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J. lnst. Brew., September-October, 1993, Vol. 99, pp. 385-388

A RAPID SPECTROPHOTOMETRIC METHOD TO DETERMINE ESTERASE ACTIVITY OF YEAST CELLS IN AN AQUEOUS MEDIUM By L. Bardi, V. Dell'oro, C. Delfini

(Sezione di Microbiologia, Istituto Sperimeniale per I'Enologia, MAF, Via Pielro Micca 35, 14100 Asli, 1) AND

M. Marzona

(Dipartimento di Chimica Generate e Organica Applicaia, Universitd di Torino, C.so Massimo d'Azeglio, 48, 10100 Torino, I) Received 30 November 1992

A new rapid method to determine the total esterase enzymatic activity of yeast cells is proposed. In a sodium phosphate buffer a p-naphthol synthetic ester is hydrolized by cells, and the released pnaphthol Is coupled with a diazonium salt (Fast Garnet GBC) in the presence of sodium dodecyl sulfate. The whole procedure Is carried out in an aqueous buffer medium, and the resulting azo dye is directly evaluated by absorbance measurement at 524 nm. The analytical results from different assays were

adjusted to a fixed cell concentration with a statistical procedure. The method shows good repeatability, reproducibility and detectability, and it requires simple equipment and instruments. It is therefore suitable both for routine analysis, as industrial yeast strain screening, and for yeast physiological studies, in order to improve the aromatic quality of fermented drinks.

Key Words; Yeast, Saccharomyces cerevisiae, esterase, enzy matic activity, flavour. Introduction

An important quality parameter of fermented drinks is the aromatic composition. It is well known that yeast contributes to form the finished product's flavour, particularly by produc ing carboxylic acid esters. Some hydrolytic enzymes, the carboxyl esterases (E. C. 3.1.1.1), are produced by yeast; they can regulate ester concentration during alcoholic fer

mentation2-4 -7-*10.

Esterase enzymatic activity is therefore important in tech nological processes, and more knowledge on influencing fac tors may be useful to improve the aromatic quality of fer mented drinks. A rapid method to determine cellular estcrase activity is indispensable both for physiological studies and to select industrial yeast strains with different esterase activities. Methods to determine qualitatively and quantitiatively

esterase enzymatic activity are reported in literature2""13.

Generally, they require the utilisation of chromogenic syn thetic substrates, as a- and /3-naphthol or p-nitrophenol esters. After enzymatic hydrolysis, the released alcohols cou ple with diazonium dyes producing coloured compounds. In an aqueous medium these coupling products form col loidal precipitation; therefore, these methods are particularly suitable and usually used only in qualitative analysis, e.g. as enzyme localisation in a solid phase (electrophoretic gels, tissues, or cells). To allow quantitative spectrophotometrical determinations, extraction of the azo dyes by organic solvents is necessary8-13; but this means using polluting compounds and lengthy analysis. The purpose of this work was to provide a new rapid and simple spectrophotometrical method to determine the total endo and extracellular esterase enzymatic activity of yeast, avoiding the use of polluting solvents. Materials and Methods Yeast Strains

The yeast strains employed for this study were Saccharomyces cerevisiae YPH 98 (a, ura3, Iys2, add, Ieu2, trpl) and S185c (wine yeast strain from the culture collection CNLBSV of Istituto Sperimentale per I'Enologia di Asti).

Media and Growth Yeast strains were maintained at 4°C on YEPD slopes containing, per litre of water: yeast extract (Difco) 10 g, bacto-peptone (Difco) 20 g, glucose (Fluka) 20 g, agar (Difco) 25 g. Cells were cultured at 28°C in YEPD broth in conical flasks on an orbital shaker at 250 rpm.

Buffers and Stock Solutions Sodium phosphate buffer 100 mM at pH 7 was obtained by mixing 10.27 g of Na2HPO4-2H2O and 6.60 g of NaH2PO4-2H:O per litre of water and it was sterilized at 120°C for 20 min. Fresh solutions of diazonium dies were made daily in absolute ethanol (2.5 mg/ml of Fast Blue RR salt and 1 mg/ ml of Fast Garnet GBC sulphate salt). Stock solution of sodium dodecyl sulfate (SDS) was made by dissolving 10 g in 80 ml of sterile water at 68°C, adjusting the pH to 7 with NaOH IN and adding sterile water up to 100 ml.

Stock solution of /3-naphthyl caprylate 100 mM was made in absolute ethanol and maintained at 4°C in hermetic con tainers. Fresh solutions of /3-naphthol were prepared at dif ferent concentrations in absolute ethanol. All reagents were of the highest purity available commer cially from Sigma. Assay Procedure

Stationary-phase cell cultures (48 h) were harvested by centrifugation (4000 g, 6 min), washed in sodium phosphate buffer 100 mM at pH 7 and resuspended in the same buffer. Final cell concentration was determined by haemocytometer count.

8 ml of cellular suspension was placed in 15 ml sterile Corning centrifuge tubes. 160 /xl of /3-naphthyl caprylate stock solution were added, and hydrolysis reaction carried out for 30 min at 28°C on an orbital shaker at 350 rpm. Reaction was stopped by adding 1 ml of SDS stock solution. 1 ml of diazonium salt stock solution was added, and the coupling reaction with p-naphthol carried out for 15 min at 28°C. Then the samples were centifugated at 3500 rpm for 10 min, and the supernatant was used to measure absorbance at 524 nm against the blank sample which was obtained with the sodium phosphate buffer without adding cells.

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Absorbances were measured using an UV-Vis Cary Spectrophotometer Varian.

analysis procedure on 10 different days, with 4 replications/ day, using the YPH 98 strain.

Calibration Curves

Results and Discussion

Curve A Increasing concentrations of /3-naphthol (37.96 /nM, 30.83 /liM, 15.41 fiM, 7.10 fiM, 3.80 /oiM) were prepared in 8 ml sodium phosphate buffer 100 niM at pH 7 to which 1 ml SDS stock solution and 1 ml Fast Garnet GBC stock solution were added. Preliminary experiments showed that these /3-naphthol concentrations give absorbance values which fall within the linearity range. For each concentration the above described procedure was executed in triplicate. Spectrophotometrical measurements were made at 524 nm. The slope of this curve was named b, coefficient.

Assay Procedure Taking into account that the coupling of /3-naphthol with

Curve B In order to calculate the regression line between final absorbance values and cellular concentrations in the hydroly sis reaction buffers, the whole assay procedure was perfor med as described with increasing cellular concentrations. Preliminary assays with the YPH 98 strain at 6 different

cellular concentrations (from 2.7 x 10s to 2.76 x 108) showed a linearity range between 8 x 10s and 8 x 107 cells/ ml. Therefore, 5 cellular concentrations were finally chosen for estimating the calibration curve B, as follows: 8.1 x 10s, 2.08 x 107, 4.08 x 107, 6.09 x 107 and 7.70 x 107 cells/ml. For each cellular concentration the whole assay procedure was performed in quadruplicate. The slope of the resulting regression line was named b2 coefficient. For the SI85c strain, the following concentrations were assayed: 1.6 x 10", 6.4 x 106, 2.6 x 107 and 1.02 x 10* cells/ml. The relative regression line showed a high corre lation coefficient (0.999), therefore it was considered that this regression line was suitable to calculate b2 coefficient. Repeatability and Reproducibility Tests The method was tested for repeatability by 10 replications made for each cellular suspension the test was executed with

1.8 x 107, 1.49 x 107 and 3.15 x 107 cells/ml for the YPH

98 strain. For the SI85c strain, we executed 4 series of 4 replications

with 1.6 x 106, 6.4 x 106, 2.6 x 107 and 1.02 x 108 cells/ ml.

Reproducibility was evaluated by repeating the whole

diazonium salt would have been carried out in the same hydrolysis reaction medium, the optimum conditions to obtain the maximum speed of hydrolytic reaction by cells

(buffer, concentration and type of substrate) were selected from the literature'-4-5-6-7-11. Hydrolysis reaction time changes according to the cellular concentration. For the YPH 98 strain 30 min was the best

time for a cellular concentration ranging from 8 x 105 to 8 x 107 cells/ml: the quantity of the reaction product formed during this time seems optimum for spectrophotometrical analysis, because the absorbance values detected are within the linear range of the calibration curve (see below). To stop the hydrolytic enzymatic activity, SDS, a surface active agent, was evaluated. Its inhibiting action appeared to be instantaneous. In fact, its addition at 1.25% wt/vol final concentration at the beginning of the reaction immediately stopped the hydrolysis from happening (Table 1). SDS also has a function during the subsequent coupling of /3-naphthol with diazonium dye, because it dissolves the reaction pro ducts which have hydrophobic components. In our SDS tests it was demonstrated that, at the end of the assay procedure, a limpid matrix is formed which can be directly employed in the spectrophotometrical analysis, while without SDS this was impossible, because of the separation of suspended solids from the liquid phase. In this method the diazonium dye utilized was the Fast Garnet GBC sulphate salt; indeed, without modifying neutral

pH and temperature conditions3, maximum dyeing was achi eved in less than 20 min (Figure 1). Fast Blue RR salt3-"13 was also tested, but it appeared to require an analytically unacceptable time (more than 120 min). As shown in Figure 2, the absorption spectrum of the azo dye produced by Fast Garnet GBC emphasized a maximum absorption at 524 nm; this wavelength was therefore chosen for all measurements. Enzymatic Activity Calculation The quantity of /3 -naphthol produced by hydrolysis was deduced by the calibration curve A, shown in Figure 3. The

parameters found are: slope (b() = 0.0330; slope standard error = 0.0002; correlation coefficient = 0.998. TABLE I.

In order to compare data obtained in different assays, a

Control of Sodium dodecil sulfate (SDS) effectiveness to stop cellular esterasc enzymatic activity

sample ml /J-NC 100 mM fi\ SDS 10% wt/vol ml

reference cellular concentration of 5 x 107 cells/ml was

B

1

II

B,

I

II

8 160 1

8 160

8 160 1

8 160 0

160

1

8 160 0

chosen. Absorbances as for that common X-value sponding regression line for the YPH 98 strain is

adjusted Y-values were calculated cellular concentration by the corre (curve B). The curve B obtained shown in Figure 4; the parameters

8 0

Incubation for 30 min at 28°C

Abaorbanco (524 nm) 1 -

0.8-

SDS 10% wt/vol ml Fast Garnet GBC 1 mg/ml ml

0 1

0

0

1

1

1

1

1 1

1

Assay response

-

-

-

-

+

+

B = blank 1 (buffer without cells) B, = blank 2 (buffer without cells) I = cellular suspension 1 (2.8 x 107 cells/ml) II = cellular suspension 2 (8.6 x 107 cells/ml) 0-NC = /3-naphthyl caprylate + : red-violet colour developed (hydrolysis occurcd) -: no colour development (no hydrolysis occured)

1

0.6-

0.4

O.2

0

S

10

IS

20

25

30

Time (min)

Fig. 1. Kinetic coupling of /3-naphthol with Fast Garnet GBC.


Vol. 99, 1993]

387

ESTERASE ACTIVITY IN YEAST

TABLE II.

Absorbance

Repeatability tests for values obtained with the YPH 98 Sacchuromyces cerevisiae yeast strain III

Scries* 0.8-

Replication number Means' Standard Deviation Coefficient of Variation (%)

0,6-

0.4-

10 0.5817 0.01802 3.10

10

0.5530 0.00596 1.08

'Cellular concentrations: I = 1.18 x 10' cells/ml

0.2

400

10 0.5929 0.00518 0.87

600

500 Wavelength (nm)

Fig. 2. Azo dye (Fast Garnet GBC—/3-naphthol) absorbance spec trum; max at 524 nm.

II = 3.15 x 107 cells/ml III = 1.49 x 107 cells/ml 'Mean values of esterase enzymatic activity C expressed as nanomolcs/ml/min and adjusted to 5 x 107 cells/ml

0.7 fiM /3-naphthol concentration due to the hydrolitical action of 8 x 105 cells/ml. Esterase enzymatic activity C, expressed as nanomoles of the substrate hydrolyzed by X,, cells per min per ml, was calculated according to the following formula:

C"

b,t

where: b| = slope of curve A; b2 = slope of curve B; A = absorbance value at 524 nm;

X = sample cellular concentration, referred to final volume (10.16 ml); X,, = cellular concentration of reference; t = hydrolysis reaction time.

Fig. 3. Calibration curve for /3-naphthol different concentrations in sodium phosphate 100 mM buffer at pH 7 with 1% SDS and 0.1 mg/ml of Fast Garnet GBC (Curve A). Data obtained by 3 replications. Intercept: 0 Slope: 0.0330 Slope standard error: 0.0002 Correlation coefficient: 0.9981

Esterase enzymatic activity C of stationary phase cultured 5 x 107 cells/ml was 0.6499 nanomoles/ml/min for the YPH 98 strain.

Repeatability and Reproditcibility The results of repeatability and reproducibility test are

shown in Tables 2 and 3. The low values of coefficient of variation, which were always lower than 6%, emphasize that the proposed method is suitable both for routine analysis, as industrial yeast strain screening, and for yeast physiological studies. However, it is necessary to harvest cells in the same growth phase to guarantee the method reproducibility: the results shown were obtained with stationary phase cultures.

TABLE III.

Reproducibility tests for values obtained with the YPH 98 Saccharomyces cerevisiae yeast strain C

Days 1 2

Fig. 4. Calibration curve for /3-naphthol produced in the Saccharomyces cerevisiae YPH 98 suspensions at different cellular con centrations in sodium phosphate 100 mM buffer at pH 7 with 1% SDS and 0.1 mg/ml of Fast Garnet GBC (Curve B). Data obtained by 4 replications. Intercept: 0 Slope: 1.2955 Slope standard error: 0.0156 Correlation coefficient: 0.9970

3 4 5 6 7 8 9 10

Mean Standard Deviation Coefficient of Variation (%)

found are: slope (b2) = 1.29SS; slope standard error = 0.0156; correlation coefficient = 0.997. Since 8 x 10s cells/ml is the minimum concentration of the curve B linearity range, analytical detectability appears to be

0.5540 0.6144 0.5958 0.6144 0.6139 0.6671 0.6511 0.6507 0.6475 0.6308

2i :t :t 2t 2t ;t 2t 2t :t ±

0.0065 0.0120 0.0117 0.0120 0.0279 0.0023 0.0265 0.0237 0.0258 0.0076

0.6240

0.0.331 5.31

*C = csterase enzymatic activity (expressed as nanomols/ml/min and adjusted to 5 x 107 cells/ml) obtained as 4 replications mean.


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Method Applications

The proposed method enables the rapid measurement of the whole endo and extracellular yeast csterase enzymatic activity. It has good repeatability, reproducibility and detectability. and requires only simple equipment and instruments, so this procedure is suitable for any microbiological labora tory.

It is possible to extend this method to all Saccluiromyces cerevisiae yeast strains, and, in theory, also to other yeast species or other unicellular organisms, by calculating the relative b2 coefficient in the linear range of a specific cali bration curve B.

The curve B was calculated for the S185c wine yeast strain and the following parameters were found: slope (b2) = 0.9944; slope standard error = 0.0130; correlation coefficient: 0.999. Esterase enzymatic activity C of stationary

phase cultured 5 x 107 cells/mfwas 0.5200 nanomols/ml/min.

Repeatability test results, shown in Table 4, confirm the precision of this method which can easily be extended to other yeast strains.

Acknowledgments. The authors would like to thank Dr. M. Castino and Prof. A. Marchesini for helpful discussions and assistance in the preparation of the manuscript. References 1. Bardi. L., Dcll-Oro, V. & Delfini, C. X Convegno Sciemifico SIMGBM, Viierbo, 1991, 145.

2. Campbell. I.. Gilmour, R. H. & Rous, P. R. Journal of the Institute of Brewing, 1972, 78, 491.

3. Castro, G. R., Stettler, A. O., Ferrero, M. A. & Sincriz, F. Journal of Industrial Microbiology, 1992, 10, 165.

4. Parkkinen. E. Cellular and Molecular Biology, 1980, 26, 147.

TABLE IV.

Repeatability tests for values obtained with the SI85c Saccharomyces cerevisiae wine yeast strain

Series*

II

HI

IV

Replication number

4444

Means' Standard deviation Coefficient of variation (%)

0.5265 0.0193 3.67

0.5428 0.0222 4.09

0.5106 0.0082 1.60

0.5002 0.0060 1.20

'Cellular concentrations: I = 1.02 x 10" cells/ml II = 2.60 x 107 cells/ml III = 6.40 x 10* cells/ml IV = 1.60 x 10" cells/ml

'Mean values of estcrasc enzymatic activity C expressed as nanom-

olcs/ml/min and adjusted to 5 x 107 cells/ml

5. Parkkinen, E., Oura, E. & Suomalaincn, H. Journal of the Institute of Brewing, 1978, 84, 5. 6. Parkkinen, E. & Suomalainen, H. Journal of the Institute of Brewing, 1982, 88, 98. 7. Parkkinen, E. & Suomalainen, H. Journal of the Institute of Brewing, 1982, 88, 34.

8. Pilz, W. & Johann. 1. Zeitschrift fur Analytische Chemie, 1965, 210, 113.

9. Schermers. F. H., Duffus, J. H. & MacLeod, A. M. Journal of the Institute of Brewing, 1976, 82, 170. 10. Strobcl, R. & Wohrmann, K. Cenetica, 1972, 43, 274. 11. Suomalaincn. H. Journal of the Institute of Brewing, 1981, 87, 296.

12. Wernimont, G. Y. Use of statistics to develop and evaluate analytical methods. Arlington: AOAC, 1985. 13. Wheeler, J. E., Colcman, R. & Finean, J. B. Biochimica et Biophysica Ada, 1972, 255, 917.
