JOURNAL OF F~RMENTATION AND BIOENGINEElUNO

Vol. 75, No. 5, 349-352. 1993

Purification and Characterization of a Novel Solvent-Tolerant

Lipase from Fusarium heterosporum
YUJI SHIMADA, l* C H I G U S A KOGA, 2 AKIO SUGIHARA, 1 T O S H I H I R O N A G A O , ' NOBUO TAKADA, 3
SUSUMU TSUNASAWA, 4 AND YOSHIO T O M I N A G A 1
Osaka Municipal Technical Research Institute, 1-6-50, Morinomiya, Joto-ku, Osaka 536,1 Sonoda Women's
College, 7-29-1, Minamitsukaguchi-cho, Amagasaki 661, 2 Katayama Chemical Inc., 1-6-27,
Higashiawaji, Higashiyodogawa-ku, Osaka 533, 3 and Institute for Protein Research,
Osaka University, 3-2, Yamadaoka, Suita 565, 4 Japan
Received 6 January 1993/Accepted 13 February 1993

A microorganism producing a solvent-tolerant lipase was identified as Fusarium (F.) heterosporum. The
lipase was purified from the culture filtrate to homogeneity as judged by disc-PAGE and SDS-PAGE. The
purification included SP-Sephadex chromatography, gel filtration and isoelectric focusing, and the recovery
yield was 38Yo. The lipase was a monomeric protein with a molecular weight of 31 kDa estimated by SDS-
PAGE, and a pI of 7.0. The optimum pH at 40°C and optimum temperature at pH 5.6 were 5.5-6.0 and 45-
50°C, respectively, when olive oil was used as the substrate. The iipase was stable over a pH range of 4-10 at
30°C for 4 h, and up to 40°C at pH 5.6 for 30 rain. Furthermore, the enzyme was not inactivated even after
incubation at 30°C in 50Yo solvent such as dimethylsuifoxide (DMSO), hexane, benzene and ether for 20 h.
The activity did not decrease in a reaction with stirring in a mixture containing 5 0 ~ DMSO or dimethyiform-
amide. The lipase preferably reacted on middle-chain fatty acid triglycerides (6<_C<12), and cleaved only
1,3-ester bonds of triolein. The enzyme had an N-terminai sequence of Ala-VaI-Thr-Vai-Thr-Thr-Gin-Asp.
Leu-Ser, which has not previously been found in any other protein. We compared the properties of lipases
from F. heterosporum and another strain F. oxysporum.

Lipase [EC 3.1.1.3] is characterized by the ability to cata-
lyze the hydrolysis of triglyceride at the interface between
oil and water (1-3). It is well known that the reaction is
reversible, and that the enzyme can catalyze ester syn-
thesis and transesterification in reaction systems containing
a low concentration of water. Furthermore, lipase can act
even in nearly anhydrous organic solvents, and catalyzes
stereoselective and regioselective reactions. These proper-
ties attract much attention, and the lipase is used prac-
tically as a catalyst in the oleochemistry and organic
chemistry fields. Since these applications of lipase are ex-
pected to expand in the near future, it is very important
to search for lipases which are peculiar to substrate spec-
ificities and stable at high temperature and in organic
solvents.
As previously reported, we isolated a filamentous fun-
gus producing a solvent-tolerant lipase from soil, and de-
termined the culture conditions for the production of the
enzyme (4). The present paper deals with the purification
and characterization of the solvent-tolerant lipase pro-
duced by the strain identified as Fusarium heterosporum.
MATERIALS AND M E T H O D S
Microorganism and culture conditions A microor-
ganism isolated from soil as a lipase producer (4) was used
and cultivated in a medium (pH 5.5) containing 3% soy-
bean oil, 4% corn steep liquor, 0.1% yeast extract, 0.1%
KH2PO4 and 0.05% MgSO4.7H20. The cultivation was
carried out at 27°C for 65 h in 500 ml shaking flasks on
a reciprocal shaker, each containing 70 ml of the above
medium.
* Corresponding author.
349
Lipase assay Lipase activity was assayed by titrating
fatty acids liberated from olive oil (Wako Pure Chemical
Ind., Osaka) with 50 mM KOH as described previously
(5). The reaction was carried out at 40°C for 60 min with
stirring at 500 rpm. One unit of lipase activity was defined
as the activity which liberated 1/Lmol of fatty acid.
Determination o f protein The amount of protein
was assayed by measuring the absorbance at 280 nm, as-
suming that the value of E 1~ is 10.0.
• Purification of fipase All the purification steps were
carried out below 15°C.
Step 1. Ammonium sulfate fractionation To remove
mycelia, the 65-h culture broth was filtrated through cot-
ton cloth, and then centrifuged at 7,000×g for 20min.
Ammonium sulfate was added to the supernatant to give
a concentration of 15% saturation, and the precipitate was
removed by filtration. The ammonium sulfate concentra-
tion was increased to 80% saturation. The resulting precipi-
tate was collected by filtration, dissolved in 10 mM acetate
buffer (pH 4.5), and dialyzed against the same buffer.
Step 2. SP-Sephadex C-50 column chromatography
The dialyzed solution was applied to a SP-Sephadex C-50
column (3 x 40 cm, Pharmacia LKB Biotechnology,
Sweden) equilibrated with 10 mM acetate buffer (pH 4.5),
and then eluted with a linear gradient of NaCI in the same
buffer (0 to 0.6 M, 600 ml). The active fractions were col-
lected and concentrated by ultrafiltration.
Step 3. Sephadex G-75 gel filtration The concentrat-
ed enzyme solution was put on a Sephadex G-75 column
(2 × 55 cm, Pharmacia LKB Biotechnology, Sweden) equili-
brated with 20 mM phosphate buffer (pH 7.5) containing
0.2 M NaC1, and eluted with the same buffer. The active
fractions were collected, concentrated by ultrafiltration,
and dialyzed against 20 mM phosphate buffer (pH 7.5).
350 SHIMADA ET AL. J. FERMENT.BIOENO.,

Step 4. lsoelectric focusing Isoelectric focusing was 4 -

carried out according to Vesterberg and Svensson (6). The
enzyme solution was subjected to the electrophoresis in a

t
column (110 ml) with Pharmalyte (pH gradient between 3
and 10, Pharmacia LKB Biotechnology, Sweden). The 3-
7 3
applied potential was 300V for the first 18 h, and then Q
increased gradually. After the potential reached 500 V, X ••o°
electrophoresis was continued at this voltage for another ~2- 2 7
24 h. The active fractions were combined, concentrated >

by ultrafiltration, and subjected to Sephadex G-75 gel

filtration as described in Step 3 to remove the carrier
ampholyte. ~1 1 5
Analytical methods for the lipase Disc-PAGE was D
carried out according to Davis (7). SDS-PAGE was done
on a 12% separation gel under the conditions described by o
I0 20 30 40 50
Laemmli (8). Molecular weight marker proteins were pur- Fractlon number ( 2 m l )
chased from Boehringer Mannheim, Germany, After the
electrophoresis, proteins were stained with Coomassie FIG. 1. IsoelectricfocusingofF. heterosporumlipase using Phar-
Brilliant Blue. malyte 3-10. The fractions (nos. 32-34) were pooled. Symbols: ©,
The measurement of molecular weight by gel filtration A2so; • , lipase activity; ...... , pH.
was done using a Sephadex G-75 column, as described
above. The column was standardized with ribonuclease ant lipase producer (4). From its appearance and taxonom-
A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin ical characteristics (Table 1), the strain was identified as
(45 kDa) and bovine serum albumin (68 kDa), which were Fusarium (F.) heterosporum according to Booth (1 I), and
obtained from Pharmacia LKB Biotechnology, Sweden. Gerlach and Nirenberg (12).
The carbohydrate content of the lipase was measured Purification of lipase The enzyme was purified from
by the phenol-sulfate method (9) using mannose as a 1,500 ml of the culture supernatant by ammonium sulfate
standard. fractionation, cation exchange chromatography, gel filtra-
Thin-layer chromatography The positional specifi- tion and isoelectric focusing. The lipase activity curve of
city of the lipase was examined by thin-layer chromatog- isoelectric focusing showed the presence of a shoulder in
raphy of the reaction products obtained with pure triolein the earlier fractions of the main peak (Fig. 1), suggesting
as the substrate. The purification of triolein, preparation that F. heterosporum might produce a main component
of the reaction products and thin-layer chromatography with a pI of 7.0 and a minor component with a lower pI.
were carried out according to our previous paper (10). Table 2 summarizes the results of purification of the main
Amino acid composition and N-terminal amino acid component. The enzyme was purified 2,100-fold starting
sequence analyses The purified lipase was further sub- from the culture filtrate, and the activity yield was 38%.
jected to an Asahipack C4P-50 column ( 4 . 6 × 2 5 0 m m , The purified enzyme showed only one protein band on
Asahi Chemical Industry, Kawasaki, Kanagawa) connect- disc-PAGE and SDS-PAGE (Fig. 2).
ed to a Shimadzu LC-6A H P L C system. The sample was Molecular weight The molecular weight of the sub-
eluted with a 10-70% gradient of acetonitrile in 0.05% unit was estimated to be 31 kDa by SDS-PAGE (Fig. 2).
trifluoroacetic acid. The hydrolysis and amino acid analy- Gel filtration on a Sephadex G-75 column indicated a mo-
sis of the lipase were described in our previous paper (10). lecular weight of 30 kDa. These results showed that F.
The N-terminal amino acid sequence was determined heterosporum lipase was a monomeric enzyme.
with an Applied Biosystems 470A gas-phase protein se- Effects of pH and temperature Effects of p H on the
quencer. activity and stability were tested using Britton-Robinson
Chemicals Simple triglycerides were purchased from buffers of different pHs. The maximum activity at 40°C
Tokyo Chemical Ind. Co., Tokyo. All the other reagents was obtained at pH 5.5-6.0, and the enzyme was stable in
were of special grade. a pH range of 4-10 at 30°C for 4 h.
The enzyme exerted its maximum activity at 40-50°C
when assayed in 50 mM acetate buffer (pH 5.6), and was
RESULTS AND DISCUSSION stable up to 40°C at pH 5.6 for 30 min. These results sug-
Identification of lipase-producing fungus We have gested that the inactivation of lipase was slightly prevented
isolated a filamentous fungus from soil as a solvent-toler- in the presence of the substrate.
Effect of metal ions and reagents To examine the
TABLE 1. Growth and morphological characteristics of effects of various metal ions on its stability, the enzyme
filamentous fungus producing a solvent-tolerant lipase
Growth rate on malt extract agar medium: TABLE 2. Purification of lipase from F. heterosporum
greater than 9 cm in diameter at 25°C for 4 d
Microconidia: Total Total Specific Activity
could not be observed Step activity protein activity yield
Macroconidia: (U) (mg) (U/rag) (%)
heterogeneous in size; with pedicellate foot cell; formed from Culture filtrate 62,700 66, I00 0.95 100
simple phialide Ammonium sulfate 5 8 , 3 0 0 4,390 13.3 93
Culture: SP-Sephadex C-50 46,000 227 203 73
pale pink to pale peach; red or reddish-brown pigment not Sephadex G-75 43,000 43.4 991 69
produced Isoelectric focusing 23,700 11.8 2,010 38
VOL. 75, 1993 PURIFICATION OF F. HETEROSPORUM LIPASE 351

A B TABLE 3. Effects of organic solvents on stability and activity

of F. heterosporum
| 2
Activityb
Solvent StabiHty"
10°~ 50%
Methanol 73% 116~o 620//oo
Ethanol 54 94 58
Isopropanol 6 116 1
n-Butanol 4 26 0
Acetone 34 99 19
DMF 56 93 92
DMSO 105 113 98
Acetonitrile 0 100 0
Dioxane 4 111 16
Hexane 131 93 32
Benzene 127 56 1
Ether 145 125 20
None 100 100 100
The enzyme was incubated at 30°C in 50% solvents for 20 h, and
the remaining activity was measured under the standard conditions.
b The activity was measured in reaction mixtures containing 10%
or 50% solvent.

FIG. 2. PAGE of F. heterosporum lipase. (A) Disc-PAGE. (B)
SDS-PAGE. Lane 1, molecular markers; macroglobulin (170 kDa),
phosphorylase b (97.4 kDa), glutamate dehydrogenase (55.4 kDa),
lactate dehydrogenase (36.5 kDa), trypsin inhibitor (20.1 kDa); lane
2, F. heterosporum lipase.
was incubated at 30°C for 30 min with 1 m M metal chlo-
rides in 50 m M acetate buffer (pH 5.6), and the remaining
activity was measured under the standard conditions. As
in the cases o f other solvent-tolerant lipases from Pseu-
d o m o n a s (10, 13) and Bacillus (5), Cu 2+ inactivated the
enzyme strongly, and Fe 3+, Co 2+ and Hg 2+ showed 60%
inactivation. E D T A did not affect the enzyme activity,
suggesting that it was not a metaUoenzyme.
The catalytic triad o f lipase is known to be composed o f
Ser, His and A s p / G l u residues (14-16). The present lipase
was incubated for 30min at 30°C with phenylmethyl-
sulfonyl fluoride (PMSF) in 100mM Tris-HCl buffer
(pH 8.0), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate (CMC) in 100 m M glycine-HCl
buffer (pH 5.5), and diethyl pyrocarbonate (DEP) in 100
m M phosphate buffer (pH 6.0). The enzyme was com-
pletely inactivated by 1 m M DEP, and 70% by 5 m M
CMC. However, P M S F did not affect the enzyme activity.
These results suggested that His and G l u / A s p residues in
the enzyme played an important role in its activity.
Effects of organic solvents on stability and activity
Attempts have been made to utilize lipase action in hydro-
lysis, ester synthesis and transesterification in reaction mix-
tures containing organic solvents. To investigate the effect
of organic solvents on its stability, the remaining activity
was measured after incubation at 30°C for 20 h in 50 m M
acetate buffer (pH 5.6) containing 50% concentrations of
various solvents (Table 3). Since benzene, hexane, ether
and n-butanol are water-immiscible solvents, the enzyme
was incubated with these solvents in a biphasic system.
The present lipase was stable in most o f the water-immisci-
ble solvents, and stable even in the Water-miscible solvent
dimethylsulfoxide (DMSO). This lipase was strikingly
stable as compared with the Geotrichum candidum and
R h i z o p u s delemar lipases, which were completely inacti-
vated by incubation at 30°C in 50 m M acetate buffer (pH
5.6) containing 50% DMSO for 20 h (data not shown).
The effects of the organic solvents on the lipase activity
are shown in Table 3. The activity was assayed using reac-
tion mixtures containing 10% and 50% of organic sol-
vents. The activity decreased in 10% o f n-butanol and
benzene, but was not affected or slightly increased in 10%
concentrations of the other solvents. A n increase in activ-
ity in the presence of a low concentration o f solvent was
also observed with other solvent-tolerant lipases from
P s e u d o m o n a s (10) and Bacillus (5). The enzyme activity
did not decrease even in the reaction system containing
50% DMSO or dimethylformamide (DMF).
Substrate specificity Figure 3 shows the hydrolytic
activity toward various simple triglycerides. The enzyme
hydrolyzed tricaprylin and tricaprin at 30°C with a much
higher velocity than others. The presence of 50% DMSO
increased the activity toward the shorter fatty acid chain
triglycerides such as tricaproin and tricaprylin, while eleva-
tion of the reaction temperature increased the activity
toward the longer fatty acid chain triglycerides such as
trilaurin, trimyristin and tripalmitin.
25O
200
.~ 150
lOO
so[ I
o 1
2 3 4 6 8 I0 12 14 16 18 18:
Carbon number of t r l g l y c e r i d e
FIG. 3. Substrate specificityof F. heterosporum lipase. The activ-
ities toward various simple triglycerides were measured at 30°C ( ~ )
and 50°C ( I ) in the absence of organic solvent, and at 30°C in the
presence of 50% DMSO (t~). Each activity was expressed as a
percentage for that of triolein at 30°C in the absence of organic sol-
vent.
352 SHIMADA E T AL.
1 2 34 56 "/ 8
FIG. 4. Thin-layer chromatography of the products obtained by
hydrolysis of triolein. A reaction mixture containing 0.1 g triolein was
incubated at 40°C for 10-20 rain. Then, 7 ml of ether was added to ex-
tract the reaction products, and aliquots of the ether layer were sub-
jected to thin-layer chromatography. The extent of hydrolysis of the
samples applied to lanes 1 and 2 was 10 and 14%, respectively. Lane
3: l(3)-monoolein; lane 4: 2-monoolein; lane 5: 1,2(2,3)-diolein; lane
6: 1,3-diolein; lane 7: oleic acid; lane 8: triolein.
Positional specificity Figure 4 shows a thin-layer
c h r o m a t o g r a m of the reaction products with triolein as a
substrate. The extents o f hydrolysis were 10 and 14%.
Spontaneous acyl migration was considered negligible
because of the short reaction time. 1,3-Diolein and l(3)-
m o n o o l e i n were not detected in the hydrolysis products,
suggesting that the enzyme was 1,3-specific lipase. So far,
there have been few reports dealing with the positional
specificity of solvent-tolerant lipases; Pseudomonas cepa-
cia produces nonspecific lipase (10, 13), and Bacillus sp.
produces a lipase hydrolyzing 1,3-positioned ester bonds
in preference to the 2-positioned ester b o n d (5).
N-Terminal sequence The 10 a m i n o acid residues
from the N-terminus o f F . heterosporum lipase were deter-
mined by protein sequencer to be Ala-Val-Thr-Val-Thr-
Thr-Gln-Asp-leu-Ser. The N B R F and S W I S S P R O T data
bases were searched for a protein carrying a homologous
a m i n o acid sequence, b u t n o n e was found.
Comparison with F. oxysporum Hpase A n extracellu-
lax lipase from F. oxysporum has been purified and char-
acterized (17), and we compared the properties of the F.
heterosporum and F. oxysporum lipases. The molecular
weights of these two lipases were very similar (F. oxyspo-
r u m lipase; 30 kDa), a n d their positional specificities were
1,3-specific. O n the other h a n d , the F. oxysporum lipase
was a glycoprotein, whereas the F. heterosporum one
seemed to contain no carbohydrate as judged by the
phenol-sulfate method. With regard to fatty acid speci-
ficity, the F. heterosporum lipase hydrolyzed triolein with
J. FERMENT.BIOENG.,
a higher velocity than tristearin, but the F. oxysporum
lipase hydrolyzed tristearin with a higher velocity than tri-
olein. The lipase from F. heterosporum was not so inac-
tivated by Z n 2+ and P b 2+, whereas that from F. oxyspo-
r u m was strongly inactivated by these metals. Further-
more, the F. oxysporum lipase did not contain Met, but
that of F. heterosporum contained 1 or 2 Met residues.
REFERENCES
1. Brockerfoff, H. and Jensen, R.G.: Kinetics of lipolysis, p. 10-
23. In Brockman, H. and Jensen, R. G. (ed.), Lipolytic enzymes.
Academic Press, New York (1974).
2. Brockman, H.L.: General feature of lipolysis, p. 3-46. In
Borgstrom, B. and Brockman, H.L. (ed.), Lipases. Elsevier,
Amsterdam (1981).
3. Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson,
G. G., Lawson, D. M., Turkenburg, J. P., BJorkllng, F., Huge-
Jensen,B., Patkar, S. A., and Thim, L.: A model for interfacial
activation in lipases from the structure of a fungal lipase-inhibi-
tor complex. Nature, 351, 491-494 (1991).
4. Shimada, Y., Koga, C., Suglhara, A., Nagao, T., Ital, T., and
Tominaga, Y.: Isolation and culture conditions of a fungus
producing a solvent tolerant lipase. Kagaku to Kogyo, 66,
370-374 (1992). (in Japanese)
5. Sugihara, A., Tani, T,, and Tomin-ga, Y.: Purification and char-
acterization of a novel thermostable lipase from Bacillus sp. J.
Biochem., 109, 211-216 (1991).
6. Vesterberg, O. and Svensson, H.: Isoelectric fraction, analysis,
and characterization of ampholytes in natural pH gradients. Acta
Chem. Scand., 20, 820-834 (1966).
7. Davis, B. J.: Disk electrophoresis. II. Method and application to
human serum proteins. Ann. N.Y. Acad. Sci., 121, 404-427
(1964).
8. Laemmli, U. K.: Cleavageof structural proteins during the assem-
bly of the head of bacteriophage T4. Nature, 227, 680-685
(1970).
9. Dubois, M., GUles, K. A., Hamilton, J. K., Rebers, P.A., and
Smith, F.: Colorimetric method for determination of sugars and
related substances. Anal. Chem., 28, 350-356 (1956).
10. Sngihara, A., Ueshlma, M., Shlmada, Y., Tsunasawa, S., and
Tominaga, Y.: Purification and characterization of a novel ther-
mostable lipase from Pseudomonas cepacia. J. Biochem., 112,
598--603 (1992).
11. Booth, C.: The genus Fusarium. Commonwealth Agricultural
Bureau, Farnham Royal, Bucks. (1971).
12. Gerlaeh, W. and Nirenberg, H.: The genus Fusarium. A pictorial
atlas. Kommissionsverlag Paul Parey, Berlin (1982).
13. Iizunfl, T., Nakamura, K., and Fekase, T.: Purification and char-
acterization of thermostable lipase from newly isolated Pseu-
domonas sp. KWI-56. Agric. Biol. Chem., 54, 1253-1258 (1990).
14. Brady, L., Brzozowski, A.M., Derewenda, Z.S., Dodson, E.,
Dodoson, G., ToHey, S., Turkenburg, J.P., Christiansen, L.,
Huge.Jensen, B., Norskov, L., Thlm, L., and Menge, U.: A ser-
ine protease triad forms the catalytic centre of a triacylglycerol
lipase. Nature, 343, 767-770 (1990).
15. Wlnider, F.K., D'Arey, A., and Hnnziker, W.: Structure of
human pancreatic lipase. Nature, 343, 771-774 (1990).
16. Sch~g, J. D., Li, Y., Wu, S., and Cygler, M.: Ser-His-Glu triad
forms the catalytic site of the lipase from Geotrichum candidura.
Nature, 351, 761-764 (1991).
17. Hoshino, T., Sasaki, T., Watanabe, Y., Nagasawa, T., and
Yamane, T.: Purification and some characteristics of extracellu-
lar lipase from Fusarium oxysporum f. sp. lini. Biosci.
Biotech. Biochem., 56, 660-664 (1992).