Enzyme reactions mechanisms and Immobilization of enzymes

Transcription

1 Enzyme reactions mechanisms and Immobilization of enzymes Lecture CHEM-E3140 Bioprocess Technology II Aalto University School of Chemical Technology Ossi Turunen 1 Reaction mechanisms 1) General acid-base catalysis A molecule other than water plays the role of a proton donor or acceptor. 2) Covalent catalysis - Also sometimes called nucleophilic catalysis - Transient formation of a catalyst-substrate covalent bond 3) Metal ion catalysis Metal ions are often used for one or more of the following: - Binding substrates in the proper orientation - Mediating oxidation-reduction reactions - Isomerizations Metal ions can serve as electrophilic catalyst, stabilizing negative charge on a reaction intermediate (electrostatic catalysis) Metalloenzymes contain tightly bound metal ions: (usually Fe +2, Fe +3, Cu +2, Zn +2, or Mn +2 ) Metal-activated enzymes contain loosely bound metal ions: (usually Na +, K +, Mg +2, or Ca +2 ) 1

2 General acid-base catalysis: Used in the hydrolysis of glycosidic bonds and ester/ peptide bonds, phosphate group reactions, addition to carbonyl groups, etc. donate a proton (act as a general acid), or accept a proton (abstract a proton, act as a general base) What side-chains can donate or accept protons? Amino Acid Aspartic acid pk a 3.90 Glutamic acid 4.07 Histidine 6.04 Cysteine 8.33 Tyrosine Lysine COO - O H C CH C 2 NH + 3 O - COO - H C CH CH 2 2 NH + 3 H COO - C CH 2 NH + 3 COO - N H C CH SH 2 NH + 3 COO - H H C CH 2 NH + 3 COO - C CH 2 NH + 3 N C -COOH O O - OH -COOH imidazole sulfhydryl phenol + CH 2 CH 2 NH 3 -amino Chymosin protease is used in cheese production to precipitate casein protein. 2

3 Proteases cleave peptide bonds in amino acid chains (polypeptides) N-terminus C-terminus Catalytic Mechanism of Serine Proteases Serine in the name means that the protease has serine as the key catalytic amino acid Catalytic Triad Asp and His help Ser to be in correctly activated state to function as nucleophile 6 3

4 7 Lipases Lipases (triacylglycerolacyl hydrolases, EC ) catalyse the cleavage (hydrolysis) of fats (lipids) or even their formation. Jaeger and Reetz. TIBTECH SEPTEMBER

5 Catalytic Mechanism of Lipases Catalytic Triad D = Asp H = His S = Ser D H S Lid Lid is shown in open conformation. Opening of the lid is required for substrate to enter the active site, located deep inside the protein. Lipases are activated upon binding to a hydrophobic surface. Most active sites in enzymes are sequestered and somewhat hydrophobic to exclude water. Green molecule shows the position of substrate. 9 Asp and His help Ser to be in correctly activated state to function as nucleophile Serine is the key catalytic amino acid also in lipases. Serine undergoes rate-determining nucleophilic addition to the carbonyl, function to form the so-called oxyanion. Covalently bonded intermediate Reetz et al. ChemBioChem 2007, 8,

6 Summary of the reaction mechanism in proteases and lipases Activated serine (terminal oxygen in the side chain has negative charge = nucleophile) attacks the peptide bond/glycerol-fatty acid bond and cleaves it and forms a covalent bond to one half of the molecule (remaining group), and the other half is liberated (leaving group). Water molecule cleaves the covalent bond between the remaining group and serine, and then, the hydrolysis is complete. 11 Glycoside hydrolases hydrolysis Non-reducing end Reducing end 6

7 Substrate binding In glycoside hydrolases, the overal binding site for substrate is formed of several subsites that are numbered from m to +n (e.g ). Stacking interactions between sugars rings and aromatic rings and hydrogen bonds bind the substrate to the active site. Zolotnitsky et al PNAS 101: Some basic concepts α and β anomers of glucose. 14 7

8 Cellobiose in chair conformation 15 alpha-1,4 linkage Starch (amylose) beta-1,4 linkage

9 Bacillus circulans GH11 xylanase Xylanase (EC ) is a glycoside hydrolase cleaving the glycosidic bonds in beta-1,4-xylan, formed of xylose units Acid/base Nucleophile Correct conformation and ionization state of the catalytic residues (acid/base and nucleophile) is maintained by bonding or long-distance interaction to nearby amino acid side chains. 9

10 Cellobiohydrolase hydrolyses cellulose by cleaving off cellobiose (Glu-Glu) from the end. Cellobiohydrolase, 7CEL Active site tunnel with two catalytic Glu amino acids (red). Long substrate binding tunnel. Retaining and inverting mechanism 10

11 5.5 Å between catalytic residues oxocarbenium ion-like transtion states For details of the mechanism, see Glycoside hydrolases, retaining mechanism Free monosaccharides Covalent intermediate stage 11

12 sugar chain Nucleophile attacks the anomeric centre to displace OR group and form a glycosyl enzyme intermediate => covalent bond between nucleophile and remaining glycosyl group. The acid catalyst protonates the glycosidic oxygen as the bond cleaves. As a result HOR (or ROH) group is liberated. = sugar chain or other group glycosidic oxygen anomeric centre (carbon) Å between the catalytic residues: therefore, there is place for water molecule between the substrate and negatively charged catalytic amino acid For details of the mechanism, see

13 Cellulose Glycoside hydrolases bind the substrate especially by hydrogen bonds (green dotted line) and planar interaction between the aromatic rings and sugar molecules

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15 Enzyme immobilization 29 Glucose measurement utilizing immobilised enzyme 15

16 Reasons for immobilization Reuse of enzyme, reducing cost Operation life time > 1000 h Continuous processing Facilitated process control Low residence time (high volumetric activity) Optimization of product yield Easy product separation and recovery Stabilization by immobilization Increase of enantioselectivity 16

17 Commercial advantages of immobilization: the enzyme is easily removed the enzyme can be packed into columns and used over a long period speedy separation of products reduces feedback inhibition thermal stability is increased allowing higher temperatures to be used higher operating temperatures increase rate of reaction 33 Limitations Cost of carriers and immobilization Mass transfer limitations Problems with cofactors and regeneration Problems with multienzyme systems Changes in properties (selectivity) Activity loss during immobilization 17

18 Immobilization carriers Inorganic High pressure stability Abrasion in stirred vessel Polysaccharides Wide network structure Hydrophilic: enzyme friendly, no strong effect on enzyme Synthetic polymers High chemical stability Several slides are from the thesis of Piia Hara (Univ. of Turku, 2011): and from Buchholz et al. Biocatalysts and Enzyme Technology 18

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20 Linking by glutaraldehyde Porous silica or glass is treated with aminopropyl triethoxysilane in order to introduce amino groups. Carrier is then activated by glutaraldehyde (GA), followed by enzyme binding. GA 20

21 Optimal pore diameter Pore sizes Microporous: below 2 nm, Mesoporous: 2-50 nm Macroporous: over 50 nm Pure Appl. Chem., vol 73, no2, pp , 2001 (IUPAC) Size of lipase is 3-5 nm (= Å). In adsorption of lipases, the obtained activity depends on the pore size in the range below 100 nm. Above this value, activity is independent of the pore size. Activity of enzymes immobilized on carriers with pore size below 100 nm is lower and strongly related to enzyme loading and pore size, whereas pore size >100 nm results increased accessibility of pores. 21

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23 Preparation of Cross-linked enzyme aggregates (CLEA) Synthesis of sol-gels. tetramethoxysilane methyltrimethoxysilane Conventional sol-gel entrapment refers to a process where enzyme is mixed with sol-gel solution, followed by gelation process under the influence of ph and aging process. 23

24 Aquagel: Pores of the gel filled with water and alcohol Drying of sol-gels Shrinking of gel Supercritical CO 2 drying with or without acetone dialysis The particle size and V max of lipase PS in CLEA. Glutaraldehyde concentration [mm] Particle diameter [ m] V max [mmol mg -1 h -1 ] 10 < Large clusters

25 Protein surface can also be modified by mutations to direct the immobilization. - Covalent attachment to carrier via Lys, Arg, Asp, Glu - Attachment of protective groups (spacers) on the protein surface (increase stability) 50 25

26 Enzyme and immobilization parameters affecting the efficient immobilization. Size of an enzyme Stability of an enzyme under immobilization conditions Conformational flexibility Isoelectric point Surface functional groups Glycosylation Additives in enzyme preparation Immobilization time Immobilization ph - optimal to enzyme Immobilization temperature Immobilization buffers Nature and properties of carrier Reaction medium Optimal conditions for immobilization found typically by trial and error! In general, the activity of the immobilized enzymes can be enhanced by at least ten different effects involved in enzyme immobilization: microenvironment effect, partition effect, diffusion effect (reducing the ph), conformational change, flexibility of conformational change, molecular orientation, water partition (especially in organic solvent), conformation flexibility, conformation induction, and binding mode. Introduction: Immobilized Enzymes: Past, Present and Prospects 26

27 Stability of immobilised enzyme depends on the properties of its interaction with the carrier, the binding position and the number of the bonds, the freedom of the conformation change in the matrix, the microenvironment in which the enzyme molecule is located, the chemical and physical structure of the carrier, the properties of the spacer (for example, charged or neutral, hydrophilic or hydrophobic, size, length) linking the enzyme molecules to the carrier, and the conditions under which the enzyme molecules were immobilized. Rigidity of enzyme -> Smaller mobility restricts flexibility in the active» affects reaction accuracy (enantioselectivity, side reactions) the mobility increases at elevated temperatures, and the side reactions are also increased Shakeri et al 2010: Enantioselectitivity (E) may decrease at high temperature» 22 o C : over 500» 90 o C: only

28 CLEC crosslinked enzyme crystals Pure crystallized enzymes are crosslinked, e.g. by glutaraldehyde -> Insoluble High operational stability in both aquaeous and organic systems Diffusion of substrates and products through microscopic channels of 2-5 nm diameter Reaction and separation on the same column There can be diffusion limitation Barbosa et al. RSC Adv., 2014, 4, Preparation of crosslinked enzyme crystals (CLECs)

29 Preparation of crosslinked enzyme aggregates (CLEAs). Barbosa et al. RSC Adv., 2014, 4,