Front Matter

11.4 Immobilized lipases

Lipases are normally used in an immobilized form in industry because reuse or

continuous use of the lipase is made possible, and the separation of the lipase

easy. The stability of the lipase is often increased by immobilization. The advan-

tages of the various types of available enzyme reactors can also be more readily

exploited by using immobilized lipases, especially the use of packed-bed reac-

tors. However, there are some shortcomings of immobilization. Some enzyme ac-

tivity may be permanently lost during immobilization, due to irreversible denatura-

tion generated during the immobilization procedure, especially if this is achieved

chemically. Immobilization is an additional operation, and adds cost to a process;

the immobilized biocatalyst particles also always occupy a larger volume in reactors

than do the equivalent amounts of free enzyme due to the presence of the carrier. The

effect of immobilization on the activity in general can be expressed as ‘effectiveness

factor’ (g). This represents the activity of the immobilized enzyme divided by the

enzyme activity of an equivalent quantity of free enzyme assayed under the same

conditions. An effectiveness factor of 1.0 indicates good reaction control, with no

appreciable reduction in the activity of the enzymes by immobilization or diffusional

restrictions. Values less than 1.0 give an indication of the amount of activity lost

during immobilization, plus the extent of diffusion limitation on enzyme activity.

Four main factors have been identified which modify the intrinsic properties of en-

zymes, either during or after immobilization:

1. Conformational effects are caused by the chemical modification of the enzyme

protein during immobilization. These effects can be especially serious on the

enzyme activity when amino acid residuals, which form part of the active site

or are important in maintaining the tertiary structure of the enzyme, are involved.

2. Steric effects occur because some of the enzyme molecules are often immobilized

in a position that the active site is relatively inaccessible to substrate molecules.

3. Microenvironmental effects occur because of the difference of hydrophobicity

and electric charges that cause partitioning effects between different components

in the reaction mixture towards the enzyme support. For example, hydrophilic

substrates will be selectively attracted to the surface and pores of hydrophilic

support. Similarly positively charged substrates will be attracted into negatively

charged supports, giving a local high substrate concentration and low pH inside

the support.

4. Lastly, diffusional restrictions occur because the substrate must diffuse to the

immobilized enzyme before reaction can take place. Diffusional limitations

on the activity of immobilized enzymes are of the greatest importance because

they considerably affect the design of enzyme reactors and process operations

(Gacesa and Hubble, 1987; Prenosil et al., 1987; Chaplin and Bucke, 1990).

Theexternal diffusional limitationsare due to the restricted rate of diffusion of sub-

strate in the thin film of poorly mixed fluid surrounding each enzyme particle. Ex-

ternal diffusional restrictions can be decreased by increasing the degree of agitation

in stirred-tank reactors or the flow rate in packed-bed reactors, or by using less vis-

11.4 Immobilized lipases 199