26
Biochimlca et Biophysics Acta, 1044 (1990) 26-33 Elsevier
BBALIP
53388
Purification and characterization of two distinct lipases from Geotrichum candidum Kannappan
Veeraragavan,
Tracey Colpitts
and Bernard
F. Gibbs
Protein Engineering Section, Biotechnology Research Institute, National Research Council of Canada, Montreal (Canada) (Received
Key words:
Lipase;
Isoenzyme;
15 January
Enzyme purification;
1990)
Enzyme
characterization;
(G. candidum)
Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidurn were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.
Introduction Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are a class of enzymes which hydrolyse ester bonds at the interface between water and immiscible liquid organic phase. Although lipases hydrolyse water-soluble substrates of esterases, the rate of the lipase catalyzed reaction is 103- to 104-times faster with emulsified substrates [l]. This unique nature of hydrolysing lipid substrates at the interface separates the lipases from the rest of the esterases. Lipases are produced by animals, plants and microorganisms. In particular, lipases from microorganisms were widely diversified in their enzymatic properties and substrate specificities. The enzymes differ in their molecular weights, pH optima, thermal and pH stabilization, and isoelectric points. Consequently, the enzymes from bacteria, yeast and fungi have potential applications in various industries, including pharmaceutical [2,3], textile [4], chemical [5,6] and food [7-91 industries. Also, lipases have been successfully used to
Correspondence: K. Veeraragavan, Biochemical Engineering Section, National Research Council of Canada, 6100 Royalmount Ave, Montreal, Quebec, Canada, H4P 2R2. 0005-2760/90/$03.50
0 1990 Elsevier Science
Publishers
B.V. (Biomedical
produce specific fatty acids [5,10], triacylglycerols [l l161 and steroid esters [17]. With numerous microbial lipases available in the literature, very few have been purified to homogeneity and characterized [18]. Previous studies on the purification of lipases from Aspergillus niger [19], G. candidurn [20], and Cundidu rugosa [21] showed that the enzyme was homogeneous and some have been crystallized [19,20,22,23]. However, recent publication and our work showed that the lipases from A. niger [24], C. rugosu [25] and G. cundidum (present work) exist in more than one form. In this paper, we report the results of investigations of the purification and characterization of the extracellular lipases produced by G. cundidum.
Materials and Methods Fungul strain. G. cundidum ATCC 34614 was obtained from American Type Culture Collection, U.S.A. Chemicals. The following were purchased from BioRad Laboratories, Toronto, Canada: Tris, Coomassie brilliant blue, sodium dodecyl sulfate (SDS), acrylamide, N, N ‘-methylenebisacrylamide and low-molecular-weight standards. Triolein (Glyceryl trioleate, 99%) oleic acid (cis-Poctadecenoic acid, 99%) and bovine Division)
27 serum albumin (BSA, 98-998) were purchased from Sigma (St. Louis, U.S.A.). Sephacryl-200 high resolution (HR) high resolution anion-exchange mono Q HR 16/10 column, Polybuffer exchanger 94 (PBE 94), Polybuffer 74 and Pharmalyte 4-6.5 were obtained from Pharmacia (Montreal, Canada). All HPLC grade organic solvents were obtained from Fisher, Montreal, Canada. Production of extracellular lipase. G. candidum was grown in a medium containing 5 g peptone, 1 g glucose, 0.1 g NaNO,, 0.1 g KH,PO,, 0.05 g MgSO, and 1 g oleic acid in 100 ml of distilled water (pH 7.0) as recommended by Iwai et al. [26]. Initially, the cells were subcultured in shake flasks containing 100 ml medium at 28” C with agitation at 120 rpm and 2% of this culture medium was used for inoculating 20 1 of medium in a 40 1 fermentor. The medium in the fermentor maintained at 28” C was purged with air at a rate of 15 l/min and stirred at 200 rpm. At the time of maximum lipase activity, the mycelium and its debris were removed by a series of filtrations through Whatman 541, Whatman 1 and 0.45 pm filters. The cell-free medium was then concentrated to about 1 1 by an Amicon DClOL filtration system containing the Amicon Spiral Cartridge SlOY3 (M, 3000 cutoff) filter (Amicon Division, Danvers, MA, U.S.A.). The concentrated sample was lyophilized and stored at - 20 o C for later use. Lipase assay. The lipase activity was estimated using an emulsified substrate in a final volume of 450 ~1. This was prepared by shaking 111 pmol triolein, 1% gum arabic and 50 mM phosphate buffer (pH 6.8) in an Eppendorf shaker (model 5432) (120 rpm) for 5 min at 30 o C. The emulsified substrate was mixed with 50 ~1 of sample and the tubes continued shaking at 30°C. After the incubation period, 1 ml of chloroform/methanol mixture (1: 1, v/v) was added and vortexed for 2 min to stop the reaction and simultaneously extract the products. The tubes were centrifuged at 12000 x g for 2 min in an Eppendorf centrifuge and an aliquot of the lower phase (chloroform) was taken for the oleic acid estimation [27]. However, lipase activities of various column fractions were estimated by incubating an aliquot of the fraction with 111 pmol triolein in 50 mM phosphate buffer (pH 6.5) as described above. After the incubation period, an aliquot of the lipid phase obtained after centrifugation was mixed with acetone and analysed by high-performance liquid chromatography [27]. Molecular weight determination. The molecular weights of the purified lipases were determined by molecular sieving using Sephacryl-200 HR as matrix. About 2 mg of lipase was loaded at 30 ml/h on a 15 X 100 cm Sephacryl-200 HR column equilibrated with 50 mM phosphate buffer (pH 7.5) containing 0.1 M NaCl. 4-ml fractions were collected and their protein concentration and lipase activity were determined. Protein determination. Protein concentration was de-
termined by the method of Bradford [28] using bovine serum albumin as the standard. Ethanol extraction. 25 g of crude lipase powder was dissolved in 100 ml of 25 mM Tris-HCl (pH 7.5) (buffer A). The supernatant obtained after centrifugation at 20000 X g for 20 rnin at 4’ C was treated with 2 vol. of ice-cold ethanol drop-wise. The solution was constantly stirred at 0 o C during the addition of ethanol and for 1 h afterwards. The precipitated proteins were collected after centrifugation and dissolved in 75 ml of buffer A. Amino acid analysis. About 3 mg of the purified lipases were extensively dialysed against HPLC grade water. An aliquot (about 100 pg) was placed in Corning culture tubes (Cat. No. 9820, 6 X 50 mm) which were previously muffled at 450 o C overnight. The tubes were placed in Waters reaction vial and the samples were dried in the Waters Pica-Tag Work Station (Waters, Division of Millipore). Constant boiling HCl (200 ~1) containing 1% phenol was added to the vial and alternately purged (with dried nitrogen) and evacuated. After three purges, the vial was heated at 150°C for 1, 2, 3 and 4 h under vacuum. The values for ten amino acids stable to acid hydrolysis were not corrected. Values for Ser, Thr and Tyr were extrapolated to zero time, and Ile and Val were extrapolated to infinity. Tryptophan was determined by alkaline hydrolysis [29] and cysteine was detrmined as cysteic acid after performic acid oxidation [30]. Methionine was averaged throughout the timecourse. The analysis was performed on a Beckman System 6300 High Performance Analyser according to the general procedures of Spa&man et al. [31]. Electrophoresis. The purity of the enzyme was observed by non-denaturing (native) and SDS-denaturing polyacrylamide gel electrophoresis (PAGE) containing 4% stacking and 7.5% running gels. Slab gels (thickness of 1.5 mm) were run at 70 V until the tracking dye (Bromophenol blue) had penetrated the running gel and at 150 V until the Bromophenol blue had reached the 1 cm mark at the bottom of the slab [32]. Protein was detected by staining the gel with silver-stain solution (Bio-Rad). Non-denaturing gel electrophoresis was carried out at 15 o C and the gel was cut into two portions. One portion of the gel was stained, bands were identified and the areas corresponding to the bands in the other part of the gel were excised into small pieces. These pieces were directly incubated for assaying lipase activity. Isoelectric focusing. Isoelectric focusing was performed on 70 X 50 mm slab gels at 4“ C using ampholytes 4-6.5 (Pharmalyte 4-6.5) as recommended by the manufacturer. Prefocusing was done at 200 V for 10 min and samples (lo-25 pg) were applied in the middle of the gel containing 0.5 vol. of 10% acrylamide-0.3% bis-acrylamide, 0.134 vol. of glycerol, 0.064 vol. of Pharmalyte 4-6.5, 0.006 vol. of 2.3% ammonium persulfate and 0.29 vol. of distilled water. Gels were run at
28 1400 V for 2 h. After isoelectric focusing, gels were cut into small pieces (2 mm width) and each piece was incubated with 0.5 M phosphate buffer for 2 h. Triolein was added to the above buffer and lipase activities were estimated as described above. The pH gradient generated during isoelectric focusing was measured after incubating similar bits in 200 ~1 of distilled water for 4 h. Optimum pH determination. Purified lipase activity was measured at pH values ranging from 5.8 to 8.0 in 50 mM phosphate buffer as described above. Stability at different PH. Purified lipase (25 pg/ml) was incubated in 0.1 M citric acid/Na,HPO, buffer (pH 2.6-5.8) 0.1 M Na,HPO,/NaH,PO, buffer (pH 6.0-8.0) and 0.1 M H,BO,/NaOH buffer (pH 8.510.0). After a 30 min incubation at 4 o C, lipase activities were determined as described above.
Results Purification of extracellular lipases Extraction of lipase. All procedures were carried out at 4” C. Precipitation of lipase by ethanol treatment removed most of the coloured impurities present in the lyophilized medium. A 3-fold purification with a specific activity of 20.22 ~mol/min per mg protein was achieved by this extraction method (Table I). Chromatography on Sephacryl-200 HR. The precipitated proteins obtained by ethanol treatment were dissolved in 75 ml of buffer A and directly loaded on the Sephacryl-200 HR column (5 X 100 cm) (Fig. 1). Two main peaks of materials absorbing at 280 nm were detected and lipase activity was observed in the earlier shoulder of the second peak. After chromatography,
TABLE
I
Purification Purification steps a
1. Crude solution 2. Ethanol precipitate 3. Sephacryl200 HR 4. Ethanol precipitate 5. Mono Q 6. Chromatofocusing peak I peak II
of lipase
from Geotrichum candidum
Total activity
Total protein
( pmol/ min)
(mg)
411
78.25
6.09
1
1298
64.20
20.22
3
2 249
55.80
40.31
1
100
1835 1646
33.60 8.26
54.60 199.36
9 33
82 13
1398 393
Specific activity (gmol/min
Purification factor
Recovery (W)
300
I 200
100
0
20
40
60
FRACTION
80 NUMBER
100
120
Fig. 1. Chromatography on Sephacryl-200 HR. After dissolving the ethanol precipitated proteins in 25 mM Tris-HCl buffer, sample was loaded on a Sephacryl-200 HR column (5 X 100 cm), equilibrated in the same buffer. The flow rate was adjusted to 70 ml/h and 20 ml fractions were collected. Lipase activity is expressed as pmol/min.
active fractions were combined and concentrated by Amicon filtration containing a YM 3 membrane. Separation by molecular sieving increased lipase specific activity to 40.31 pmol/min per mg protein. Since the total lipase activity was increased by ethanol extraction and Sephacryl-200 HR chromatography steps, we considered the total lipase activity in Sephacryl-200 HR as 100%. Chromatography on mono Q. After concentration, the protein sample was subjected once more to ethanol precipitation before being loaded on a mono Q column equilibrated in buffer A. After washing the column to remove the unbound proteins, the lipase bound to the column was eluted by a salt gradient in buffer A. Lipase was eluted as a single peak of activity at 0.16 M NaCl (Fig. 2). Active fractions were pooled and concentrated by Amicon filtration. This anion-exchange chromatography augmented the specific activity to 199.36 ~mol/rnin per mg protein (a 33-fold purification) with a 73% recovery of the initial lipase activity.
b
per mg protein)
6.65 0.69
210.20 570.25
35 94
a All operations were performed at 4’ C. ’ % Recovery was calculated from Sephacryl-200 wards. For details refer to Results section.
FRACTION
62 18
HR
column
on-
NUMBER
Fig. 2. Chromatography on mono Q. Active fractions from the Sephacryl-200 HR column were pooled, concentrated, ethanol precipitated and dialysed against 25 mM Tris-HCl (pH 7.5). The sample was then applied on the mono Q 16/10 anion-exchange column equilibrated in Tris-HCl buffer. The sample was loaded at a flow rate of 60 ml/h and washed at a flow rate of 120 ml/h with same buffer. Lipase was eluted at a flow rate of 120 ml/mm with a linear salt gradient. Lipase activity is expressed as pmol/h.
29 r
0.5 ,
3000
T 6.0
the two isoenzymes. Lipase I and II had specific activities of 210.20 and 570.25 pmol/min per mg protein with purification factors of 35 and 94, respectively.
z F 2000 2 z 03
5.5
US >E 5"
0.2
5.0
I P
1000 iii 4.5 0.1
i
0.4 0.0
5
ch
I
4.0
FRACTION No. Fig. 3. Chromatofocusing on Polybuffer exchanger 94. Following concentration of active fractions from mono Q chromatography, the pooled fractions were dialysed against 25 mM Piperazine-HCl (pH 6.0) and applied on a colunm of Polybuffer exchanger 94 (1 X 30 cm) equilibrated in the same buffer. The flow rate was adjusted at 15 ml/h and 2 ml fractions were collected. The column was eluted at the same flow rate with 500 ml of 13-fold diluted Polybuffer 74 adjusted at pH 4.0. Lipase activity is expressed as pmol/h.
Chromatofocusing on Polybuffer Exchanger 94 (PBE 94). After dialysis at pH 6.0 with 25 mM piperazine-HCl, the sample was loaded on a PBE 94 column (1.0 X 30 cm) equilibrated in the same buffer. The column was then eluted with 13-fold diluted Polybuffer 74 adjusted to pH 4.0. Two peaks with lipase activities eluting between 4.68 and 4.44 were observed (Fig. 3). Active fractions from each peak were pooled separately, concentrated by filtration and dialysed against 10 mM phosphate buffer (pH 6.5). Chromatofocusing separated
Evaluation of purity by electrophoresis The sample obtained after mono Q and chromatofocusing columns were analysed by SDS-denaturing gel electrophoresis (Fig. 4A). The figure indicated the presence of a single band having a molecular weight of 56000, based on the molecular weight of standard proteins run under the same electrophoretic conditions. Also the sample obtained from mono Q column under non-denaturing condition showed the presence of a single band (Fig. 4B). Although the sample obtained from mono Q column showed only one band under SDS-denaturing and non-denaturing conditions, there were two proteins observed by isoelectrophoresis. Final separation of these proteins was achieved by chromatofocusing on PBE 94 column. Molecular weight of lipase The molecular weights of the lipases were determined by molecular sieving on Sephacryl-200 HR. Using the correlation between the K,, and the logarithm of the molecular weight of protein markers, the molecular weight of both lipases was estimated at 56000.
TABLE
II
Amino acid composition of lipase I and II a
A
Amino
B 2
1
3
4
92.5 K
66.2 K 92.5
K
66.2 K
._+_.ae W
WWWlrw *---
I
45.0K
45.0K
:
31.0 K
‘.:
31.OK
21.5 K 14.4 K
-
:
-”
21.5 K
14.4K Fig. 4. Polyacrylamide gel electrophoresis of the lipase samples. Electrophoresis in the presence of SDS (A) and without SDS (B) were performed as described in the Methods section. Gel A: 1, molecular weight standards; 2, lipase from mono Q column; 3, lipase I; and 4, lipase II from chromatofocusing column. Gel B: lipase from mono Q column under non-denaturing condition.
Asx Thrb Serb Gbi Gly Ala Val Met Ile Leu Tyr b Phe His Lys Trp’ Arg Pro cys *
acid
Lipase I (mo18)
Lipase II (mol%)
14.0 5.7 6.3 8.6 10.6 8.0 5.6 1.0 4.2 9.4 3.7 5.4 2.5 4.1 0.2 3.0 6.7 1.0
12.8 4.0 6.0 9.2 13.3 8.2 4.5 1.0 3.7 8.7 3.2 4.8 2.9 3.6 0.2 3.1 9.2 1.6
Proteins were gas-phase hydrolysed in 6 M HCl containing 0.1% phenol for 1, 2, 3 and 4 h at 150 OC. Determined by extrapolation to zero time. Determined by alkaline hydrolysis with 12 M NaOH at 110°C for 16 h [29]. Determined as cysteic acid by performic acid oxidation prior to acid hydrolysis [30].
30
3 10
15
20
GEL SLICE Fig. 5. Isoelectric Pharmalyte 4-6.5
25
30
0.0
35
0
10
GEL SLICE
No.
20
30
No.
point determination of lipase. Isoelectro-focusing of the purified lipase I and II was performed in polyac&rnide gel using as described in Materials and Methods. A, hpase I and B, lipase II. Lipase activity is expressed as pmol of oleic acid formed over a period of 2 h.
Amino acid composition
The amino acid composition of the purified lipase I and II are shown in Table II. They were quite homologous. The acidic residues (Asx and Glx) of lipase I and II constituted 22.6 and 22% of total amino acids, respectively. Basic amino acids (Arg, Lys and His) of both proteins contributed 9.6% of total amino acids indicating that the proteins had a very close net charge. Glycine, the predominant neutral amino acid, accounted for up to 10.6 and 13.3% of the total amino acids in lipase I and II, respectively. Isoelectric point
The isoelectric points of the lipases were determined by focusing on polyacrylamide gels using Pharmalyte 4-6.5 to generate a pH gradient. After isoelectrofocusing, gels were sliced and lipase activities were measured. The isoelectric points of 4.56 and 4.46 were observed for lipase I and II, respectively (Fig. 5). Optimum pH
To evaluate the optimum pH for lipases, enzyme activities were measured in 50 mM phosphate buffer at
pH ranging from 5.8 to 8.0 using triolein as substrate. The ester bond hydrolysis was maximum at pH 6.0 and 6.8 for lipase I and II, respectively (Fig. 6). Stability to different pH
Lipase I and II were pre-incubated at 4 o C in buffers of different pH. After 30 min, enzyme activities were measured at pH 6.0 and 6.8, respectively. For lipase I, the pre-incubation at pH 4 to 8 did not affect the enzyme activity and there was a slow decrease in activities below pH 4 and above pH 8 (Fig. 7A). Similarly for lipase II, the pre-incubation at pH 6.0 to 8.0 did not affect the enzyme activity and decrease in activities were observed below pH 6.0 and above pH 8.0 (Fig. 7B). Effect of various salts
The effect of various salts were tested on lipase activities. For these experiments, 50 mM phosphate buffer, pH 6.0 and 6.8 containing various salts were incubated with lipase I and II, respectively. NaCl and KCl, at concentrations up to 1.0 M, had no effect on lipase I activity. Divalent salts, such as CaCl,, MgCl,
65 = IE
50
40
> g 0 a
45
c > F
30
z
20
? c a
10
3 a 5
6
7
a
9
0 5
I
I
I
6
7
a
9
PH PH Fig. 6. Optimum pH for lipase activity. Lipase was incubated with 50 mM phosphate buffer (pH 5.8-8.0). A, lipase I; and B, lipase II. Lipase activity is expressed as pmol of oleic acid formed over a period of 2 h. Values are mean + SD. of four determinations.
31
a
101 . 2
# 4
I
6
-
I
.
I
.
2
12
10
a
4
6
12
10
a
PH
PH Fig. 7. pH stability of Iipase. Lipase was incubated in 0.1 M citric acid/Na,HPO,, Na,HPO,/NaH,PO, and H,BO,/NaOH buffers at pH ranging from 2.6 to 10.0. After a 30 min incubation at 4O C, the remaining activity was determined as described in Materials and Methods. A, lipase I and B, lipase II. Lipase activity is expressed as pmol of oleic acid formed over a period of 2 h. Values are mean f SD. of four determinations.
TABLE
III
Effects of various salts on lipase activity Lipase
Molarity
Lipase activity
(W of control)
a
(mM)
NaCl
KC1
EDTA
MgCI2 94 100 94 4
117 100 115 3
0 0 0 0
107 106 0 0
90 80 0 0
0 0 0 0
Lipase I
10 50 200 1000
116 105 116 104
115 110 114 101
78 73 70 3
91 23 0 0
Lipase II
10 50 200 1000
98 100 107 112
93 99 95 90
110 80 0 0
65 0 0 0
a Salts, at the final concentration indicated, were added at the beginning of the Iipase reaction. Materials and Methods and activity was expressed as % of control (no salts added). b The highest concentration was 500 mM.
and MgSO, had progressive inhibition at concentrations above 50 mM. SDS at a concentration of 10 mM completely inhibited the enzyme activity (Table III). Similar results were observed for lipase II with NaCl and KCl, divalent salts and SDS, except that the divalent salts at concentrations above 50 mM completely inhibited the enzyme activity. Discussion
We have reported a purification procedure for two lipases from G. candidum, lipase I and II, yielding a 35and 94-fold purification with 62% and 18% recoveries in the enzyme activities, respectively. The purified enzymes can be stored for weeks at 4’ C in 50 mM phosphate buffer (pH 6.5) without significant loss of enzyme activities. In general, both lipases appear to be stable only between pH 6.0 and 8.0. At acidic (below 5.0) and alkaline pH (above 9.0) the lipases are completely denatured.
b
CaCl 2
Lipase
MgSQ
activity
was measured
SDS
as described
in
Purification was essentially complete after anion-exchange chromatography (mono Q) as illustrated by a single band with SDS-PAGE. Isoelectrophoresis, however, proved that this sample was essentially two components having molecular weight 56000 and a difference in pI of 0.1 units. The separation of these components was accomplished by chromatofocusing with PBE 94. The isoelectric points were determined to be 4.56 and 4.46 for lipase I and II, respectively. The molecular weight obtained by molecular sieve chromatography under non-denaturing condition for both lipases was 56 000, a value consistent with the molecular weight deduced from SDS electrophoresis. Monovalent ions, like NaCl and KCl, had no effect whereas EDTA at concentrations above 10 mM significantly inhibited both enzymes. On the other hand, divalent ions, at concentrations above 10 mM,.caused a progressive inhibition of both enzyme activities reaching up to 100% at 1 M. SDS completely inhibited the enzyme activities at 10 mM concentrations.
32 The amino acid composition of these lipases were very similar, containing both cysteine and methionine unlike the lipase purified by Tsujisaka et al. [20]. Many other lipases have been purified and found to have molecular weights in the 50000-60000 region, but with little characterization of the enzymatic activity. The enzymes in this work have specificity for the 1 and 3 positions of the triolein. Among the fungal lipases characterized so far, Penicillium &opium lipase is more active on short-chain than on long-chain triacylglycerols. Lipases from A. niger and Rhizopus delemar show higher activities toward triacylglycerols of medium chain length, whereas G. candidum lipase is fairly specific for triacylglycerols containing unsaturated fatty acids with a double bond at position cis-9 [18]. Also we found that both enzymes from G. candidum have specificity for the 1,3 position of the triolein. There is very little similarity when the physical properties like molecular weight, isoelectric point, optimum pH, optimum temperature, pH stability and thermostability of these fungal lipases were compared [18]. Mammalian lipases, viz. hepatic lipase, lipoprotein lipase, pancreatic lipase, gastric lipase and lingual lipase, differ in several aspects from fungal lipases. The role of mammalian lipases is very complex as compared to fungal or bacterial lipases. Hepatic [33] and lipoprotein lipases [34] have broad substrate specificity in hydrolysing tri-, di- and monoacylglycerol, acyl-CoA thioesters and phospholipids. Human gastric lipase hydrolyses both short- and long-chain triacylglycerols and differs from pancreatic lipase in several major respects including optimum pH, molecular weight and activation and inactivation by various detergents [21,35-381. The lipase from G. candidum purified by Tsujisaka et al. [20] was homogeneous with a molecular mass of 53 000-55 000 and a pI of 4.33. There was no methionine and cysteine in this protein. However, the lipase obtained from the present purification method did not agree with some of the properties mentioned above. Also Jacobsen et al. [39] in their work with G. candidum observed lipases of two molecular weights, 61000 and 57000. Among these species are three lipases, with pI values of 4.42, 4.53 and 4.67, showing identical immunological responses. We have observed similar results, having isolated two lipases of 56 kDa with pZ values of 4.46 and 4.56. Recently, the presence of multiple enzymes were reported from different strains of G. candidum [39,40]. There is only a slight difference between the two proteins in their amino acid composition (more than 85% homology), pI and molecular weight. Therefore, it is speculated that G. candidum may have homologous genes encoding two or more lipases differing by a few amino-acid substitutions. This was confirmed by the recent work in which the genes responsible for these two
proteins were isolated (Shimada, Y., personal communication). A similar phenomenon was also reported with the lipases from Candida cylindracea [41]. Work is in progress to elucidate the processing and functional role of the two proteins. Acknowledgements The authors wish to thank J. Plamondon for her assistance in optimizing the culture growth conditions and initial purification steps. References 1 Chapus, C., Semeriva, M., Bovier-Lapierre, C. and Desnuelle, P. (1976) Biochemistry 15, 4980-4987. 2 Gu, D.M., Chen, C.S. and Sih, C.J. (1986) Tetrahedron Lett. 27, 176331766. 3 Kloosterman, M., Elferink, V.H.M., Lersel, J., Roskam, J.-H., Meijer, E.M., E.M., Hulshof, L.A. and Sheldon, R.A. (1988) TIBTECH 6, 251-256. 4 Fuji, T., Tatara, T. and Minagawa, M. (1986) J. Am. Oil Chemists Sot. 63, 796-799. 5 Kodera, Y., Takahashi, K., Nishimura, H., Matsushima, A., Saito, Y. and Inada, Y. (1986) Biotech. Lett. 8, 881-884. 6 Gil, G., Ferre, E., Meou, A., LePetit, J. and Triantaphylides, C. (1987) Tetrahedron Lett. 28, 1647-1648. 7 Sood, V.R.K. and Kasikowski, F.V. (1979) J. Dairy Sci. 62, 18651867. 8 Gillies, B., Yamazaki, H. and Armstrong, D.W. (1987) Biotech. Lett. 9, 709-714. 9 Langrand, G., TriantaphyIides, C. and Baratti, J. (1988) Biotech. Lett. 10, 549-554. 10 Striton, A.J. (1964) Bailey’s Industrial Oil and Fat products, 3rd Edn. Wiley, New York. 11 Cambou, B. and Khbanov, A.M. (1984) Appl. B&hem. Biotechnol. 9, 255-260. 12 Bello, M., Thomas, D. and Legoy, M.D. (1987) Biochem. Biophys. Res. Commun. 146, 361-367. 13 Dahod, S.K. and Siuta-Mangano, P. (1987) Biotechnol. Bioeng. 30, 995-999. 14 Goderis, H.L., Ampe, G., Feyten, M.P., Fouwe, B.L., Guffens, W.M. and Van Canwenbergh, SM. (1987) Biotechnol. Bioeng. 30, 258-266. 15 Kloosterman, M., Mosmuller, E.W.J., Schoemaker, H.E. and Meijer, E.M. (1987) Tetrahedron Lett. 28, 2989-2992. 16 Abraham, G., Murray, M.A. and John, V.T. (1988) Biotech. Lett. 10, 555-558. 17 Njar, V.C.O. and Caspi, E. (1987) Tetrahedron Lett. 28,6549-6552. 18 Iwai, M. and Tsujisaka, Y. (1984) in Lipases (Borgstrom, B. and Brockman, H.L., ids.), p. 443, Elsevier, New York. 19 Fukumoto, J., Iwai, M. and Tsujisaka, Y. (1963) J. Microbial. 9, 353-361. 20 Tsujisaka, Y., Iwai, M. and Tominaga, Y. (1973) Chem. 37, 1457-1464. 21 Tomizuka, N., Ota, Y. and Yamada, K. (1966) Agric. 30, 576-584. 22 Suguira, M., Gikawa, T., Hirano, K. and Inukai, T. chim. Biophys. Acta 488, 353-358. 23 Isobe, K., Akiba, T. and Yamaguchi, S. (1988) Agric. 52, 41-47. 24 Hofelman, M.. Hartmann, J., Zink, A. and Schreier, Food Sci. 50, 1721-1731.
Gen.
Appl.
Agric.
Biol.
Biol. Chem. (1977) BioBiol. Chem. P. (1985) J.
33 25 Veeraragavan, K. and Gibbs, B.F. (1989) Biotechnol. Lett. 11, 345-34s. Y. and Fukumoto, J. (1973) 26 Iwai, M., Tsujisaka, Y., Okamoto, Agric. Biol. Chem. 37, 929-931. 27 Ergan, F. and Andre, G. (1989) Lipids 24, 76-78. 28 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 29 Hugli, T.E. and Moore, S. (1972) J. Biol. Chem. 247, 2828-2834. 30 Glazer, A.N., De Lange, R.J. and Sigman, D.S. (1975) in Chemical modification of proteins. Selected methods and analytical procedures, p. 23, Elsevier, New York. 31 Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206. P. (1977) J. Biol. Chem. 32 Krueger, B.K., Fom, J. and Grccngard, 252, 2764-2773. 33 Datta, S., Luo, C., Li, W., Tuines, P.V., Ledbetter, D.H., Brown, M.A., Chen, S., Liu, S. and Chan, L. (1988) J. Biol. Chem. 263, 1107-1110.
B. and 34 Smith, L.C. and Ponall, H.J. (1984) in Lipases (Borgstrom, Brockman, H.L., eds.), p. 263, Elsevier, New York. 35 Gargouri, Y., Pieroni, G., Reviere, C., Sauniere, J.F., Lowe, P.A., Sarda, L. and Verger, R. (1986) Gastroenterology 86, 919-915. 36 Noma, A. and Borgstrom, B. (1971) Stand. J. Gastroenterol. 6, 217-223. 31 Brockerhoff, H. (1971) J. Biol. Chem. 246, 5828-5831. 38 Semeriva, M. and Dufour, C. (1972) Biochim. Biophys. Acta 260, 393-400. 39 Jacobsen, T., Olsen, J. and Allermann, K. (1989) Enzyme Microbiol. Technol. 11, 90-95. 40 Baillargeon, M.W., Bistline, R.G., Jr. and Sonnet, P.E. (1989) Appl. Microbial. Biotechnol. 30, 92-96. 41 Kawaguchi, Y., Honda, H., Taniguchi-Morimura, J. and Iwasaki, S. (1989) Nature 341, 164-166.
Biochimlca et Biophysics Acta, 1044 (1990) 26-33 Elsevier
BBALIP
53388
Purification and characterization of two distinct lipases from Geotrichum candidum Kannappan
Veeraragavan,
Tracey Colpitts
and Bernard
F. Gibbs
Protein Engineering Section, Biotechnology Research Institute, National Research Council of Canada, Montreal (Canada) (Received
Key words:
Lipase;
Isoenzyme;
15 January
Enzyme purification;
1990)
Enzyme
characterization;
(G. candidum)
Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidurn were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.
Introduction Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are a class of enzymes which hydrolyse ester bonds at the interface between water and immiscible liquid organic phase. Although lipases hydrolyse water-soluble substrates of esterases, the rate of the lipase catalyzed reaction is 103- to 104-times faster with emulsified substrates [l]. This unique nature of hydrolysing lipid substrates at the interface separates the lipases from the rest of the esterases. Lipases are produced by animals, plants and microorganisms. In particular, lipases from microorganisms were widely diversified in their enzymatic properties and substrate specificities. The enzymes differ in their molecular weights, pH optima, thermal and pH stabilization, and isoelectric points. Consequently, the enzymes from bacteria, yeast and fungi have potential applications in various industries, including pharmaceutical [2,3], textile [4], chemical [5,6] and food [7-91 industries. Also, lipases have been successfully used to
Correspondence: K. Veeraragavan, Biochemical Engineering Section, National Research Council of Canada, 6100 Royalmount Ave, Montreal, Quebec, Canada, H4P 2R2. 0005-2760/90/$03.50
0 1990 Elsevier Science
Publishers
B.V. (Biomedical
produce specific fatty acids [5,10], triacylglycerols [l l161 and steroid esters [17]. With numerous microbial lipases available in the literature, very few have been purified to homogeneity and characterized [18]. Previous studies on the purification of lipases from Aspergillus niger [19], G. candidurn [20], and Cundidu rugosa [21] showed that the enzyme was homogeneous and some have been crystallized [19,20,22,23]. However, recent publication and our work showed that the lipases from A. niger [24], C. rugosu [25] and G. cundidum (present work) exist in more than one form. In this paper, we report the results of investigations of the purification and characterization of the extracellular lipases produced by G. cundidum.
Materials and Methods Fungul strain. G. cundidum ATCC 34614 was obtained from American Type Culture Collection, U.S.A. Chemicals. The following were purchased from BioRad Laboratories, Toronto, Canada: Tris, Coomassie brilliant blue, sodium dodecyl sulfate (SDS), acrylamide, N, N ‘-methylenebisacrylamide and low-molecular-weight standards. Triolein (Glyceryl trioleate, 99%) oleic acid (cis-Poctadecenoic acid, 99%) and bovine Division)
27 serum albumin (BSA, 98-998) were purchased from Sigma (St. Louis, U.S.A.). Sephacryl-200 high resolution (HR) high resolution anion-exchange mono Q HR 16/10 column, Polybuffer exchanger 94 (PBE 94), Polybuffer 74 and Pharmalyte 4-6.5 were obtained from Pharmacia (Montreal, Canada). All HPLC grade organic solvents were obtained from Fisher, Montreal, Canada. Production of extracellular lipase. G. candidum was grown in a medium containing 5 g peptone, 1 g glucose, 0.1 g NaNO,, 0.1 g KH,PO,, 0.05 g MgSO, and 1 g oleic acid in 100 ml of distilled water (pH 7.0) as recommended by Iwai et al. [26]. Initially, the cells were subcultured in shake flasks containing 100 ml medium at 28” C with agitation at 120 rpm and 2% of this culture medium was used for inoculating 20 1 of medium in a 40 1 fermentor. The medium in the fermentor maintained at 28” C was purged with air at a rate of 15 l/min and stirred at 200 rpm. At the time of maximum lipase activity, the mycelium and its debris were removed by a series of filtrations through Whatman 541, Whatman 1 and 0.45 pm filters. The cell-free medium was then concentrated to about 1 1 by an Amicon DClOL filtration system containing the Amicon Spiral Cartridge SlOY3 (M, 3000 cutoff) filter (Amicon Division, Danvers, MA, U.S.A.). The concentrated sample was lyophilized and stored at - 20 o C for later use. Lipase assay. The lipase activity was estimated using an emulsified substrate in a final volume of 450 ~1. This was prepared by shaking 111 pmol triolein, 1% gum arabic and 50 mM phosphate buffer (pH 6.8) in an Eppendorf shaker (model 5432) (120 rpm) for 5 min at 30 o C. The emulsified substrate was mixed with 50 ~1 of sample and the tubes continued shaking at 30°C. After the incubation period, 1 ml of chloroform/methanol mixture (1: 1, v/v) was added and vortexed for 2 min to stop the reaction and simultaneously extract the products. The tubes were centrifuged at 12000 x g for 2 min in an Eppendorf centrifuge and an aliquot of the lower phase (chloroform) was taken for the oleic acid estimation [27]. However, lipase activities of various column fractions were estimated by incubating an aliquot of the fraction with 111 pmol triolein in 50 mM phosphate buffer (pH 6.5) as described above. After the incubation period, an aliquot of the lipid phase obtained after centrifugation was mixed with acetone and analysed by high-performance liquid chromatography [27]. Molecular weight determination. The molecular weights of the purified lipases were determined by molecular sieving using Sephacryl-200 HR as matrix. About 2 mg of lipase was loaded at 30 ml/h on a 15 X 100 cm Sephacryl-200 HR column equilibrated with 50 mM phosphate buffer (pH 7.5) containing 0.1 M NaCl. 4-ml fractions were collected and their protein concentration and lipase activity were determined. Protein determination. Protein concentration was de-
termined by the method of Bradford [28] using bovine serum albumin as the standard. Ethanol extraction. 25 g of crude lipase powder was dissolved in 100 ml of 25 mM Tris-HCl (pH 7.5) (buffer A). The supernatant obtained after centrifugation at 20000 X g for 20 rnin at 4’ C was treated with 2 vol. of ice-cold ethanol drop-wise. The solution was constantly stirred at 0 o C during the addition of ethanol and for 1 h afterwards. The precipitated proteins were collected after centrifugation and dissolved in 75 ml of buffer A. Amino acid analysis. About 3 mg of the purified lipases were extensively dialysed against HPLC grade water. An aliquot (about 100 pg) was placed in Corning culture tubes (Cat. No. 9820, 6 X 50 mm) which were previously muffled at 450 o C overnight. The tubes were placed in Waters reaction vial and the samples were dried in the Waters Pica-Tag Work Station (Waters, Division of Millipore). Constant boiling HCl (200 ~1) containing 1% phenol was added to the vial and alternately purged (with dried nitrogen) and evacuated. After three purges, the vial was heated at 150°C for 1, 2, 3 and 4 h under vacuum. The values for ten amino acids stable to acid hydrolysis were not corrected. Values for Ser, Thr and Tyr were extrapolated to zero time, and Ile and Val were extrapolated to infinity. Tryptophan was determined by alkaline hydrolysis [29] and cysteine was detrmined as cysteic acid after performic acid oxidation [30]. Methionine was averaged throughout the timecourse. The analysis was performed on a Beckman System 6300 High Performance Analyser according to the general procedures of Spa&man et al. [31]. Electrophoresis. The purity of the enzyme was observed by non-denaturing (native) and SDS-denaturing polyacrylamide gel electrophoresis (PAGE) containing 4% stacking and 7.5% running gels. Slab gels (thickness of 1.5 mm) were run at 70 V until the tracking dye (Bromophenol blue) had penetrated the running gel and at 150 V until the Bromophenol blue had reached the 1 cm mark at the bottom of the slab [32]. Protein was detected by staining the gel with silver-stain solution (Bio-Rad). Non-denaturing gel electrophoresis was carried out at 15 o C and the gel was cut into two portions. One portion of the gel was stained, bands were identified and the areas corresponding to the bands in the other part of the gel were excised into small pieces. These pieces were directly incubated for assaying lipase activity. Isoelectric focusing. Isoelectric focusing was performed on 70 X 50 mm slab gels at 4“ C using ampholytes 4-6.5 (Pharmalyte 4-6.5) as recommended by the manufacturer. Prefocusing was done at 200 V for 10 min and samples (lo-25 pg) were applied in the middle of the gel containing 0.5 vol. of 10% acrylamide-0.3% bis-acrylamide, 0.134 vol. of glycerol, 0.064 vol. of Pharmalyte 4-6.5, 0.006 vol. of 2.3% ammonium persulfate and 0.29 vol. of distilled water. Gels were run at
28 1400 V for 2 h. After isoelectric focusing, gels were cut into small pieces (2 mm width) and each piece was incubated with 0.5 M phosphate buffer for 2 h. Triolein was added to the above buffer and lipase activities were estimated as described above. The pH gradient generated during isoelectric focusing was measured after incubating similar bits in 200 ~1 of distilled water for 4 h. Optimum pH determination. Purified lipase activity was measured at pH values ranging from 5.8 to 8.0 in 50 mM phosphate buffer as described above. Stability at different PH. Purified lipase (25 pg/ml) was incubated in 0.1 M citric acid/Na,HPO, buffer (pH 2.6-5.8) 0.1 M Na,HPO,/NaH,PO, buffer (pH 6.0-8.0) and 0.1 M H,BO,/NaOH buffer (pH 8.510.0). After a 30 min incubation at 4 o C, lipase activities were determined as described above.
Results Purification of extracellular lipases Extraction of lipase. All procedures were carried out at 4” C. Precipitation of lipase by ethanol treatment removed most of the coloured impurities present in the lyophilized medium. A 3-fold purification with a specific activity of 20.22 ~mol/min per mg protein was achieved by this extraction method (Table I). Chromatography on Sephacryl-200 HR. The precipitated proteins obtained by ethanol treatment were dissolved in 75 ml of buffer A and directly loaded on the Sephacryl-200 HR column (5 X 100 cm) (Fig. 1). Two main peaks of materials absorbing at 280 nm were detected and lipase activity was observed in the earlier shoulder of the second peak. After chromatography,
TABLE
I
Purification Purification steps a
1. Crude solution 2. Ethanol precipitate 3. Sephacryl200 HR 4. Ethanol precipitate 5. Mono Q 6. Chromatofocusing peak I peak II
of lipase
from Geotrichum candidum
Total activity
Total protein
( pmol/ min)
(mg)
411
78.25
6.09
1
1298
64.20
20.22
3
2 249
55.80
40.31
1
100
1835 1646
33.60 8.26
54.60 199.36
9 33
82 13
1398 393
Specific activity (gmol/min
Purification factor
Recovery (W)
300
I 200
100
0
20
40
60
FRACTION
80 NUMBER
100
120
Fig. 1. Chromatography on Sephacryl-200 HR. After dissolving the ethanol precipitated proteins in 25 mM Tris-HCl buffer, sample was loaded on a Sephacryl-200 HR column (5 X 100 cm), equilibrated in the same buffer. The flow rate was adjusted to 70 ml/h and 20 ml fractions were collected. Lipase activity is expressed as pmol/min.
active fractions were combined and concentrated by Amicon filtration containing a YM 3 membrane. Separation by molecular sieving increased lipase specific activity to 40.31 pmol/min per mg protein. Since the total lipase activity was increased by ethanol extraction and Sephacryl-200 HR chromatography steps, we considered the total lipase activity in Sephacryl-200 HR as 100%. Chromatography on mono Q. After concentration, the protein sample was subjected once more to ethanol precipitation before being loaded on a mono Q column equilibrated in buffer A. After washing the column to remove the unbound proteins, the lipase bound to the column was eluted by a salt gradient in buffer A. Lipase was eluted as a single peak of activity at 0.16 M NaCl (Fig. 2). Active fractions were pooled and concentrated by Amicon filtration. This anion-exchange chromatography augmented the specific activity to 199.36 ~mol/rnin per mg protein (a 33-fold purification) with a 73% recovery of the initial lipase activity.
b
per mg protein)
6.65 0.69
210.20 570.25
35 94
a All operations were performed at 4’ C. ’ % Recovery was calculated from Sephacryl-200 wards. For details refer to Results section.
FRACTION
62 18
HR
column
on-
NUMBER
Fig. 2. Chromatography on mono Q. Active fractions from the Sephacryl-200 HR column were pooled, concentrated, ethanol precipitated and dialysed against 25 mM Tris-HCl (pH 7.5). The sample was then applied on the mono Q 16/10 anion-exchange column equilibrated in Tris-HCl buffer. The sample was loaded at a flow rate of 60 ml/h and washed at a flow rate of 120 ml/h with same buffer. Lipase was eluted at a flow rate of 120 ml/mm with a linear salt gradient. Lipase activity is expressed as pmol/h.
29 r
0.5 ,
3000
T 6.0
the two isoenzymes. Lipase I and II had specific activities of 210.20 and 570.25 pmol/min per mg protein with purification factors of 35 and 94, respectively.
z F 2000 2 z 03
5.5
US >E 5"
0.2
5.0
I P
1000 iii 4.5 0.1
i
0.4 0.0
5
ch
I
4.0
FRACTION No. Fig. 3. Chromatofocusing on Polybuffer exchanger 94. Following concentration of active fractions from mono Q chromatography, the pooled fractions were dialysed against 25 mM Piperazine-HCl (pH 6.0) and applied on a colunm of Polybuffer exchanger 94 (1 X 30 cm) equilibrated in the same buffer. The flow rate was adjusted at 15 ml/h and 2 ml fractions were collected. The column was eluted at the same flow rate with 500 ml of 13-fold diluted Polybuffer 74 adjusted at pH 4.0. Lipase activity is expressed as pmol/h.
Chromatofocusing on Polybuffer Exchanger 94 (PBE 94). After dialysis at pH 6.0 with 25 mM piperazine-HCl, the sample was loaded on a PBE 94 column (1.0 X 30 cm) equilibrated in the same buffer. The column was then eluted with 13-fold diluted Polybuffer 74 adjusted to pH 4.0. Two peaks with lipase activities eluting between 4.68 and 4.44 were observed (Fig. 3). Active fractions from each peak were pooled separately, concentrated by filtration and dialysed against 10 mM phosphate buffer (pH 6.5). Chromatofocusing separated
Evaluation of purity by electrophoresis The sample obtained after mono Q and chromatofocusing columns were analysed by SDS-denaturing gel electrophoresis (Fig. 4A). The figure indicated the presence of a single band having a molecular weight of 56000, based on the molecular weight of standard proteins run under the same electrophoretic conditions. Also the sample obtained from mono Q column under non-denaturing condition showed the presence of a single band (Fig. 4B). Although the sample obtained from mono Q column showed only one band under SDS-denaturing and non-denaturing conditions, there were two proteins observed by isoelectrophoresis. Final separation of these proteins was achieved by chromatofocusing on PBE 94 column. Molecular weight of lipase The molecular weights of the lipases were determined by molecular sieving on Sephacryl-200 HR. Using the correlation between the K,, and the logarithm of the molecular weight of protein markers, the molecular weight of both lipases was estimated at 56000.
TABLE
II
Amino acid composition of lipase I and II a
A
Amino
B 2
1
3
4
92.5 K
66.2 K 92.5
K
66.2 K
._+_.ae W
WWWlrw *---
I
45.0K
45.0K
:
31.0 K
‘.:
31.OK
21.5 K 14.4 K
-
:
-”
21.5 K
14.4K Fig. 4. Polyacrylamide gel electrophoresis of the lipase samples. Electrophoresis in the presence of SDS (A) and without SDS (B) were performed as described in the Methods section. Gel A: 1, molecular weight standards; 2, lipase from mono Q column; 3, lipase I; and 4, lipase II from chromatofocusing column. Gel B: lipase from mono Q column under non-denaturing condition.
Asx Thrb Serb Gbi Gly Ala Val Met Ile Leu Tyr b Phe His Lys Trp’ Arg Pro cys *
acid
Lipase I (mo18)
Lipase II (mol%)
14.0 5.7 6.3 8.6 10.6 8.0 5.6 1.0 4.2 9.4 3.7 5.4 2.5 4.1 0.2 3.0 6.7 1.0
12.8 4.0 6.0 9.2 13.3 8.2 4.5 1.0 3.7 8.7 3.2 4.8 2.9 3.6 0.2 3.1 9.2 1.6
Proteins were gas-phase hydrolysed in 6 M HCl containing 0.1% phenol for 1, 2, 3 and 4 h at 150 OC. Determined by extrapolation to zero time. Determined by alkaline hydrolysis with 12 M NaOH at 110°C for 16 h [29]. Determined as cysteic acid by performic acid oxidation prior to acid hydrolysis [30].
30
3 10
15
20
GEL SLICE Fig. 5. Isoelectric Pharmalyte 4-6.5
25
30
0.0
35
0
10
GEL SLICE
No.
20
30
No.
point determination of lipase. Isoelectro-focusing of the purified lipase I and II was performed in polyac&rnide gel using as described in Materials and Methods. A, hpase I and B, lipase II. Lipase activity is expressed as pmol of oleic acid formed over a period of 2 h.
Amino acid composition
The amino acid composition of the purified lipase I and II are shown in Table II. They were quite homologous. The acidic residues (Asx and Glx) of lipase I and II constituted 22.6 and 22% of total amino acids, respectively. Basic amino acids (Arg, Lys and His) of both proteins contributed 9.6% of total amino acids indicating that the proteins had a very close net charge. Glycine, the predominant neutral amino acid, accounted for up to 10.6 and 13.3% of the total amino acids in lipase I and II, respectively. Isoelectric point
The isoelectric points of the lipases were determined by focusing on polyacrylamide gels using Pharmalyte 4-6.5 to generate a pH gradient. After isoelectrofocusing, gels were sliced and lipase activities were measured. The isoelectric points of 4.56 and 4.46 were observed for lipase I and II, respectively (Fig. 5). Optimum pH
To evaluate the optimum pH for lipases, enzyme activities were measured in 50 mM phosphate buffer at
pH ranging from 5.8 to 8.0 using triolein as substrate. The ester bond hydrolysis was maximum at pH 6.0 and 6.8 for lipase I and II, respectively (Fig. 6). Stability to different pH
Lipase I and II were pre-incubated at 4 o C in buffers of different pH. After 30 min, enzyme activities were measured at pH 6.0 and 6.8, respectively. For lipase I, the pre-incubation at pH 4 to 8 did not affect the enzyme activity and there was a slow decrease in activities below pH 4 and above pH 8 (Fig. 7A). Similarly for lipase II, the pre-incubation at pH 6.0 to 8.0 did not affect the enzyme activity and decrease in activities were observed below pH 6.0 and above pH 8.0 (Fig. 7B). Effect of various salts
The effect of various salts were tested on lipase activities. For these experiments, 50 mM phosphate buffer, pH 6.0 and 6.8 containing various salts were incubated with lipase I and II, respectively. NaCl and KCl, at concentrations up to 1.0 M, had no effect on lipase I activity. Divalent salts, such as CaCl,, MgCl,
65 = IE
50
40
> g 0 a
45
c > F
30
z
20
? c a
10
3 a 5
6
7
a
9
0 5
I
I
I
6
7
a
9
PH PH Fig. 6. Optimum pH for lipase activity. Lipase was incubated with 50 mM phosphate buffer (pH 5.8-8.0). A, lipase I; and B, lipase II. Lipase activity is expressed as pmol of oleic acid formed over a period of 2 h. Values are mean + SD. of four determinations.
31
a
101 . 2
# 4
I
6
-
I
.
I
.
2
12
10
a
4
6
12
10
a
PH
PH Fig. 7. pH stability of Iipase. Lipase was incubated in 0.1 M citric acid/Na,HPO,, Na,HPO,/NaH,PO, and H,BO,/NaOH buffers at pH ranging from 2.6 to 10.0. After a 30 min incubation at 4O C, the remaining activity was determined as described in Materials and Methods. A, lipase I and B, lipase II. Lipase activity is expressed as pmol of oleic acid formed over a period of 2 h. Values are mean f SD. of four determinations.
TABLE
III
Effects of various salts on lipase activity Lipase
Molarity
Lipase activity
(W of control)
a
(mM)
NaCl
KC1
EDTA
MgCI2 94 100 94 4
117 100 115 3
0 0 0 0
107 106 0 0
90 80 0 0
0 0 0 0
Lipase I
10 50 200 1000
116 105 116 104
115 110 114 101
78 73 70 3
91 23 0 0
Lipase II
10 50 200 1000
98 100 107 112
93 99 95 90
110 80 0 0
65 0 0 0
a Salts, at the final concentration indicated, were added at the beginning of the Iipase reaction. Materials and Methods and activity was expressed as % of control (no salts added). b The highest concentration was 500 mM.
and MgSO, had progressive inhibition at concentrations above 50 mM. SDS at a concentration of 10 mM completely inhibited the enzyme activity (Table III). Similar results were observed for lipase II with NaCl and KCl, divalent salts and SDS, except that the divalent salts at concentrations above 50 mM completely inhibited the enzyme activity. Discussion
We have reported a purification procedure for two lipases from G. candidum, lipase I and II, yielding a 35and 94-fold purification with 62% and 18% recoveries in the enzyme activities, respectively. The purified enzymes can be stored for weeks at 4’ C in 50 mM phosphate buffer (pH 6.5) without significant loss of enzyme activities. In general, both lipases appear to be stable only between pH 6.0 and 8.0. At acidic (below 5.0) and alkaline pH (above 9.0) the lipases are completely denatured.
b
CaCl 2
Lipase
MgSQ
activity
was measured
SDS
as described
in
Purification was essentially complete after anion-exchange chromatography (mono Q) as illustrated by a single band with SDS-PAGE. Isoelectrophoresis, however, proved that this sample was essentially two components having molecular weight 56000 and a difference in pI of 0.1 units. The separation of these components was accomplished by chromatofocusing with PBE 94. The isoelectric points were determined to be 4.56 and 4.46 for lipase I and II, respectively. The molecular weight obtained by molecular sieve chromatography under non-denaturing condition for both lipases was 56 000, a value consistent with the molecular weight deduced from SDS electrophoresis. Monovalent ions, like NaCl and KCl, had no effect whereas EDTA at concentrations above 10 mM significantly inhibited both enzymes. On the other hand, divalent ions, at concentrations above 10 mM,.caused a progressive inhibition of both enzyme activities reaching up to 100% at 1 M. SDS completely inhibited the enzyme activities at 10 mM concentrations.
32 The amino acid composition of these lipases were very similar, containing both cysteine and methionine unlike the lipase purified by Tsujisaka et al. [20]. Many other lipases have been purified and found to have molecular weights in the 50000-60000 region, but with little characterization of the enzymatic activity. The enzymes in this work have specificity for the 1 and 3 positions of the triolein. Among the fungal lipases characterized so far, Penicillium &opium lipase is more active on short-chain than on long-chain triacylglycerols. Lipases from A. niger and Rhizopus delemar show higher activities toward triacylglycerols of medium chain length, whereas G. candidum lipase is fairly specific for triacylglycerols containing unsaturated fatty acids with a double bond at position cis-9 [18]. Also we found that both enzymes from G. candidum have specificity for the 1,3 position of the triolein. There is very little similarity when the physical properties like molecular weight, isoelectric point, optimum pH, optimum temperature, pH stability and thermostability of these fungal lipases were compared [18]. Mammalian lipases, viz. hepatic lipase, lipoprotein lipase, pancreatic lipase, gastric lipase and lingual lipase, differ in several aspects from fungal lipases. The role of mammalian lipases is very complex as compared to fungal or bacterial lipases. Hepatic [33] and lipoprotein lipases [34] have broad substrate specificity in hydrolysing tri-, di- and monoacylglycerol, acyl-CoA thioesters and phospholipids. Human gastric lipase hydrolyses both short- and long-chain triacylglycerols and differs from pancreatic lipase in several major respects including optimum pH, molecular weight and activation and inactivation by various detergents [21,35-381. The lipase from G. candidum purified by Tsujisaka et al. [20] was homogeneous with a molecular mass of 53 000-55 000 and a pI of 4.33. There was no methionine and cysteine in this protein. However, the lipase obtained from the present purification method did not agree with some of the properties mentioned above. Also Jacobsen et al. [39] in their work with G. candidum observed lipases of two molecular weights, 61000 and 57000. Among these species are three lipases, with pI values of 4.42, 4.53 and 4.67, showing identical immunological responses. We have observed similar results, having isolated two lipases of 56 kDa with pZ values of 4.46 and 4.56. Recently, the presence of multiple enzymes were reported from different strains of G. candidum [39,40]. There is only a slight difference between the two proteins in their amino acid composition (more than 85% homology), pI and molecular weight. Therefore, it is speculated that G. candidum may have homologous genes encoding two or more lipases differing by a few amino-acid substitutions. This was confirmed by the recent work in which the genes responsible for these two
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