Front Matter

position and arrangement mimic those of human milk fat lipids (Quinlan and Moore,

1993). However, at the time that the research described here was initiated, the

R. delemar(Rd) lipase and other 1,3- specific enzymes were not generally available

commercially, and very little was known concerning the basic biochemical features

or capabilities of lipases in general. The research program described here was under-

taken to fill these gaps. It was one of the first of what have now become several

thorough studies of lipolytic enzymes, and illustrates the strengths of combining

biochemical and molecular biological approaches to achieve advances in basic

and applied enzymology.

4.2 Simplification of lipase production

Lipases are enzymes of secondary metabolism, designed by nature to initiate the

metabolism of lipids when they become available in the environment. Therefore,

lipase production is often regulated in response to the presence of lipids in the

growth medium. In the production of microbial lipases it is standard practice to

grow the microorganism in liquid medium containing fats or oils to induce lipase

production. This approach has disadvantages, among them the limitation of nutrient

and energy availability due to the low solubility of the carbon source, difficulties in

maintaining the lipid in an emulsified state in the culture, and the fact that the lipids

make the growth medium turbid, which hinders optical or spectral assessment of the

growth of the organism. In addition, residual glycerides and free fatty acids from the

medium contaminate the lipase preparation and can complicate characterization or

purification. We postulated that metabolites of glycerides might serve as inducers of

lipase production, and explored the ability of glycerol, a fully water-soluble ultimate

product of glyceride hydrolysis, to foster lipase production. UsingR. delemarATCC

34612 (American Type Culture Collection, Rockville, MD, USA), it was found that

the use of glycerol as a carbon source not only supported lipase production, but

stimulated it by as much as three-fold relative to the use of glucose or triglyceride

(Haas and Bailey, 1993). This provided a simple method for the ready production of

crude lipase preparations that were uncontaminated by lipid and thus optimally sui-

ted for subsequent biochemical characterization of the lipase.

4.3 Purification and characterization of the Rd lipase

Complete purification of an enzyme may not be necessary for its use as an applied

catalyst. However, purification and characterization can identify reaction conditions

that give optimal activity. Such information facilitates efficient biotechnological

application of the enzyme. Thus, the development and implementation of a purifica-

tion scheme for the Rd lipase was undertaken (Haas et al., 1992).

Affinity chromatography played a key role in purification of the enzyme. An

affinity resin was produced by coupling oleic acid molecules through their car-

boxylic acid groups to free amino groups of a polyacrylamide gel resin. The result-

4.3 Purification and characterization of the Rd lipase 71