15/64 Common targets of protein enginee □ enzyme stability □ enzyme activity □ enzyme substrate specificity □ enzyme enantioselectivity Protein Engineering 16/64 Enzyme stability □ thermodynamic x kinetic stability Definitions of various stability parameters. Measure Symbol Type of stability Definition Free energy of unfolding Melting temperature Unfolding equilibrium constant Half-concentration Observed deactivation rate constant Half-life Temperature of half-in activation Optimum temperature Total turnover number T, in Ci /2 rtd,obs ^50 1 opt TTN Thermodynamic Thermodynamic Thermodynamic Thermodynamic Kinetic Kinetic Kinetic Kinetic Kinetic Change in Gibbs free energy going from the folded to unfolded state The temperature at which half of the protein is in the unfolded state The concentration of unfolded species divided by the concentration of folded species The concentration of denaturant needed to unfold half of the protein {chemical equivalent of Tm) Overall rate constant for going from native to deactivation species Time required for residual activity to be reduced to half Temperature of incubation to reduce residual activity by half during a defined time period Temperature leading to highest activity Moles of product produced over the lifetime of the catalyst Protein Engineering 17/64 Enzyme stability 18-15- 12- 9 t 61 (-)MRE/u 3 0 -3 -9 180 —r— 195 CD spectra Half-life 210 225 /Vom -*■ 240 255 100 ~i-1-1-1-1-r 0 5 100 200 300 400 500 600 t/h — Melting temperature Half-concentration Fal°/< 40 CoMSo/% Protein Engineering 18/64 Enzyme activity □ enzyme property measured by the increase in reaction rate □ reaction rate - concentration of product produced per time o '■4—> i_ cu u d o u 4—> u ■a o Time Protein Engineering 19/64 Enzyme activity Units of enzyme activity □ SI unit, katal (kat) - amount of enzyme that catalyzes conversion of 1 mole of substrate per second (mol.s-1) □ activity unit (U) - amount of enzyme that catalyzes conversion of lpmol of substrate per minute ((jmol.min-1), 1U = 16.67 nkat □ specific activity - activity of enzyme per milligram of total protein ((jmol.s^.mg-1) Protein Engineering 20/64 Enzyme substrate specificity □ definition of substrate specificity - discrimination between several substrates competing for enzyme active site □ commonly used meaning - enzyme activity with alternative substrate in the absence of specific (native) substrate □ enzyme activity measured towards a broad range of substrates under similar conditions □ fingerprint of enzyme ability to convert various substrates □ usually compared for different enzymes □ quantitative comparison of enzyme data - statistical analysis Protein Engineering 21/64 0.15 DpcA 0.10 0.05 A I l.l.ll. Substrates Enzyme substrate specificity □ statistical analysis of substrate specificity data - sorting of enzymes according to their preference to different substrates □ identification of unique SSGs within one enzyme family B LU "O CD -*—> ü CD non-homologous mutagenesis •epPCR •SeSaM homologous mutagenesis • gene shuffling screening genejsj of interest mutagenesis gene library CD _c CD CD C CD C LU c B o fx c w CD Q Öj c o • co E CD en I ..... information about hot spots saturation mutagenesis • ISM • CASTing Computational tools • HotSpot Wizard • QSAR • SCHEMA • "small but smart" mutagenesis gene library co c g 1 rr protein structure computer supported jaredjctais^nchTTu^ac^^ specific point mutations chimeric genes ene variant determination of specific activity, enantioselectivity, etc. Protein engineering □ altering the structure of existing protein to improve its properties □ overcome limitations of natural enzymes as catalysts □ basic understanding of how enzymes fuction and have evolved □ already point to many industrial successes □ *in the past, an enzyme-based process was designed around the limitations of the enzymes; today the enzyme is engineered to fit the process specifications " Protein Engineering 30/64 Protein engineering □ three main approaches of protein engineering - rational design - directed evolution - semi-rational design Protein Engineering 31/64 Rational design RATIONAL DESIGN 1. Computer aided design 2. Site-directed mutagenesis Individual mutated gene 3. Transformation 4. Protein expression 5. Protein purification 6. not applied IMPROVED ENZYME Constructed mutant enzyme 7. Biochemical testing Protein Engineering 32/64 Rational design □ site-specific changes on the target enzyme □ few amino-acid substitutions that are predicted to elicit desired improvements of enzyme function □ based on detailed knowledge of protein structure, function and catalytic mechanism □ relatively simple characterization of constructed variants □ factor limiting general application of rational design - complexity of structure-fuction relationship in enzymes Protein Engineering 33/64 Directed evolution DIRECTED EVOLUTION 1. not applied IMPROVED ENZYME 2. Random mutagenesis u Library of mutated genes ( > 10,000 clones ) 3. Transformation 4. Protein expression 5. not applied 6. Screening and selection - stability selectivity affinity 7. Biochemical testing Selected mutant enzymes Protein Engineering 34/64 Directed evolution □ large numbers of mutants randomly generated □ mimicking natural evolution processes □ evolution without knowledge of enzyme structure and function □ identification of functionally improved variants required powerful screening or selection □ limitation - necessity of developing a high-throughput screening Protein Engineering 35/64 Semi-rational design RATIONAL DESIGN DIRECTED EVOLUTION 1. Computer aided design 1. not applied 2. Site-directed mutagenesis Individual mutated gene 3. Transformation 4. Protein expression 5. Protein purification 6. not applied IMPROVED ENZYME Constructed mutant enzyme 7. Biochemical testing 2. Random mutagenesis u Library of mutated genes ( > 10,000 clones ) 3. Transformation 4. Protein expression 5. not applied 6. Screening and selection - stability selectivity affinity Selected mutant enzymes Protein Engineering 36/64 Semi-rational design □ also called focused directed evolution □ based on knowledge of structure and fuction of target enzyme □ combine advantages of rational and random approaches □ selection of promising target sites □ creation of small focused smart libraries □ elimination the need of high-throughput screening □ increase likelihood of beneficially modifying property Protein Engineering 37/64 Protein engineering approache: Comparison of protein engineering approaches Rational design Directed evolution Semi-rational design high-throughput screening/selection not essential essential advantageous but not essential structural and/or functional information both essential neither essential either is sufficient sequence space exploration low high, random moderate, targeted probability to obtain synergistic mutations moderate low high Protein Engineering 38/64 Process design criteria □ higher activity at process conditions □ increased process stability □ increased thermostability to run at higher temperatures □ stability to organic solvents □ absence of substrate and/or product inhibition □ increased selectivity (enantio-, regio-, chemo-) □ accept new substrate □ catalyse new reactions Protein Engineering 39/64 Target enzyme family □ haloalkane dehalogenases (HLDs) □ microbial enzymes - a/(3 hydrolases1 □ hydrolytic cleavage of C-X bond H H R H Enz HH R Enz OH H H Enz R □ broad substrate specificity □ high enantioselectivity □ potential applications: biodegradation, biosensing, biosynthesis 'Ollis, D.L. et al. : Protein Eng. 5, 197-211 (1992) Protein Engineering 40/64 Studied HLD DhaA from Rhodococcus rhodochrous Protein Engineering 41/64 Directed evolution epPCR DhaA 7 000 colonies ^ 4 positive hits Protein Engineering 42/64 Mutant resistant to DMSO DhaA 57 Protein Engineering 43/64 Mutant resistant to DMSO Protein Engineering 44/64 melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h) melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h) DhaA DhaA61 melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h) Gray, K.A. et al.: Adv. Synth. Catal. 343, 607-617 (2001) melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h) 1Gray, K.A. et al.: Adv. Synth. Catal. 343, 607-617 (2001) DhaA 82 melting temperature in buffer (°C) half-concentration of DMSO (%) half-life in 40% DMSO at 37 °C (h) 1Gray, K.A. et al.: Adv. Synth. Catal. 343, 607-617 (2001) Protein Engineering 50/64 Protein Engineering 51/64 Mutant resistant to DMSO DhaA wt DhaA 57 DhaA 80 Protein Engineering 52/64 □ resistance towards organic cosolvents correlates with thermostability □ mutations lining access tunnel modulate occupancy of active site by solvent and can stabilize protein □ robust catalysts (DhaA80) were developed: 4 point mutations, 7~m | 19 °C, t1/2 (40% DMSO) min days □ engineering of access tunnels represents novel strategy for engineering of robust catalysts Protein Engineering 53/64 DhaA80 □ 4 point mutations: T148L, G171Q, A172V and C176F □ r1/2 (40% DMSO) improved 4000-fold □ A7"m = 16 °c □ very low activity in buffer Protein Engineering 54/64 Protein Engineering 55/64 DhaA80 DhaA106 Protein Engineering 56/64 Stability and specific activity1 Variant DhaAwt DhaA63 DhaA80 DhaA106 Tm (°C) 50.4 ± 0.3 68.3 ± 0.3 66.8 ± 0.2 62.7 ± 0.1 Aqueous environment 32* 250-, 200- :> b 8 I 150- o CO =5 Ö 100- Q- c co — 50-0- 1 DhaA DhaA63 DhaA80DhaA106 40% DMSO 40-, :f £ 30- o E cd ^ o co 20 H— _1_ Ü o X E a. c 10- 0. 10x DhaA DhaA63DhaA80DhaA106 Measured with 1,2-dibromoethane Protein Engineering 57/64 Substrate specificity DhaA80 190 180 I % 60 o E & v 50 o co II 40 Q- c CO — 30 20 10 30 halogenated compounds Protein Engineering 58/64 Substrate specificity DhaA80 DhaA106 190 180 I % 60 o E & v 50 o tu II 40 Q- c CO — 30 20 10 Uli I j. i i I 1 1 L 30 halogenated compounds Protein Engineering 59/64 Substrate specificity DhaA80 DhaA106 DhaAwt 190 180 I % 60 o E & v 50 o tu II 40 Q- c CO — 30 20 10 ^4 ü ll 30 halogenated compounds Protein Engineering 60/64 7179 Protein Engineering 61/64 DhaAwt Bottelneck radius (Á) ND CO □ enzyme catalytic performance enhanced by fine-tuning the geometry and flexibility of its access tunnel □ tunnel residues are good targets to balance activity-stability trade-off of enzymes Protein Engineering 63/64 Helpful references □ Faber, K. (2011) Biotransformations in Organic Chemistry, Springer □ Soetaert, W. & Vandamme, EJ. (2010) Industrial Biotechnology, Wiley-VCH Verlag GmbH & Co. KGaA □ Kazlauskas, RJ. & Bornscheuer, U.T. (2009) Finding better protein engineering strategies, Nat. Chem. Biol. 5: 526-529 □ Davis, T. et al. (2013) Strategies for the Discovery and Engineering of Enzymes for Biocatalysis, Curr. Opin. Chem. Biol. 17: 1-6 □ Koudelakova, T. et al. (2013) Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel, Angew. Chem. Int. Ed. 52: 1959-1963 □ Liškova, V. et al. (2015) Balancing the stability-activity trade-off by fine-tuning dehalogenase assess tunnels, ChemCatChem 7: 648-659 Protein Engineering 64/64