Enzyme immobilization: fundamentals and application

1. Enzyme immobilization: fundamentals and application Xiaoli wang 2013.11.30

2. Enzyme Immobilization Advantages • enhanced stability • ease of separation Disadvantages • lowered activity • additional costs

3. Immobilization of enzymes on mesoporous silicate materials Microporous (<2nm), mesoporous (2-50nm), macroporous (>50nm) BET: adsorption–desorption isotherms obtained can be classified into six types, I–VI. TEM TEM can provide information on the size of pores and of the pore walls as well as the degree of short range and long range order of the material. XRD XRD can be used with materials containing ordered mesoporous channels to ascertain if the structure is hexagonal, cubic, lamellar or disordered.

4. Immobilization of enzymes on mesoporous silicate materials Advantages of mesoporous materials • ordered pore structures, • narrow pore size distributions, • high surface areas • high stability and can be • chemically modified with various functional groups. Methods Enzymol., 1976, 44, 776.

5. controlled pore glass (CPG) The first report on the immobilisation of enzymes on controlled pore glass (CPG) was described by Weetall. CPG with pore sizes ranging from 10 to 300 nm are commercially available, and can be prepared with particle sizes of 100 mm, a size suitable for use in packed bed reactors or columns. A major disadvantage of the material was the surface area rapidly decreases with increasing pore size. Methods Enzymol., 1976, 44, 776.

6. Sol–gel materials Sol–gels are formed via hydrolysis and condensation of a precursor species such as Si(OC2H5)4, Enzyme encapsulation occurs by placing the enzyme in the reaction mixture. While the production of ethanol may be detrimental to the activity of the enzyme. Encapsulation introduces a diffusion barrier which can reduce the rate of delivery of substrate to the enzyme and the rate of removal of the product. Anal. Chim. Acta, 2002, 461, 1–36.

7. Mesoporous silicates Zeolites are among the most commercially important porous materials and in widespread use in a range of industrially important processes. The synthesis of mesoporous silicates (MPS) was first described in 1971. MPS are formed using surfactants, which act as structure directing agents. Principles and Practice of Heterogeneous Catalysis, VCH, Weinheim, 1997. US Pat., 3556725, 1971.

8. Functionalised mesoporous materials • In addition to pure silicate materials, a wide range of functionalised • MPS can be prepared. • Post-synthesis modification • Direct functionalisation • Incorporation of bridged silesquioxanes

9. Immobilisation of enzymes on poroussilicate materials • Adsorption • Adsorption of enzymes on to MPS supports is controlled by the pore dimensions, surface charge and composition of the support together with the size, surface charge distribution and hydrophilic/hydrophobic nature of the enzyme. • Covalent immobilisation of enzymes • Covalent attachment of the enzyme to the surface can obviate the problem of leaching, increase the stability and enable reuse of the immobilised enzyme.

10. Conclusions Mesoporous silicates are attractive materials for use as supports of the enzymes immobilisation. The synthesis of MPS is relatively straight forward and produces materials with well defined and ordered pore structures, high surface areas and good mechanical and chemical stability. Surface functionalisation of MPS can be utilised to produce a material that can be tailored to suit the immobilisation of a particular enzyme.

11. Enzymatic reactions on immobilised substrates The following analytical techniques were available to monitor enzyme activity on surfaces. Radioactive labelling Fluorescence Electrochemical detection Mass spectrometry Surface plasmon resonance (SPR) Atomic force microscopy (AFM) Quartz crystal microbalance (QCM) Chem. Soc. Rev., 2013, 42, 6378--6405

12. Enzymatic reactions on immobilised substrates Not all enzymes are suitable for reactions on solid support, in particular where active sites are deeply buried in the enzyme structure. So far, there are no general rules that would allow predictions on activity on the surface. However, certain classes of enzymes have emerged as being very successful. Chem. Soc. Rev., 2013, 42, 6378--6405

13. An overview of all successful enzymatic transformations on surfaces is provided as followed. • DNA polymerase, Hydrolases, Transferases • Kinases, Phosphatases, Proteases • Acetylases, Methyltransferases, Ligases • Glycosyltransferases • Lipases • Other hydrolases This review has tried to compile the most common enzymatic reactions performed on surfaces and address issues of surface material and analytical readout. Chem. Soc. Rev., 2013, 42, 6378--6405

14. Immobilisation and application of lipases in organic media The natural reaction of lipases is to hydrolyse ester bonds. lipases are excellent examples of ‘‘promiscuous’’ enzymes, catalysing reactions quite different from the normal ones. Lipase-catalysed aldol addition, racemisation and epoxidation constitute a few interesting examples. Chem. Soc. Rev., 2013, 42, 6406--6436

15. Effects of reaction conditions on lipase-catalysed reactions Factors cause the differences in enzyme activity The catalytic activity of enzymes in organic solvents is sometimes several orders of magnitude lower than in water, making practical applications unattractive. However, the situation can be improved by choosing the appropriate conditions for biocatalysis in organic solvents. • Method of enzyme preparation (immobilisation, etc.) • The quantity of water • The kind of solvent • The pH

16. Lipase immobilisation Lipases can be immobilised using most of the methods developed for enzyme immobilisation in general. The major types of immobilisation: • Adsorption • Entrapment • Covalent coupling

17. Adsorption Physical adsorption is the simplest method of enzyme immobilisation. In the case of lipases, hydrophobic interaction is most common, but ionic interactions with ionexchange materials, etc., can also be useful. • Adsorption on inorganic supports. • Adsorption on mesoporous silica. • Adsorption on organic polymers. • Organic solvent rinsing as an alternative drying method. • Protein-coated microcrystals. Adsorption on porous hydrophobic supports is a very useful method for lipase immobilisation.

18. Entrapment • Entrapment in sol–gel materials. • Entrapment in organic polymers. • Covalent coupling Covalent coupling to a solid support is the most typical way of immobilising enzymes, and a variety of such methods has been described. There are several commercially available activated support materials intended for covalent enzyme immobilisation. • Cross-linking Typical cross-linked preparations contain the enzyme as the main constituent, but cross-linking is also used in combination with other immobilisation methods, such as adsorption, to prevent enzyme leakage. Cross-linked enzyme aggregates Cross-linked enzyme crystals

19. Surfactant-based lipase preparations Surfactants can activate lipases in different immobilisation procedures, probably by increasing the fraction of the open, active lipase conformation. In addition, surfactants can be used as the main agent for the preparation of lipases for use in organic media. These methods can be classified into: • Ion-paired lipases • Surfactant-coated lipases • Lipases in microemulsions • Microemulsion-based organogels

20. Water dependence of lipase-catalysedreactions Water is always present, even when using organic solvents as reaction media for lipase-catalysed reactions. It was observed that the catalytic activity of enzymes correlated well with the amount of water bound to the enzyme. There is a large variation among the enzymes concerning how the water in organic media influences their catalytic activity.

21. Water dependence of lipase-catalysedreactions Water has both positive and negative effects on the rate of lipase-catalysed reactions. • Positive effects of water • General activation due to increased internal flexibility of the enzyme • Increased active site polarity • Increased proton conductivity • Functions as substrate (increases hydrolysis only) • Negative effects of water • Inhibition (interference with substrate binding) • Formation of a diffusion barrier for hydrophobic substrates • Causes hydrolysis which competes with the desired reaction (esterification, transesterification)

22. Applications involving lipase-catalysedreactions • Ester synthesis The majority of applications of lipases in organic media are in the preparation of esters, and a variety of esters has indeed been prepared. • Triacylglycerols • Structured lipids, Interesterification of triacylglycerols Lipases are excellent tools for the synthesis of triacylglycerols or the modification of existing triacylglycerols in organic media.

23. Applications involving lipase-catalysedreactions • Enantiomer resolution The lipase converts one of the enantiomers into a product that can easily be separated from the unreacted isomer. • Fatty-acid enrichment Lipases can also be used for the enrichment of other isomers or closely related substances, provided that they have the appropriate substrate specificity. • Biodiesel Triacylglycerols are the normal substrates, lipases express high activity in biodiesel production. • Phospholipid conversion • Carbohydrate modification • Increasing the lipophilicity of bioactive compounds

24. In which applications are lipases attractive? • Lipase-catalysed reactions have proven to be better than other approaches for the preparation of many products. The most thoroughly studied applications are those that have been realised on an industrial scale. • The regioselectivity of lipases makes them the natural choice as catalysts for the production of structured lipids. • The mild reaction conditions which lead to fewer side reactions, higher product yields and less waste. • Product purification is simplified by the reduction in by-products. • Less environmental impact than alternative methods, which is naturally desirable.

25. Which is the best method of lipase immobilisation? • The following are important in obtaining an immobilised lipase preparation that uses the enzyme as efficiently as possible. • No enzyme inactivation should occur during immobilisation • No enzyme leakage should occur after immobilisation • Be present in fully activated form • Mass transfer limitations should be negligible The first two are sometimes difficult to combine. Covalent coupling to a support or covalent cross-linking are the best ways to avoid enzyme leakage, but they often cause enzyme inactivation. Non-covalent immobilisation is sufficient because the lipase is not soluble in the reaction medium, but remains in the immobilised preparation. The best approach may be not to immobilise the lipase, but to use surfactants to form hydrophobic ion pairs, surfactant-coated lipases or microemulsions. However, in these cases there is a need for a more sophisticated separation step to separate the enzyme from the product mixture.

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