A simple and sensitive method for the estimation of microbial lipase activity

ANALYTICALBIOCHE~~STRY

186,301~305

(1990)

A Simple and Sensitive Method for the Estimation of microbian Lipase Activity Kannappan

Veeraragavan

Biochemical Engineering Section, Biotechnology Montreal, Quebec, Canada H4P 2R2

Received

June

Research Institute,

ho.

The potential applications of lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) have attracted several biotechnological companies, including those in the food, chemical, pharmaceutical, and agricultural industries. Lipase has a characteristic property of acting with a substrate at the interface between the aqueous and the lipid phase (1) and hydrolyzing triglycerides into diglycerides, monoglycerides, glycerol, and fatty acids. Although several methods have been reported in the literature (2), a simple and sensitive method is still needed for estimating the lipase activity from the batch cultures and column fractions. The water-soluble substrates used in most of the methods are not suitable for determining true lipase activity since they are also hydrolyzed by esterases. The most frequently used method is titration, in which the fatty acids produced by the enzyme are titrated by standardized NaOH (3). However, ~03-2697/90 Copyright AU rights

Research Council of Canada,

20,1989

A simple and sensitive method for the estimation of microbial lipase activity is described. In this method, lipase (EC 3.1.1.3) is incubated with an emulsified substrate and the fatty acid formed is estimated by highperformance liquid chromatography. The emulsified substrate, triolein, is prepared with various solubilizers, bovine serum albumin, gelatin, ovalbumin, gum arabic, Triton X-100, and n-octyl-glucopyranoside, in suitable buffers. The oleic acid, one of the products formed in the reaction, is separated on an ODS column with a premixed acetone-acetonitrile solvent system. Results with various lipase activities showed gum arabit to be the best among various solubilizers used to prepare the emulsified substrate. Also the effects of mono- and divalent ions on microbial lipase activities are analyzed. Finally, this method is compared with two other methods, titration and reversed micelles, and found to be simple and more sensitive. Q ISSO Academic Press,

National

$3.00 0 1990 by Academic Press, of reproduction in any form

this method is time consuming, less sensitive than radioactive methods (4,5), and impractical for titrating several samples obtained from batch cultures and purification steps. Other methods involve long procedures such as substrate preparation, solvent extraction, chemical modification, or tedious sample preparation. In this paper, a simple and sensitive method using an emulsified substrate is described. In addition the effects of various protein and nonprotein solubilizers on lipases are discussed. MATERIALS

AND

METHODS

Chemicals. Triolein (glyceryl trioleate, 99%), oleic acid (cis-9-octadecenoic acid, 99%), bovine serum albumin (BSA)’ (9899%), gelatin (porcine skin), and lipases from Pseudomonas species and Rhizopus arrhizus were purchased from Sigma Chemical Co. Lipase from Candidu rugosa (Candida cyl~ndr~eu) and n-octyl-glucopyranoside (1-o-n-octyl-~-D-glucopyranoside) were obtained from Boehringer-Mannheim Biochemicals, Montreal, Canada. Gum arabic (acacia) and HPLCgrade solvents were purchased from Fisher Chemical Co., Montreal, Canada. Lipase from Geotrichum candidum was grown in-house and partially purified. Apparatus. High-performance liquid chromatography analyses were performed with a 1090 HP liquid chromatograph system (Hewlett-Packard) equipped with a diode-array detector. Titration experiments were done with a Radiometer (PHM 82 standard pH meter, TTT 80 titrator and ABU 80 autoburette) Copenhagen, Denmark. Incubations were performed in an Eppendorf shaker (Model 5432; 120 rpm). Lipase assay. Lipase activity was estimated using an emulsified substrate in a final volume of 0.5 ml. This was prepared by shaking triolein, 1% gum arabic, and phos1 Abbreviation

used: BSA,

bovine

serum

aIbumin. 301

Inc. reserved.

302

~NNAPPAN

VEERARAGAVAN

0

0.6 ACID

OLEIC FIG. 2.

Linearity of absorbance oleic acid concentration. Changes tive to the area of the peak.

R8T8NtlOll

TINE

(ml4

FIG. 1. HPLC separation of monoolein (l), oleic acid (2), diolein (3), and triolein (4) observed from one of the samples in which the optimum conditions were used. The peaks were monitored at 208 nm with 2.0 absorbance units full scale.

phate buffer in the Eppendorf shaker for 5 min at 30°C. The emulsified substrate was mixed with 100 ~1 of lipase solution and the tubes continued shaking at 30°C for various incubation times. After the incubation period, 1 ml of chloroform-methanol mixture (l:l, v/v) was added and vortexed for 2 min to stop the reaction and simultaneously extract the products. The tubes were centrifuged at 12,000g for 2 min and an aliquot of the organic phase (lower) was taken for HPLC analysis. The oleic acid, monoolein, dioleins (both forms), and remaining triolein were separated by HPLC as described elsewhere (6). An ODS column (Cat. No. CSC-S ODS2, Chromatography Sciences Co., Inc., Montreal, Canada) maintained at 35°C was used to elute the above components with a premixed acetone-acetonitrile (l:l, v/v) solvent system. A stepwise flow rate of 0.8 ml/min from 0 to 10.00 min, 4 ml/min from 10.01 to 18.00 min, and 0.8 ml/ min from 18.01 to 20.00 min was used and the components eluted were monitored at 208 nm. The titration and reversed-micelle assays were performed as described by Peled and Krenz (3) and Han et al. (7), respectively. Lipase activity was expressed as micromoles of oleic acid formed under the incubation conditions. Protein co~e~trut~~. The protein concentration was determined by the method of Lowry et al. (8) using BSA as standard. The data were averages of four to six samples. RESULTS

The optimization experiments outlined in this paper were performed with lipase from C. rugosz. However,

1.2 ipmol)

change at 208 nm as a function of in absorbance were expressed rela-

similar experiments were also carried out with other lipases and the results were consistent. Figure 1 is a typical chromatogram of the lipase-catalyzed reaction. Acetone or chloroform alone when injected as a blank gave an artifact at 3.2 min. Monoolein, oleic acid, diolein(s), and triolein had retention times of 4.3, 4.8, 9.8 (lO.O), and 16.1 min, respectively. The relative areas obtained with 0.1 to 1.0 pmol of oleic acid prepared in acetone were linear (Fig. 2). The oleic acids produced in all the experiments were estimated in this range. Using various concentrations (0.1 to 5.0%) of gum arabic, a 1% concentration was adequate to obtain maximum lipolytic activity with 0.222 M triolein (data not shown). Consequently 1% gum arabic (final concentration) was used throughout the experiments. An optimum pH of 6.2 was observed by measuring the rate of triolein hydrolysis at various pH intervals for the lipase from C. rugosa (Fig. 3). Similarly optimum pHs of

800 -j

0 !

I

I

1

5

6

7

8

PH FIG, 3. Effect of pH on lipase activity. Incubations were carried out in a final volume of 0.5 ml containing 50 mM phosphate buffer, pH range from 5.5 to 8.0, with 1% gum arabic, 0.222 M triolein, and 2 &g of Iipase at 30°C for 30 min.

CHROMATOGRAPHIC 40

ESTIMATION

OF

LIPASE

303

ACTIVITY

8007

1

Y

0

20

60

40 TIME

.

I

*

I

0.2

0

(min)

TRIOLEIN

-

1

0.4 (M)

FIG. 4. Effect of incubation period on lipase activity. Assays were carried out in a final volume of 0.5 ml containing 0.222 M triolein, 2 pg of lipase, and 50 mM phosphate buffer, pH 6.2, with 1% gum arabic at 30°C.

FIG. 6. Effect of substrate (triolein) concentration on lipase activity. Incubations were carried out in a final volume of 0.5 ml containing 2 pg of lipase, triolein (0.037 to 0.37 M), and 50 mM phosphate buffer, pH 6.2, with 1% gum arabic at 30°C for 30 min.

6.8, 7.0, and 7.6 were observed for the lipases from G. candidum, Pseudomonas species, and R. arrhizus, respectively. Incubations were carried out at 30°C since at 37°C and above the reaction rate for various lipases declined with time (3,9-11). The effects of incubation period with enzyme activity are shown in Fig. 4. All subsequent incubations were performed within this linear time range. Linearity of the method was observed as a correlation between enzyme activity and lipase concentration (Fig. 5). The enzyme activity was linear up to 3 pg lipase per incubation (6 pg/ml). Incubation of 2 pg lipase with various concentrations (0.037 to 0.37 M) of substrate showed that the enzyme was saturated with 0.222 M triolein (Fig. 6).

The results obtained with the samples of 0, 1, 2, 4,8, and 16 h after the addition of the chloroform-methanol mixture indicated that the lipase-catalyzed reaction was completely terminated (data not shown). The effect of monovalent and divalent ions on microbial lipase activities is shown in Table 1. Although calcium was used to activate mammalian lipases (l2), its effects on microbial enzymes were not well documented. The present study showed that the presence of calcium ions up to 50 mM concentration in the assay medium without emulsifier

TABLE Effects

Enzyme Candida rugosa Geotrichum candidum Pseudomonas species Rhizopus arrhizus

1

ENZYME

2 CONC. (pg)

3

FIG. 5. Effect of enzyme concentration. Assays were carried out in a final volume of 0.5 ml containing various concentrations of lipase (0.5 to 3.0 fig/assay), 0.222 M triolein and 50 mM phosphate buffer, pH 6.2, with 1% gum arabic at 30°C for 30 min.

Salts on Lipase

Salt concentration bM)

-

0

of

10 50 200 10 50 200 10 50 200 10 50 200

1

Lipase

Activity” activity

(% of control)*

NaCl

KC1

CaCl,

100 100 100 100 102 101 104 98 95 100 89 90

100 100 100 loo 100 109 113 122 101 100 94 90

109 109 80 91 90 0 110 73 0 100 100 91

MgClz 121 131 175 94 106 94 100 100 110 100 100 90

a Values are average of four samples. Lipases from C. rugosa, G. candidum, Pseudomonas species, and R. arrhizus (5, 131, 12, and 1 pg, respectively) were incubated with 0.222 M triolein and various concentrations of salts mentioned above in 250 mM phosphate buffer containing no solubilizer for 30 min at 30°C. * The control experiments were performed without salts under identical conditions.

304

KANNAPPAN

VEE~~AGAVAN

did not affect lipase activities, whereas higher concentrations (200 mM and above) actually inhibited most of the enzyme activities. Lipases from C. rugosa, G. candidum, Pseudomonas species, and R. arrhizus were analyzed for their activities with the emulsified triolein prepared by various solubilizers, BSA, gelatin, ovalbumin, gum arabic, T&on X100, and n-octyl-glucopyranoside. It was observed that gum arabic was the best solubilizer in this system and the only one that reproducibly activates all of the enzymes tested. In addition, other solubilizers such as BSA and gelatin did not have much effect and Triton X-100 and ~-octyl-glucopyranoside were generally inhibitory. Also it is observed that all but n-octyl-glucopyranoside were useful in the assay with the R. arrhizus enzyme (Table 2). The lipase activities obtained by the gum arabic method were compared with the titration and reversedmicelle methods (Table 3). For the same concentration of enzyme, the present method gave an activity range higher than that with other methods. It was also observed that 0.025 Fmol of oleic acid can easily be measured, whereas in the other methods (3,7) the range was 5 to 50 f*mol. DISCUSSION

The data obtained with this method indicated that the procedure is simple and more sensitive than other methods used. The method was developed with (a) gum arabit, a compound known to produce emulsion systems with various lipid substrates (13-16); (b) triolein, a good substrate for most of the microbial lipases (17); and (c) a sensitive method for estimating the products (fatty

TABLE

2

Effect of Various Solubilizers on Lipase Activitya Lipase activity

Lipase candida ?Tgosa Geotrichum candidurn Pseudomonus

BSA

Gelatin

GUllI arabic

(X of control)’

Ovalbumin

T&ton X-100

nOctylglucopyrsnoside

50-62

7484

180-212

34-40

36-42

5

90-105

95-120

200-230

40-50

SO-93

5

60-68

72-79

104-115

65-75

18-19

161-187

229-238

476-493

212-246

170-178

45-51

species Rhizopus arrhizus

5

fi Lipases from C. rugosu, G. candidurn, Pseudomonas species, andR. nrrhizus (2, 30, 12, and 1 pg, respectively) were incubated in a final volume of 0.5 ml containing 0.222 M triolein and 250 rnM phosphate buffer with 1% solubilizer for 30 min at 30°C. b The control experiments were performed without solubilizer under identical conditions.

TABLE

3

Comparisonof Lipase Activities by Different Methods” Lipase activity (pmo1/30 min)

Method Titration* Reversed HPLCd

33.0 t 0.8 10.1 f 0.4 64.5 + 2.8

micelles’

’ In all the methods, 7 Mg of lipase from C. rugosa was incubated with triolein for 30 min and the product formed was estimated as described according to each method. b Peled and Krenz (3). ’ Han et al. (7). * As described in Table 2.

acids) formed. The present method is three- to sixfold more sensitive than previously reported methods (3,7). This is the first report describing the optimum conditions for preparing emulsified substrate for microbial lipases with the best solubilizer, gum arabic. It is very important to understand not only the degree of dispersion but also the nature of emulsifiers. Earlier studies showed that lipases from various mammalian sources were activated (9), inactivated (l&19), or unaffected (20) by bile salts. Albumin inhibited porcine pancreatic lipase by unfolding the enzyme at the watersubstrate interface (19). Unlike mammalian lipases, the effect of emulsifiers on microbial lipases was not extensively studied. Hence, the present work was carried out to study various emulsifiers and to select the best for microbial lipases. All the lipases used had higher velocities with gum arabic. Monovalent as well as divalent ions up to 200 mM did not affect lipase activities, except for the lipase from C. rugosa, in which there was an increase with MgClz, suggesting that there was no inactivatio,~ of the enzyme or product interference by ions. Also the activities of microbial lipases, unlike those from mammalian sources (12), were not increased by calcium. This technique is very useful for measuring the hydrolytic reactions of lipases using emulsified substrates. Due to the differences in the retention times of fatty acids and their glycerides, this method is appropriate for analyzing the substrate specificities of lipases. ACKNOWLEDGMENTS The author thanks Dr. A. Storer for his technical assistance.

for his suggestions

and B. F. Gibbs

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