Food Lipids: Chemistry, Nutrition, and Biotechnology

ation. Fungal lipases typically exist as monomers with molecular masses ranging
from about 30 to 60 kDa. They vary in specificity, specific activity, temperature
stability, and other properties; however, dimeric lipases have been reported (Table
1) (16–17).
III. PRODUCTION, ISOLATION, AND PURIFICATION
A. Lipase Production
Microbial extracellular lipase production for laboratory study is typically carried out
under liquid shake culture conditions or in small fermentors. Conditions for optimum
lipase production seem to be variable depending on the species. Typically, triacylglycerols
(olive or soybean oil are commonly used) are placed in the culture medium
to induce lipase production (17,28,29), but fatty acids may also induce lipase production
(30–32). However, lipase production has been studied in the absence of
lipids (e.g., with sugars as the carbon source) (33). Chang et al. (34) reported that
Tween 80 and Tween 20 in the culture medium promoted lipase production and a
change in the multiple forms of lipase produced by Candida rugosa. The following
is a specific example of conditions for the maximal production of lipases by Geotrichum
candidum: growth for 24 hours in liquid medium containing 1% soybean
oil, 5% peptone, 0.1% NaNO3, and 0.1% MgSO4 at pH 7.0, 30�C and shaking at
300 rpm (11).
B. Isolation and Purification
Extracellular lipases have been isolated, purified, and characterized from numerous
microbial species. Some examples of recently characterized lipases are illustrated in
Table 1. The specific isolation and purification methodology differs in detail from
study to study, but it generally involves the ammonium sulfate precipitation of proteins
from the culture medium after removal of cells or mycelium, and then fractionating
the proteins through a series of ion exchange, affinity, and gel filtration
columns. Several recent publications can be consulted for specific procedures for
isolating lipases (16–21,35,36). The following is given as an example of a lipase
from Neurospora sp. TT-241 that was isolated and purified 371-fold (22).
1. Ammonium sulfate fractionation. Add solid ammonium sulfate to the clarified
aqueous extract (or culture medium) up to 60% saturation. The resulting
precipitate is collected by centrifugation at 12,000 � g for 30
minutes and dissolved in a minimal volume of buffer A (50 mM phosphate
buffer, pH 7.0). The enzyme solution is dialyzed overnight against a 50fold
excess of the same buffer.
2. Sephadex G-100 gel filtration chromatography. The enzyme solution (70
mL) is applied to a Sephadex G-100 column (4 cm � 120 cm), eluted
with buffer A at a flow rate of 40 mL/h, and 8 mL fractions are collected.
3. Toyopearl phenyl-650M column chromatography. Add ammonium sulfate
to the pooled active fractions from gel filtration to a final concentration of
1 M. The enzyme solution is then applied to a Toyopearl phenyl-650M
column (2.0 cm � 18 cm) that has been preequilibrated with buffer A
containing 1 M ammonium sulfate. The column is washed with 300 mL
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.