Front Matter

solution, can affect the lid conformation. The lid appears to be in dynamic motion,

exposing the active site even in the absence of an oil-water interface. Stabilization of

the open configuration by such an interface, or by similar chemical structures, results

in the appearance of appreciable lipolytic activity. Thus, these studies of the Rd

lipase structure refined and expanded notions of the dynamics of lid conformation

in the lipases, and helped to unify previously disparate theories regarding the roles

and importance of enzyme structure and substrate structure in regulating lipase ac-

tivity.

4.5 Cloning and characterization of an expressed

R. delemarlipase gene

Perhaps the most effective method of increasing the availability of an enzyme is

through the cloning and expression of its corresponding gene. Cloned genes are

also the basis of enzyme modification via directed mutagenesis. For these rea-

sons, the cloning of the Rd lipase gene was undertaken. From the outset, the

goal was not only to isolate the gene but also to achieve its expression, and in

this the project differed from other lipase cloning projects, which achieved cloning

but lacked expression.

The initial approach involved construction, inE. coli, of a library of totalR. de-

lemarDNA and screening for direct expression of the lipase gene. However, direct

expression was not detected (Haas et al., 1990). This most likely was due to an

inability ofE. colito read the transcriptional or translational control signals of

the fungus, since subsequent work (W. Friesen, and W. Baker, unpublished re-

sults) demonstrated the absence of intervening sequences in the lipase gene.

Subsequently, a complementary DNA (cDNA) cloning strategy was undertaken

(Haas et al., 1991). Bulk RNA was prepared from anR. delemarculture growing on

glycerol and producing lipase. The messenger fraction isolated from this RNA by

affinity chromatography served as the template for synthesis of double-stranded

cDNA. DNA linkers containing recognition sites for the restriction enzymeEcoRI

were ligated onto the ends of these cDNA fragments, which were then joined by

ligation to bacteriophagekgt11 arms.E. coliY1090r-was infected with intact bac-

teriophage produced byin vitropackaging of these DNA molecules, and plated as a

lawn on solid growth media containing olive oil and rhodamine B. [Rhodamine B is a

pink fluorescent dye. In the presence of free fatty acids its fluorescence wavelength is

shifted and the fluorescence intensity increases, resulting in a strikingly ‘hot pink’

signal that is an unmistakable indication of the presence of lipolytic activity (Kouker

and Jaeger, 1987)]. Since the color intensity increases with continued enzyme ac-

tivity, it is useful for the detection of very low levels of lipase. In addition to its

application in gene screening, we have employed the dye to locate lipase quickly

in fractions collected from chromatography columns, and to identify lipolytic pro-

tein bands on nondenaturing electrophoresis gels, as shown in Figure 2.

Following overnight incubation of rhodamine B-olive oil plates containingk-in-

fectedE. coli, no fluorescence could be detected among the lytic plaques formed in

the bacterial lawn. However, after additional days of incubation a few fluorescent

76 4 Cloning, Mutagenesis, and Biochemical Properties