Purification and characterization of two distinct lipases from Candida cylindracea

Biochimica et Biophysica Acta, 1156 (1993) 181-189 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00

181

BBAGEN 23747

Purification and characterization of two distinct lipases from Candida cylindracea M. Luisa Rfia a, Teresa Diaz-Maurifio b, Victor M. Fernfindez a, Cristina Otero a and Antonio Ballesteros a a Instituto de Catdlisis, CSIC, UniL,ersidad Autdnorna and b lnstituto de Qulmica-F[sica 'Rocasolano" CSIC, Madrid (Spain)

(Received 5 August 1992)

Key words: Lipase; Isoenzyme; Enzyme purification; Enzyme characterization; Candida cylindracea; Candida rugosa

We have purified and characterized two isoenzymes from a commercial lipase preparation of Candida cylindracea. The purification procedure includes ethanol precipitation and DEAE-Sephacel and Sephacryl HR 100 chromatographies. Lipase A and lipase B were purified ll-fold with a 5% and 21% recovery in activity, respectively. The enzymes have similar amino acid content, N-terminal sequence and molecular weight, but differ on neutral sugar content, hydrophobicity, presence of isoforms and stability to pH and temperature. They also show some differences in the substrate specificity. Introduction

Lipases (EC 3.1.1.3) are enzymes which hydrolyze triacylglycerols into fatty acids and glycerol. Lipases are produced by animals, plants and microorganisms. In particular, extracellular lipases from microorganisms have received much attention for their potential use in biotechnology, mainly due to their availability and high stability. Lipase activity is greatly increased at the lipid-water interface, a phenomenon kwown as interracial activation [1]. It has been postulated that in this activation process lipases undergo a conformational change. Structural evidence for it has been recently reported by Brzozowski et al. [2]. The lipase produced by Candida cylindracea has been one of the most used in research, owing to its high activity in hydrolysis [3-6] as well as in synthesis [7]. Several studies dealing with the properties of immobilized derivatives of the enzyme have also appeared [8-12]. A lipase gene has been identified in C. cylindracea. It has been cloned, sequenced, and the amino acid sequence of the protein deduced from the cDNA [13]. This lipase is related to the lipase produced by Geotricum candidum and to the cholinesterases. They all have in common the catalytic triad, Ser-His-Glu Correspondence to: M. Luisa Rfia, Instituto de CatAlisis, CSIC, Universidad Aut6noma, 28049 Madrid, Spain. Abbreviations: Mes, 4-morpholineethanesulphonic acid; Bes, 2[bis(2-hydroxyethyl)amino]ethanesulphonic acid; Con A, concanavalin A; THF, tetrahydrofuran.

[14], with Glu replacing the usual Asp found in other lipases and serine proteases [15]. In 1966, Tomizuka et al. [17] purified and characterized a single extracellular lipase from C. cylindracea. Recently, at least two lipases have been identified in extracellular cultures of the yeast [18,19]. However, the molecular properties reported by these authors and others [20-22] are not in agreement, probably due to the presence of contaminants associated with the lipases or to the different methods followed in their purification. In the present work we have purified and characterized two extracellular lipases from C. cylindracea. The results obtained suggest that they are related enzymes which share some molecular properties but differ in the sugar content, hydrophobicity and substrate specificity. Materials and Methods

Lipase type VII from C. cylindracea - now named C. rugosa - p-nitrophenyl butyrate, tributyrin (99%), triolein (99%), Concanavalin A, a-methyl-o-glueopyranoside, Fast Blue BB salt, a-naphthyl acetate and Nonidet P-40 were purchased from Sigma (St Louis, MO, USA). Iodogen was from Pierce Eurochemie (Rotterdam, Netherlands). Extra virgin olive oil (0,7°), from Carbonell, C6rdoba, Spain. DEAE-Sephacel, Sephacryl HR 100, phenyl- and octyl-Sepharose CL-4B, molecular weight markers for electrophoresis and gel filtration chromatography, were obtained from Pharmacia (Sweden). All other chemicals used were of the purest grade available. Enzyme assays Lipase activity was assayed in a Radiometer pH-stat at 30°C in 1 mM Tris-HC1 (pH 7.2)

182 containing 0.1 M NaCI and 0.1 M CaC12. Three substrates were used. (i) Tributyrin. The reaction mixture consisted of 15 ml Tris buffer, 0.30 ml tributyrin and 0.45 ml acetonitrile. The kinetic constants were determined varying the tributyrin concentration between 2 and 390 mM. (ii) Triolein. 15 ml Tris buffer were mixed with 0.6 ml of a triolein solution (between 5 and 500 tzM) in acetone, to give a final acetone concentration of 4%. (iii) Olive oil. The olive oil emulsion consisted of 25% of olive oil and 75% of a 10% (w/v) gum arabic solution in water, mixed in a homogeneizer on an ice-bath for 3 x 5 min (this emulsion was stable at 4°C for a week). The reaction mixture was prepared by mixing 4 ml of olive oil emulsion with 11.75 ml of Tris buffer. In all cases, the reaction was started by addition of the enzyme preparation (between 5 and 100 /zl). One unit (U) is the amount of enzyme that liberates 1 tzmol of fatty acid per min under the above conditions (1 U = 16.67 nanokatals). The esterase activity using p-nitrophenyl butyrate as substrate was followed spectrophotometrically at 30°C in a Varian Cary 210 spectrophotometer equiped with magnetic stirring. The assay mixture (5 ml) consisted of 0.2 ml of p-nitrophenyl butyrate (between 2.9 and 440 # M ) in acetone and 0.1 M sodium phosphate buffer (pH 7.2) containing 0.1 M NaC1. The final acetone concentration of the mixture was 4%. The reaction was started by addition of the enzyme preparation (between 3 and 50 /zl). Initial rates were estimated by measuring the increase in the absorbance at 346 nm isosbestic point of the p-nitrophenol/p-nitrophenoxide couple - and considering the molar extinction coefficient as 4800 M - l cm-~ [23]. In all cases the initial rate was followed up to 10% conversion. One unit (U) is the amount of enzyme that liberates 1 tzmol of p-nitrophenol/min under the above conditions. Electrophoresis. Polyacrylamide gel electrophoresis in denaturing conditions (in the presence of SDS) was performed as described Laemmli [24], in 10% polyacrylamide gel slab with stacking gels containing 4.5% polyacrylamide. Phosphorylase B (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa) were used as molecular weight markers. Samples and controls were boiled for 3 rain in the sample buffer, which contained 5% 2-mercaptoethanol. Electrophoresis in the absence of SDS was carried out in 7.5% polyacrylamide gels in Tris-glycine buffer (pH 8.3) [25]. Electrophoresis was performed on a vertical slab mini gel apparatus (Model SE 200; Hoefer Scientific, San Francisco) at 150 V for 2-3 h. Two-dimensional electrophoresis was carried out essentially as described O'Farrel [26] with the minor modifications of Bravo [27]. For isoelectric focusing, gels (0.3 × 17 cm) were

prepared using a combination of ampholines from LKB in the pH ranges 3.5-10 and 5-7, in the ratio 1:5, in 4% acrylamide, 8 M urea and 2% (w/v) Nonidet P-40. The gels were prerun at 300 V for 15 min, 400 V for 15 min and 700 V for 45 rain, on a Hoefer GT3 apparatus. After the prerun, the samples (3 tzg of protein) were loaded and the gels run for 18 h at 800 V. The pH gradient of focused gels was monitored immediately at the end of each run by cutting a control gel into pieces of 1 cm width. After shaking with distilled water for 1 h the pH was measured [27]. The pH gradients were typically from 4.4 to 7. After isoelectric focusing the gels were gently shaken in 5 ml of equilibration buffer for 10 min and stored at - 7 0 ° C until used for the second dimension. Equilibrated first dimension gels were quickly thawed, loaded onto the slab gel and immediately layered with warm 1% agarose dissolved in equilibration buffer containing 0.002% bromophenol blue. The second dimension was performed in 12% acrylamide gels (26 × 19 × 0.1 cm) in the presence of SDS, according to Laemmli [24], with 1 cm of stacking gel on the top. Proteins were stained with Coomassie Brillant Blue R-250 and silver [28]. Detection of hydrolytic activity with a-naphthyl acetate was performed by coupling with Fast Blue BB [29]. Lectin binding assays. Concanavalin A (Con A) in 0.1 M sodium acetate buffer (pH 5.0) was labelled with ~25I in the presence of 0.1 M a-methyl-D-glucopyranoside, using Iodogen as instructed by the manufacturer. The labelled lectin was indistinguishable from the corresponding unlabelled protein by denaturing electrophoresis and autoradiography. Lipase preparations were subjected to SDS electrophoresis and the proteins transferred to nitrocellulose sheets essentially according to Towbin et al. [30]. Protein binding sites on the nitrocellulose sheets were blocked by incubation with 0.5% bovine serum albumin in 50 mM Tris-HC1 (pH 7.5) containing 0.15 M NaC1 and 0.1% Tween, under agitation for 1 h at room temperature. The samples were then incubated with ~25I-Con A in 50 mM Tris-HC1 (pH 7.5) containing 0.15 M NaC1 for 1 h at room temperature, washed in the same buffer, dried and autoradiographied. For gels running in the absence of SDS, the interaction with ]25I-Con A was done according to Beeley [31]. Amino acid analysis. The enzymes were hydrolyzed with 6 N HCI at 110°C for 20 h in an argon atmosphere and in the presence of 5 mM thioglycollic acid. The amino acids were quantified in a Biotronic LC 7000 analyzer. Amino terminal sequence analysis. Amino terminal sequence analysis was performed in a Beckman 890C sequencer, following the manufacturer's instructions. Identification of the PTH-amino acids was carried out according to Lottspeich [32].

183

Other methods. Protein concentration was determined by the Lowry method [33] with bovine serum albumin as standard. The content in neutral sugars of the different enzyme preparations was estimated by the phenol-sulfuric acid method [34] using xylose as standard.

Activity (U/ml)

A 280 nm

- - 1

300

1"6 l

i

L

1.2

200

Results

Lipase purification. 10 g of crude powder were suspended in 100 ml of 25 mM Tris-HC1 buffer (pH 7.5), kept stirring for 90 min and centrifuged at 17 000 x g for 20 min at 4°C. The supernatant (crude extract) was treated with two volumes of ice-cold ethanol. The solution was constantly stirred at 0°C during the addition of ethanol and kept stirring for 1 h. The precipitate which contained the enzymatic activity was collected by centrifugation at 17000 x g for 20 min at 4°C, dissolved in buffer and dialyzed overnight against the same buffer. The clear solution obtained (5 mg protein/ml) was loaded on a DEAE-Sephacel column (4.5 x 14 cm) equilibrated with Tris-HC1 buffer and eluted at 4°C at 100 ml/h. Fractions of 6 ml were collected and measured for absorbance at 280 nm, esterase activity (using p-nitrophenyl butyrate as substrate) and lipase activity (using olive oil emulsion and tributyrin). After elution of unbound material without activity, two main peaks with activity (lipase A and lipase B) were eluted with buffer containing 60 mM and 100 mM NaCI, respectively (Fig. 1). The fractions with higher activity were separately pooled and concentrated by ultrafiltration through Amicon PM30 membranes. Concentrated aliquots of lipase A and B were separately loaded on a Sephacryl HR 100 column (2.6 x 80 cm) equilibrated with 0.1 M phosphate buffer (pH 7.2)

A/280

nm

,~

1000

A

~ 1o0

0.4

0~ - ~ ~ - ~ = ~ - 0 25 50 75

100

125,

0 150

Fraction Number Fig. 1. Chromatography on DEAE-Sephacel. After dissolving the ethanol precipitate proteins in 25 mM Tris-HCI buffer (pH 7.5), sample was loaded on a DEAE-Sephacel column (4.5 x 14 cm) equilibrated in the same buffer. The flow rate was adjusted to 100 ml/h and 6 ml fractions were collected. Bound fraction was eluted by buffer containing 60 mM NaCl (A) and 100 mM NaCI (B). The chromatography was carried out at 4°C. ([]). Absorbance at 280 nm; (*) activity with tributyrin; ( [] ) activity with olive oil emulsion.

containing 0.1 M NaC1, and eluted with the same buffer. Both enzymes came out of the column at the same volume, suggesting that they have similar molecular weight (Figs. 2A and 2B). The specific activities (measured with tributyrin as substrate) of lipase A and B were very similar: 750 and 744 /~mol/min per mg

Activity (U/ml)

0.8

B

A1

0.8

0

A/280 nm . 8 - -

Activity (U/ml) 1000

B

800

8OO

0.6 600

6OO

]

7 o.2

~-

400

i

S

200

J o 30

50

70

90

Fraction number

110

0 130

0 30

50

70

~] 90

110

I400 200

Io 130

Fraction Number

Fig. 2. Chromatography on Sephacryl HR 100. Active fractions of lipase A (A) and of lipase B (B) from the DEAE-Sephacel column were pooled, concentrated, and aliquots loaded on a Sephacryl HR, 100 column (2.6X80 cm) equilibrated in sodium phosphate buffer (pH 7.2) containing 0.1 M NaCl. The flow rate was adjusted to 20 ml/h, and 3 ml fractions were collected. The chromatography was carried out at 4°C. Symbols as in Fig. 1.

184

123

TABLE I

Purification of lipase A and B from C. cylindracea Purification step

Total protein (mg)

Specific activity a (U/mg)

Purification (fold)

Yield (%)

Crude extract Ethanol precipitate DEAE-Sephacel Lipase A Lipase B Sephacryl HR 100 Lipase A Lipase B

1 100 500

65 120

1.0 1.8

100 84

80 154

134 230

2.0 3.5

15 49

4.5 20

750 744

11.5 11.3

5 21

94 67 43 30

Tributyrin was used as substrate

20 protein, respectively. The purification factors were close to 11 for both proteins and the yield were 5% and 21%, respectively. In all the purification steps the same pattern of enzyme activity was obtained using either olive oil, tributyrin or p-nitrophenyl butyrate as substrates. Therefore, lipases A and B are able to hydrolyze soluble esters (characteristic of esterases) as well as long chain triacyl-glycerols (natural substrates of lipases). On the other hand, the activity measured with olive oil emulsion was twice higher than the activity obtained with tributyrin. By electrophoresis in denaturing conditions lipases A and B showed, when staining for protein, a single band with similar electrophoretic mobility. Table I summarizes the data of the specific activities and yields during the purification of 10 g of commercial lipase. Molecular properties. The chromatographic behaviour of purified preparations of lipases A and B on octyl- and phenyl-Sepharose columns indicated the hydrophobic nature of these lipases. Both enzymes were bound to octyl-Sepharose and could only be eluted by Triton X-100. On the other hand, lipase B was eluted from phenyl-Sepharose by 1 mM sodium phosphate buffer whereas lipase A was eluted by 50% ethylenglycol. These results indicate that lipase A is more hydrophobic than lipase B. The molecular weight of lipase A and B, as determined by gel filtration chromatography on Sephacryl HR 100 (1 × 90 cm), was very similar: 60000 for lipase B and slightly higher for lipase A. By electrophoresis in the presence of SDS the molecular weights were 64 000 and 62 000 for lipases A and B, respectively (Fig. 3). By electrophoresis in non-denaturing conditions lipase A showed a single band when stained for activity (a-naphtyl acetate), for protein (silver) or for carbohydrate (t~5I-Con A). Only the gel obtained after anaphtyl acetate stain is shown in Fig. 4 (line 1). Lipase B was resolved in four bands after staining for protein

Fig. 3. SDS polyacrylamide gel electrophoresis. Line 1, molecular weight standards indicated in kDa; line 2; lipase A; line 3, lipase B. Gels were stained for protein with Coomassie.

(Fig 5, line 1) or for carbohydrate (Fig. 5, line 2). The relationship between the intensity of the bands stained with silver and with t25I-Con A suggests that the isoforms have different carbohydrate content. Only the two major bands, with the highest electrophoretic mobilities, gave reaction with a-naphtyl acetate (Fig. 4, line 2). Although the possibility that the two other

1

2

b a i~!ii~!iiii~i~iii~~i~i

~5~ ~ ~

~

i ~ii!;;i;ili ¸¸¸

Fig 4. Polyacrylamide gel electrophoresis in non-denaturing conditions of lipase A (line 1) and lipase B (line 2). Gels were stained for esterase activity with a-naphtyl acetate.

185 TABLE II

J

Amino acid composition of lipase A and B (mol%)

tl

el

C ~,

C

i1

!

b

b

[ i

a

8

Amino acid

Lipase A

Lipase B

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Lys His Arg Trp Cys

13.1 5.5 8.2 6.6 7.2 11.2 9.7 4.7 2.7 4.5 8.9 3.9 5.5 4.3 1.3 2.6 n.d. n.d.

12.7 6.5 8.8 7.3 7.0 10.8 9.1 4.6 2.5 4.3 8.9 4.7 5.7 3.6 0.8 2.6 n.d. n.d.

n.d., not determined

1

2

Fig 5. SDS-polyacrylamide gel electrophoresis. Gels were stained with silver (line 1) and 125I-Con A (line 2).

bands were glycosylated contaminants cannot be excluded, the homogeneity of the lipase B preparation (as assessed by the analysis of the N-terminal amino acid sequence, molecular weight . . . . ) strongly suggests that these isoforms are active lipases that cannot be easily detected with a-naphtyl acetate due to their low concentration in the gel. By two-dimensional electrophoresis lipase A also showed a single component with a p I of 5.5 and a molecular weight of 60000. Lipase B showed again four components, two major bands having p l s of 4.80, 4.84 and two minor ones having p l s of 4.95 and 5.04. All isoforms detected in lipase B and lipase A samples had the same molecular mass of 60 kDa. The neutral sugar content as determined by the phenol-sulfuric method was dependent on the preparation, the average values being 8 _+ 3 and 3.6 _+ 0.8 for lipase A and B, respectively. As shown in Table II the amino acid composition of both lipases was very similar. Also the amino terminal sequences were almost identical (Table III), with only the fifth amino acid being different (Thr in lipase B and Lys in lipase A). The sequence found for lipase B coincides with the sequence deduced by Kawaguchi et al. [13] from the cDNA of lipase I.

Effect of pH on the enzyme activity. Both isoenzymes showed similar pH-profiles using tributyrin as substrate, with optimum activity around pH 7. However, the decrease in activity at pH 8 was more pronounced in lipase B, whereas lipase A was less active at pH 5. Effect of organic solvents. The effect of organic solvents on the activity of lipases A and B was studied using tributyrin as substrate. A clear stimulation was observed by the addition of up to 1 - 2 % (v/v) acetonitrile. As the acetonitrile concentration increased the activity dropped and virtually no activity was obtained above 17% (v/v). Similar results were obtained with acetone as cosolvent. In contrast, tetrahydrofuran (THF), a water immiscible solvent, inhibited the activity of the enzyme in a concentration dependent manner even at the lowest concentrations studied (1%). pH and thermal stability. The stability was studied by incubating samples of lipases A and B at different pH values (7.2, 8.0 and 10.0) and temperatures (4°C and 30°C). At regular intervals, aliquots were removed and assayed for esterase activity using p-nitrophenyl butyrate as substrate. Lipase A was more stable to both pH and temperature. Thus, at pH 7.2 the time required to produce a 50% decrease in the activity of lipase A was about 50 days at 4°C and 4 days at 30°C, TABLE III

Amino terminal sequences of C. cylindracea lipases A and B, and of lipase I (deduced by Kawaguchi et al. [13]) Lipase A Lipase B Lipase I

APTAKLANGD APTATLANGD APTATLANGD

186 TABLE IV

v * 10 ÷7 (M s - l )

Kinetic parameters of lipases A and B using p-nitrophenyl butyrate as substrate

15°°v/[E ] 4O 1000.

5oo~

E

Lipase A Lipase B

30 0

8

16

kcat (s - I )

Km 0zM)

kc~t ~Kin (s J M -1)

1010±75 > 1600

39.2±3.6 > 400

2.51+0.18.10 +7 0.42 ± 0.09.10 + 7

24

(v/[EIISl) " 10-6

20

10

f'

o/ 0

t t

0.1

0.2

[pNPB]

0.3

0.4

(mM)

Fig. 6. Dependence of the rate of hydrolysis of p-nitrophenyl butyrate by lipase A (t~) and lipase B (*), on the ester concentration. The solutions were buffered with 0.1 M sodium phosphate (pH 7.2), containing 0.1 M NaCI and 4% (v/v) acetone. The assay temperature was 30°C. The concentrations of lipase A and lipase B were 1.8 nM and 2.7 riM, respectively. Inset: Eadie-Hofstee transformation of the data.

data obtained for lipase B fall on a line parallel to the ordinate; under these conditions k2 ° was constant, and kca t and K m could not be determined. Activity on neutral lipids .Plots of the rate of triolein hydrolysis catalyzed by lipase A and by lipase B against substrate concentration gave sigmoidal curves typical of lipases (Fig. 7). Lipases display low activity when the substrate is in the monomeric state [35]. At triolein concentrations up to 20 /xM lipases A and B act on substrate monomers with a relatively low and similar activity. At concentrations above the solubility limit, a break in the activity pattern was observed, being the enhancement of the rate of hydrolysis specially remarkable for lipase B (five times higher than the rate with lipase A at the highest substrate concentration tested). This behaviour indicates the presence of a much larger lipid-water interphase, which is a requirement for full expression of the lipolytic activity. The K m values

v * 10 ÷6 (M s - l )

while that of lipase B was 44 days and 1.5 days, respectively. The difference was more noticeable at higher pH values; at 8.0 and 30°C the pseudo half-life of lipase A was 100 h while that of lipase B was less than 2 h. Under these conditions lipase B also lost its activity using tributyrin as substrate.

•

2.5

i

j///

)l~" //

Kinetic parameters Activity on soluble esters. The rate of p-nitrophenyl butyrate hydrolysis as a function of substrate concentration up to 440 lzM (maximum solubility of pnitrophenyl butyrate in the experimental conditions used) was studied. The plot of the rate of p-nitrophenyl butyrate hydrolysis catalysed by lipase A against substrate concentration showed saturation kinetics, while the one corresponding to lipase B did not deviate substantially from linearity (Fig. 6). The behaviour of lipase B could be consistent with an enzyme which still obeys Michaelis-Menten kinetics but with a K m value for p-nitrophenyl butyrate much higher than the ester solubility at the experimental conditions. Inset in Fig. 6 represents a modified Eadie-Hofstee plot of the data. The data obtained with lipase A could be fitted to a straight line (correlation coefficient = 0.946), whose extrapolation to the ordinate and abscissa axes yielded kc, t and k2 ° = (kcat/K m) (Table IV). The experimental

/s /

1.5

/J ~c 7~

/ r

0.5

/ ,~ .... , /'

S

0

0

0.1

t

I

I

_

0.2

0.3

0.4

0.5

[Triolein]

(mM)

Fig. 7. Dependence of rate of hydrolysis of triolein by lipase A ([]) and lipase B (*), on the triglyceride concentration. The solutions were buffered with 1 mM Tris/HC1 (pH 7.2), containing 0.1 M NaCI, 0.1 M CaCI 2 and 4% (v/v) acetone. The assay temperature was 30°C. The concentrations of lipase A and lipase B were 3.5 nM and 3.1 nM, respectively.

187 estimated using tributyrin as substrate were 0.4 mM and 8.4 mM for partially purified lipases A and B, the Vmax values being 280 and 770 U / m g protein, respectively. Discussion

There are several reports on the multiple forms of lipase produced by microorganisms [36-38]. This multiplicity has been ascribed either to post-transcriptional processing like partial proteolysis [39] or deglycosylation [35,36], or to the synthesis of different lipases [40]. In this work, we have separated by ion-exchange chromatography two major populations of lipases from a commercial preparation of C. cylindracea The lipases, called A and B, have been further purified by gel filtration, and some of their structural and functional properties studied. The purification factor achieved, close to 11 for both lipases A and B, was higher than previously reported values: Veeraragavan and Gibbs [18], for example, obtained 5.8 and 1.9 for lipases A and B, respectively whereas Wu et al. [21] reported a purification factor of 3. The amino acid composition of the two enzymes was found to be very similar. The composition was also similar to that deduced from the base sequence of the lipase I gene cloned by Kawaguchi, assuming that C. cylindracea the codon CUG is read as Ser instead of Leu [13]. The N-terminal sequences are almost identical with only the fifth amino acid of the ten determined being different. The lipase B sequence found coincides with the one deduced for lipase I by Kawaguchi et al. [13]. The two forms behave differently in hydrophobic chromatography, being lipase A more hydrophobic than lipase B. They also differ on the neutral sugar content (estimated by the phenol-sulfuric method): approximately 8 + 3% and 3.6 + 0.8% for lipase A and lipase B, respectively, although we have found some differences on the neutral sugar content of the lipase A purified from different commercial batches. This could explain the discrepancy between our results and those reported by Kawaguchi and Honda [41]. They found that the two extracellular lipases produced by the yeast C. cylindracea contain 7% carbohydrate. Both lipases strongly interact with the lectin Con A, suggesting the presence of mannose on the carbohydrate chains. The molecular weight of both lipases, estimated by analytical gel filtration, was similar and close to 60 kDa. However, by native electrophoresis and isoelectric focusing, lipase B was resolved in a mixture of several isoforms with pls ranging from 4.8 to 5.0, while lipase A showed only one major band with a pI of 5.5. The molecular masses of the two isoenzymes determined by different authors by denaturing electrophoresis [18-22] were very similar to the values here re-

ported. However, Veeraragavan et al. [18] found that in non-denaturing conditions the molecular masses were 92 and 58 kDa for lipases A and B, respectively. Also the pls reported by these authors - 5.6 and 5.8 and by Brahimi-Horn et al. [19] - 4.3 and 4.7 respectively, are in disagreement with our data. These discrepancies could be attributed to preparations that have not been sufficiently purified since we have observed that lipases can associate glycosylated impurities present in the crude extracts. This leads to the formation of high-molecular-weight aggregates with anomalous pI. In addition, this process seems also to modify the catalytic properties of these lipases (unpublished data). The different treatments followed during the purification of the enzymes could also account for the different values reported. Thus, it has been described that the isoelectric point of different proteins could be modified by treatment with phospholipids [42] or sugars [43]. Also, treatment with bile salts [44,45] or detergents [46,47] can give rise to protein aggregates. This could explain the molecular mass of 120 kDa described by Tomizuka et al. [17] who used sodium deoxycholate during the purification, and also the molecular masses of 362, 200 and 143 kDa reported by Shaw et al. [20], who used SDS. The different isoforms of lipase B have apparently the same Mr, the same N-terminal sequence and similar hydrophobicity (as they co-elute on phenyl-Sepharose columns). Its heterogeneity could be attributed to a diversity in the glycosylation pattern of the core protein. In fact, we observed some differences in the reactivity of the mannose specific lectin, Con A, with the isoforms separated by non-denaturing electrophoresis. This could be in agreement with the resuits reported by Baillargeon [48] and Spener et al. [49]. They found that when the carbohydrate was removed from the Geotricum candidum lipase, the several bands which appeared in isoelectric focusing, were reduced to a single one. However, the possibility that these forms are the product of different genes cannot be ruled out. Thus, Kawaguchi et al. [13] found in C. cylindracea homologous genes that encode several homologous lipases I which differ only in a few amino acid substitutions. Although both enzymes can hydrolyse p-nitrophenyl butyrate, lipase A has a higher affinity for this esterasic substrate than lipase B. The K m value of lipase B is higher than the substrate solubility in the reaction conditions and, therefore, could not be determined. Both enzymes are more active on triolein emulsion than on monomolecularly dispersed molecules. Thus, the affinity of the two lipases for single triolein molecules is rather similar but at high substrate concentration, in which triglyceride droplets may be formed, the activity of lipase B is five times higher than

188 that of lipase A. Moreover, lipase B has lower affinity for the soluble substrate (p-nitrophenyl butyrate) and also for the short-chain triglyceride (tributyrin). All these results indicate that lipase B should be considered as a better catalyst for lipolytic substrates than lipase A, whereas lipase A is a more specific catalyst for esterasic substrates. The influence of the pH and organic solvents on the activity of both lipases was quite similar. Using tributyrin as substrate, the two lipases showed a broad maximum of activity between pH 6 and 7.5. Organic solvents had a notable influence on lipase activity of both purified forms. It is well known that lipase activity depends on the properties of the oil-water interface (nature of the lipidic substrate, orientation and conformation of the constitutive lipids, molecular and charge density, etc) [50]. It has been reported that tributyrin at concentrations above 0.25 mM produce a lipid interface [51,52]. Therefore, the increase in the activity observed at acetonitrile concentrations below 2% could be due to the modification of the oil-water interface which facilitates the enzymatic action [53], while the loss of activity at higher acetonitrile concentrations could be due to denaturation of the enzymes. The apolar organic solvent, THF, could also dilute the interracial substrate concentration producing a decrease in the activity [54]. Although both lipases are very stable at neutral pH, lipase A is more stable to changes in pH and temperature than lipase B. From the structural and kinetic characterization it might be concluded that the two enzymes purified from C. cylindracea are truly different lipases. They have similar amino acid content, N-terminal sequence and molecular weight, but they differ on the neutral sugar content, hydrophobicity, presence of isoforms, and stability to pH and temperature. They also show differences in substrate specificity, being lipase A a better catalyst for the esterasic substrate whereas lipase B has a more lipasic character.

Acknowledgements We would like to thank Dr. Juan J. Calvete for the N-terminal sequences determination and Germ~in Andr6s for helping with two-dimensional electrophoresis. This work has been supported by the EEC (project No. BIOT-CT90-0176(TSTS)) and by the Spanish CICYT (No. BIO091-0861-CE).

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