Enzyme immobilization: fundamentals and application Xiaoli wang 2013.11.30

Enzyme immobilization:

fundamentals and application

Xiaoli wang2013.11.30

Enzyme ImmobilizationEnzyme Immobilization

• enhanced stability • ease of separation

Advantages

Disadvantages• lowered activity

• additional costs

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.



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.

controlled pore glass (CPG) 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.

Sol–gel materials 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.

Mesoporous silicates 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.

Functionalised mesoporous materials

Functionalised mesoporous materials

In addition to pure silicate materials, a wide range of functionalised

MPS can be prepared.

Post-synthesis modificationDirect functionalisationIncorporation of bridged silesquioxanes

Immobilisation of enzymes on poroussilicate materials

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.

Conclusions 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.

Enzymatic reactions on immobilised substrates


1. Radioactive labelling

2. Fluorescence

3. Electrochemical detection

4. Mass spectrometry

5. Surface plasmon resonance (SPR)

6. Atomic force microscopy (AFM)

7. Quartz crystal microbalance (QCM)

The following analytical techniques were available to

monitor enzyme activity on surfaces.

Chem. Soc. Rev., 2013, 42, 6378--6405



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

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

Immobilisation and application of lipases in organic media

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

Effects of reaction conditions on lipase-catalysed reactions

Effects of reaction conditions on lipase-catalysed reactions

Factors cause the differences in enzyme activity Method of enzyme preparation (immobilisation, etc.) The quantity of water The kind of solvent The pH

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.

Lipase immobilisation 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

Adsorption Adsorption

Adsorption on inorganic supports. Adsorption on mesoporous silica. Adsorption on organic polymers. Organic solvent rinsing as an alternative drying method. Protein-coated microcrystals.

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 porous hydrophobic supports is a very useful method for lipase immobilisation.

Entrapment Entrapment Entrapment in sol–gel materials. Entrapment in organic polymers.

Covalent coupling Covalent couplingCovalent 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 Cross-linkingTypical 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 aggregatesCross-linked enzyme crystals

Surfactant-based lipase preparations

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.

Ion-paired lipasesSurfactant-coated lipasesLipases in microemulsionsMicroemulsion-based organogels

These methods can be classified into:

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.



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)

Applications involving lipase-catalysedreactions


• Ester synthesisThe majority of applications of lipases in organic media are in the preparation of esters, and a variety of esters has indeed been prepared.

• TriacylglycerolsStructured lipids, Interesterification of triacylglycerols

Lipases are excellent tools for the synthesis of triacylglycerols or the modification of existing triacylglycerols in organic media.



• Enantiomer resolutionThe lipase converts one of the enantiomers into a product that can easily be separated from the unreacted isomer.

• Fatty-acid enrichmentLipases can also be used for the enrichment of other isomers or closely related substances, provided that they have the appropriate substrate specificity.

• BiodieselTriacylglycerols are the normal substrates, lipases express high activity in biodiesel production.

• Phospholipid conversion• Carbohydrate modification• Increasing the lipophilicity of bioactive compounds

In which applications are lipases attractive?

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.

Which is the best method of lipase immobilisation?

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 immobilisationNo enzyme leakage should occur after immobilisationBe present in fully activated formMass 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.

Thank you for your attention

谢谢！

Thank you for your attention

谢谢！

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Enzyme immobilization: fundamentals and application Xiaoli wang 2013.11.30