Biotechnology Letters 22: 1291–1294, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

1291

Purification and characterization of two isoforms from Candida rugosa

lipase B

Cristina López, Nelson P. Guerra & M. Luisa Rúa∗

Area de Bioquı́mica e Bioloxı́a Molecular, Facultade de Ciencias de Ourense, Universidade de Vigo,
32004 Ourense, Spain
∗ Author for correspondence (Fax: +34 988 387001; E-mail: mlrua@uvigo.es)

Received 12 May 2000; Revisions requested 23 May 2000; Revisions received 20 June 2000; Accepted 22 June 2000

Key words: Candida rugosa, characterization, isoforms, lipase B, purification

Abstract
Two isoforms of Candida rugosa lipase B (LB1 and LB2) were purified by anionic exchange chromatography. The
lipases had the same N-terminal sequence, carbohydrate content and pH and thermal stability but different pIs and
significant differences in their activities against different p-nitrophenol esters and triacylglycerides.

Introduction Materials and methods

Candida rugosa secretes a mixture of lipase isoen- Materials

zymes which have been extensively used in biotrans-
formations. Lotti & Alberghina (1996) have elucidated
seven lipase gene sequences of C. rugosa (LIP1-
LIP7), five of them (LIP1-LIP5) having been fully
characterized. Comparison of the predicted amino acid
sequences reveals a close similarity among the five
lipases, ranging between 77% and 88% identity for
pairs of proteins. However, an important variability
in several positions around the active site that could
influence interactions with the substrate have been re-
ported (Lotti & Alberghina 1996). Only three lipase
isoenzymes have been identified in C. rugosa lipase
(Sigma type VII), the commercial extract most fre-
quently used in biotransformations. Diczfalusy et al.
(1997) detected Lip2 and two closely related enzymes:
CEL1 and CEL3 both being the product of LIP1 gene.
Aditionally, Rúa & Ballesteros (1994), using a sim-
pler method, now used in several laboratories, purified
Lip3 and a mixture of 4 isoforms with different pIs
named as lipase B, which is the main fraction in Sigma
extracts (70%) (Rúa et al. 1993). Nevertheless, it
is not known whether or not the isoforms are true
isoenzymes. The aim of this work was the separa-
tion of lipase B isoforms in order to determine their
differences at molecular and activity level.
Lipase type VII from C. rugosa, endoproteinase Glu-
C and triolein emulsion and p-nitrophenyl esters used
as substrates were from Sigma. Tributyrin and tri-
acetin were from Fluka (Deisenhofen, Germany). N-
glycosidase F deglycosylation kit from Boehringer
Mannhein (Mannhein, Germany). Ion-exchange col-
umn (Mono P HR 5/5), desalting columns (PD-10)
and gels for isoelectric focusing were from Pharmacia
(Sweden). All other chemicals used were of analytical
grade.
Purification
Purification of lipase B was carried out as described by
Rúa & Ballesteros (1994). Concentrated lipase B was
loaded on a Mono P HR5/5 column equilibrated with
25 mM histidine/HCl buffer (pH 6.1). The column was
eluted applying a linear gradient of 0.150–0.275 M
NaCl in 20 ml of equilibration buffer. The obtained
lipases were desalted using PD-10 columns equili-
brated in 25 mM Tris/HCl (pH 7.5) following the
manufacturer’s instructions.
1292
Fig. 1. Chromatography on Mono P HR5/5. 500 µl lipase B was loaded onto the column equilibrated in 25 mM histidine/HCl buffer (pH 6.1).
Bound lipases were eluted with a linear gradient of NaCl (0.150–0.275 M) in 20 ml of equilibration buffer. Flow rate: 1 ml min−1 ; fractions:
0.5 ml.
Enzyme assays
Lipase activity was assayed in a Metrohm pH-stat at
30 ◦ C in 5 mM Tris/HCl (pH 7.0) containing 0.1 M
CaCl2 , using 1 M triacetin (TA), 0.11 M tributyrin
(TB) and 0.5 ml commercial emulsion of triolein (TO).
The final volume of reaction mixture was 15 ml. The
esterase activity was followed spectrophotometrically
at 348 nm using p-nitrophenol esters as substrates. The
assay was performed at 30 ◦ C with 1 mM of each p-
nitrophenol ester in 50 mM sodium phosphate buffer
(pH 7.0) containing 0.3% (w/v) Triton X-100 and 4%
(v/v) acetone. One activity unit was defined as the
quantity of enzyme necessary to release 1 µmol prod-
uct per min. To compare statistically the activity mean
values of LB1 and LB2 against different substrates, a
hypothesis test of one tail was used.
Protein concentration
Protein concentration was determinated using the
Lowry method with bovine serum albumin as stan-
dard.
Amino terminal sequence analysis
The purified proteins were sequenced directly from
solutions in a Beckman LF3000 sequencer.
Enzymatic deglycosylation of the lipases
Digestion of lipase samples was carried out using the
N-glycosidase F deglycosylation kit, according to the
manufacturer’s instructions. Samples were analysed
by SDS-PAGE in 10% polyacrylamide gels, on a ver-
tical slab mini gel apparatus (Model SE 250; Pharma-
cia Biotech) and stained for proteins with Coomassie
Brillant Blue R-250.
Isoelectric focusing (IEF)
Commercial gels in the pH-range 3.5–9.5 were em-
ployed. The electrophoresis was performed on a Mul-
tiphor Electrophoresis System (Pharmacia Biotech)
following the manufacturer’s intructions and stained
with Coomassie Brillant Blue R-250.
Peptide mapping
Proteolytic digestion of denatured lipase samples was
performed as described by Cleveland et al. (1977).
Denatured lipase samples, dissolved in 50 mM am-
monium bicarbonate (pH 7.8) containing 0.2% (w/v)
SDS were incubated for 1 h at 37 ◦ C with endopro-
teinase Glu-C, keeping the substrate to enzyme ratio
of 50. The reaction was stopped adding one volume
of electrophoresis sample buffer and boiling at 95 ◦ C
for 3 min. About 20 µl (20 µg) of each sample were
analysed by SDS-PAGE.
 1293
Table 1. Summary of the purification.

Fraction Total protein Specific activity Purification Yield

(mg) (U mg−1 ) (fold) (%)

Lipase B 2.7 1080 1 100

Mono P HR5/5
LB1 1.0 700 0.7 28
LB2 0.5 385 0.4 7

Comparison of lipase B isoforms at the molecular

level

Lipases LB1 and LB2 showed different pIs: 4.8 (LB1)

and 4.7 (LB2) but same carbohydrate content (2.5%).
The N-terminal sequences were identical up to the
20th amino acid (APTATLLANGDTITGLNAII). Ac-
cording to the published sequences, lipases LB1 and
LB2 could be the product of the genes LIP1, LIP4 and
LIP5 but not LIP3 (lysine in position 5) or LIP2 (va-
line in position 19). Isoenzymes LB1 and LB2 were
digested with endoproteinase Glu-C but the resulting
peptides were identical for both enzymes (not shown).

Effect of pH and temperature on the lipase activity

and stability

The effect of pH and temperature on the activity and

Fig. 2. Isoelectric focusing. Lipase B (lane 1); lipase LB1 (lane
2) and lipase LB2 (lane 3). Gels were stained for protein with
Coomassie Blue R-250.
Results
Purification of lipase B isoforms
Lipase B from C. rugosa was purified as described by
Rúa & Ballesteros (1994). Only the two major pro-
tein bands detected by the authors could be observed
by IEF. These proteins were separated on a Mono P
HR5/5 (Figure 1) as described in Materials and meth-
ods. Isoform LB1 (fractions 31–36) was the major one
with a 28% yield being also more active than LB2
(fractions 38–41) on tributyrin (700 vs 385 U mg−1 )
(Table 1). The purity of both fractions was checked by
IEF (Figure 2).
stability of LB1 and LB2 was studied using triolein
as substrate. No significative differences could be ob-
served between the purified lipases: the optimum pH
and temperature were 7.0 and 30 ◦ C, respectively, for
both enzymes. Both showed high stability at pH 5.0
and 7.0 whereas at pH 9.0 were completely inacti-
vated after 30 min incubation.Their thermal stabilities
at 40 ◦ C were almost identical.
Activity on triacylglycerides and p-nitrophenyl esters
LB1 and LB2 activity increased with the chain-length
of the assayed triacylglycerides (TO>TB>TA) be-
ing isoform LB1 significantly more active than LB2
(Table 2). On p-nitrophenyl esters, the catalytic dif-
ferences between the isoforms were more noticeable
than with triacylglycerides. Thus, with the exception
of pNPC6, LB2 was significantly more active than
LB1, being the differences more marked with pNPC4
and pNFC10, which were the best substrates for both
isoforms. The activity profiles passed through two
minima with pNPC3 and pNPC6 (Table 2).
1294
Table 2. Specific activity of lipases LB1 and LB2 on triacylglycerides and various
p-nitrophenyl esters and significance.
Substrate Specific activity (U mg−1 )
LB1 LB2
Triolein 2040 1860
Tributyrin 833 725
Triacetin 29 26
pNPC3 12 13
pNPC4 27 49
pNPC6 15 15
pNPC10 28 44
te: experimental Student t; ν: degree freedom; pNPC3, pNPC4, pNPC6 and
pNPC10: p-nitrophenyl propionate, butyrate, caproate and caprate, respectively.
Discussion
In this work a method was developed for the isola-
tion of two lipases from the pool of lipase B isoforms
(LB1 and LB2), using anionic exchange chromatogra-
phy. Although some characteristics of LB1 (the major
isoform) and LB2 were similar (N-terminal sequence,
molecular weight, carbohydrate content, peptide frag-
ments and stability) differences in pI and activity on
triacylglycerides and p-nitrophenyl esters were found.
Thus, LB1 showed significant higher activity on tri-
acylglycerides than LB2, whereas LB2 was more
effective to hydrolyze the p-nitrophenyl esters (with
exception of pNPC6 to whom both had identical ac-
tivity). Our results strongly resemble those reported
by Diczfalusy et al. (1997) for isoforms CEL1 and
CEL3 although the authors do not provide their pIs
thus making difficult a direct comparison with LB1
and LB2. CEL1 exhibed a markedly higher activity
than CEL3 on triolein and tributyrin, similarly to LB1
purified in this work. Also, both had the same activ-
ity on pNPC6 as isoforms LB1 and LB2. However,
although CEL1 was more active than CEL3 on those
esters with the shortest acyl chain (C < 6) the opposite
was true for the esters with the longhest acyl chains (C
> 6). Apart from this difference, LB1 might be the
same protein as CEL1 also the major protein in ex-
tracts purified by Diczfalusy et al. (1997). As LIP1 has
been cristallizated from Sigma extracts and reported to
be the major isoenzyme in this preparation (Grochul-
ski et al. 1993) it seems plausible that LB1 and CEL1
correspond to LIP1. The origin of LB2 remains uncer-
tain: as suggested by Diczfalusy et al. (1997) a result
te t(α ≤ 0.05) ν
4.5 2.8 4
8.3 4.3 2
4.5 2.8 4
3.8 2.2 10
4.9 2.2 10
0.0 2.2 10
3.5 2.2 10
of minor posttranslational modifications of LIP1 or
the product of different genes. Alberghina & Lotti
(1997)have reported the existence of at least two addi-
tional genes (LIP6 and LIP7) and perhaps up to three
more, in the lipase gene family. Thus, taking into ac-
count the significant kinetic differences found between
LB1 and LB2, it could be suggested that LB2 would
be the product of one of these genes. It should be em-
phasized that these two proteins (LB1 and LB2) have
been identified in all Sigma lots used for more than 8
years.
References
Alberghina L, Lotti M (1997) Cloning, sequencing and expression
of C. rugosa lipases. In: Rubin B, Dennis E, eds. Methods in
Enzymology, Vol. 284. San Diego: Academic Press, pp. 246–260.
Cleveland DW, Fischer SG, Kirschner MW, Laemmli U (1977)
Peptide mapping by limited proteolysis in sodium dodecyl sul-
fate and analysis by gel electrophoresis. J. Biol. Chem. 252:
1102–1106.
Diczfalusy MA, Hellman U, Alexson SEH (1997) Isolation of
carboxylester lipase (CEL) isoenzymes from C. rugosa and iden-
tification of the corresponding genes. Arch. Biochem. Biophys.
348: 1–8.
Grochulski P, Schrag JD, Douthillier F, Smith P, Harrison D, Rubin
B, Cygler M (1993) Insights into interfacial activation from an
open structure of C. rugosa lipase. J. Biol. Chem. 268: 12843–
12847.
Lotti M, Alberghina L (1996) Candida rugosa lipase isoenzymes.
In: Malcata FX, ed. Engineering of/with Lipases, Dordrecht:
Kluwer Academic Publishers, pp. 115–124.
Rúa ML, Ballesteros A (1994) Rapid purification of two lipases
from C. rugosa. Biotechnol. Tech. 8: 21–26.
Rúa ML, Díaz-Mauriño T, Fernández VM, Otero C, Ballesteros A
(1993) Purification and characterization of two distinct lipases
from C. cylindracea. Biochim. Biophys. Acta 1156: 181–189.