ing resin had a high affinity and capacity for lipase. A small column (20 mL bed
volume) quantitatively bound lipase from 20–30 L of culture supernatant. Lipolytic
activity was released from this column as a single peak by elution with a gradient of
Triton X-100 from 0 to 0.5 % (v/v), resulting in a seven-fold increase in specific
activity. Denaturing gel electrophoresis followed by silver staining indicated that
this step reduced the number of peptides in the sample from about 20, in the crude
enzyme preparation, to four (Figure 1).
Ion-exchange chromatography of the affinity-purified lipase on carboxymethyl-
Sephadex, with elution by a gradient of sodium chloride from 0 to 0.25 M, produced
a lipolytically active sample containing a single polypeptide band of molecular mass
30.3 kDa (Figure 1). Nondenaturing isoelectric focusing, followed by activity stain-
ing, verified that this polypeptide was lipolytically active, and had an isoelectric
point of 8.6 (Figure 2).
The pure lipase displayed maximum hydrolytic activity between pH 7.5 and 8.5. It
was most active between 25 and 35 8 C, and was rapidly inactivated above these
temperatures. The purified enzyme contained less than one saccharide molecule
per molecule of enzyme. This is unusual, in that extracellular fungal proteins often
contain significant degrees of glycosylation. However, it is possible that endogenous
hydrolases are present in the crude enzyme preparation, and that these deglycosy-
lated the lipase during the affinity chromatography step, which was conducted at
room temperature and took several days. The enzyme was not inhibited by disulfide
reducing agents. Cations such as calcium, barium or manganese were required for
full activity, which was manifested above a cation concentration of 10 mM. This
cation requirement is a general feature of lipases, and is often attributed to a masking
by the ion of electrostatic repulsions between the enzyme and either its emulsified
substrate or product fatty acids.
72 4 Cloning, Mutagenesis, and Biochemical Properties
Figure 1. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the extracellular Rd lipase at
various stages of purification (Haas et al., 1992). Proteins were detected by silver staining. Lane a, 2.25lg
of protein molecular mass marker preparation; lane b, 37.5lg of culture filtrate; lane c, 3lg of pooled
lipase-positive fractions eluted from oleic acid affinity chromatography column; lane d, 1lg of pooled
lipolytic material obtained by carboxymethyl–Sephadex column chromatography. The masses of the
molecular weight marker proteins (kDa) are indicated in the left margin.