Front Matter

3.3 Recovery of lipases by microemulsion-based

extraction

Water-in-oil microemulsion solutions have been employed successfully to purify and

recover proteins from complex aqueous solutions through specific (electrostatic or

bioaffinity) or nonspecific (hydrophobic) interactions in a process known as micro-

emulsion-based liquid–liquid extraction. This body of research has been recently

reviewed (Pires et al., 1996). Cabral and co-workers have employed this method

to separate two different lipases from a crude protein mixture derived fromChro-

mobacterium viscosum(Camarinha Vicente et al., 1990; Aires-Barros and Cabral,

1991). Lipase A and B differed in their molecular weight (120 kDa and 30 kDa,

respectively) and their pI values (3.7 and 7.3, respectively). These workers em-

ployed AOT/isooctane solution as extractant, indicating an electrostatic driving

force. The protein mixture was solubilized in a pH 6 buffer in order for lipase B

to have a net positive charge to promote interaction with the anionic sulfonate

groups of AOT. Upon contact with the w/o-ME solution (forward extraction),

most of the Lipase B molecules were extracted away. However, all of the Lipase

A remained in the aqueous phase, presumably because the AOT w/o-MEs were

too small to host the large proteins. Hence, the degree of separation was quite good.

However, recovery of the w/o-ME-encapsulated lipase B was difficult. Cabral and

co-workers adopted a typical back-extraction procedure, consisting of contact with

an aqueous stripping solution at a pH that encourages electrostatic repulsion between

the protein’s charged surface groups and the surfactant head group, and high ionic

strength to induce Debye shielding of the surfactant head groups. This approach was

not successful, probably because hydrophobic interactions between lipase and AOT

were not interrupted. Hence, they included ca. 5 % polar alcohol (e.g., ethanol) to

disrupt the interfacial packing of the w/o-ME interface. This led to the release of

encapsulated lipase. However, the presence of polar alcohol in aqueous medium

is known to promote inactivation. Two approaches have been applied to improve

the recovery of lipases from w/o-ME solution: (i) the employment of a mixed

AOT/polyoxyethylene fatty acid ester (Tween 85) system (Yamada et al., 1994);

and (ii) the inclusion of guanidine hydrochloride or high ionic strength in the dis-

persed aqueous phase (Nagayama et al., 1999). With regard to the first approach, the

authors hypothesize that Tween improves back-extraction by reducing the surface

charge density of the interfacial sulfonate surfactant head groups (Yamada et al.,

1994). Regarding the second approach, although guanidine is a well-known dena-

turant, its presence at low-to-moderate amounts in the w/o-ME system did not reduce

the activity of encapsulated or recovered lipases (Nagayama et al., 1999).

58 3 Lipid Modification in Water-in-Oil Microemulsions