BIOTECHNOLOGY AND BIOENGINEERING VOL. XIII, PAGES 549-560 ( I ! ) i l )

Release of Enzymes from Bakers' Yeast by Disruption in an Industrial Homogenizer

MAGGIE FOLLOWS, P. J. HETHERINGTON, P. DUNNILL, and M. D. LILLY, Biochemical Engineering Section, Chemical

Engineering Department, University College London, London, England

Summary The rates of release of 7 enzymes from bakers' yeast have been measured.

The disruption process did not cause loss of activity of these enzymes. The various operating pressures, temperatures, and initial yeast concentrations used did not affect the rates of enzyme release relative to protein release. The release of acid phosphatase and invertase was faster than the overall protein release. Alcohol, glucose-&phosphate, and 6-phosphogluconate dehydrogenases were released slightly faster or a t the same rate as the overall protein and alkaline phosphatase and fumarase were released more slowly. These observations corre- late well with the reported locations of these enzymes in the yeast cell.

INTRODUCTION

The release of intracellular enzymes from microorganisms is an important step in their isolation. Various techniques have been used for this purpose.'e2 Previously we have reported3 the release of protein from bakers' yeast by disruption in a high pressure homog- enizer. We showed that for yeast slurries not exceeding 600 g packed yeast/liter the rate of release of protein could be described by the relationship :

log (R,/(R, - R ) ) = K N P 9

where R is the amount of soluble protein released and R, is the maximum amount of soluble protein that can be released; in our experiments, R, was 96 mg protein/g packed yeast. K is a dimen- sional temperature-dependent rate constant, N is the number of

549 @ 1971 by John Wiley & Sons, Inc.

550 FOLLOWS ET A L .

times the slurry was passed through the homogenizer valve, and P was the operating pressure.

In this paper we describe the release of seven enzymes from bakers’ yeast using the same disrupting device. The choice of enzymes was based on several criteria-the cost and ease of assay, their location in the intact yeast cell, and the potential usefulness of the isolated enzymes. The enzymes were acid and alkaline phosphatases (ortho- phosphoric monoester phosphohydrolases, E.C.3.1.3.1. and E.C.- 3.1.3.2.), invertase (8-D-fructofuranoside fructohydrolase, E.C.- 3.2.1.26), fumarase(Lma1ate hydrolase, E.C.4.2.1.2.) and alcohol, glucose-6-phosphate and 6-phosphogluconate dehydrogenases (alco- hol: NAD oxidoreductase, E.C. 1.1.1.1.) D-glucose-6-phosphate: NADP oxidoreductase, E.C.1.1.1.49. and 6-phospho-D-gluconate : NADP oxidoreductase (decarboxylating), E.C.l.l. 1.44.). For each enzyme the rate of release has been correlated with the rate of re- lease of protein from yeast disrupted at several concentrations, pres- sures, and temperatures. When high yeast concentrations were dis- rupted at high operating pressures the above relationship for protein release was not obeyed. Experiments on enzyme release therefore were done at low and high yeast concentrations, 450 and 750 g packed yeast/liter. Enzymes from different parts of the yeast cell were likely to be released at rates different from the bulk of the protein. Since not only cell disruption but also solubilization of enzymes bound to subcellular particles was likely to be pressure- dependent, three different operating pressures were used. Protein release increased with temperature up to about 35°C. Above this temperature denaturation led to a reduced yield of soluble protein. Experiments on enzyme release were done at 5°C and 30°C to find out if temperature had a similar effect on the rate of release of particu- lar enzymes and to examine the stability of these enzymes during the disruption process.

METHODS

Bakers’ yeast was obtained from the Distillers Company Ltd. and stored at 4-7°C. For disruption experiments it was suspended in 0.15 M sodium chloride + 4 mM dipotassium hydrogen phosphate solution. All concentrations of yeast suspensions are expressed as g packed yeast/liter.

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DISRUPTION OF YEAST FOR ENZYME RELEASE 55 1

The Manton-Gaulin Homogenizer model 15M-8BA was supplied by the APV Company Ltd., Crawley, England. All temperatures quoted are those of suspensions entering the homogenizer. The suspensions leaving the homogenizer were 4-5°C warmer. In all experiments reported here the yeast suspension leaving the homo- genizer was passed through a plate heat exchanger to readjust the temperature rapidly (within 15 sec) before returning to the reservoir feeding the homogenizer. These recycle experiments were done with a total liquid volume of 4.4 literg. Samples for assay were with- drawn a t intervals from the reservoir. Full details of the homog- enizer and its operation have been reported el~ewhere.~

Samples of disrupted suspensions were centrifuged a t 34,000 g and 5°C for 1 hr in an angle-head laboratory centrifuge and the super- natant assayed for enzyme activity and protein concentration.

Most of the assays were done using a Technicon Autoanalyser. Samples were diluted automatically to bring them into the range of protein concentration, 0-1 mg/ml. Parts of the diluted sample stream were fed to three separate manifold systems for assay of dehydrogenase activity, phosphatase activity, and protein. The remainder of the diluted sample stream went to waste. The three dehydrogenases were assayed by passing separate sets of diluted samples through the manifold three times but with different sub- strates. Similarly the manifold for phosphatase assay was used twice, using the same substrate but a t two different pH values. Alcohol dehydrogenase and acid phosphatase (both supplied by Boehringer (London) Corporation Ltd., London, W.5, U. K.) were used to calibrate the manifold systems. The reaction conditions are given in Table I . The substrates were not in excess in all cases so that the reported activities were not necessarily maximal values. Protein was determined using Folin-Ciocalteau’s reagent, (BDH Ltd., Poole, England) with bovine serum albumen-Fraction V pow- der, (Sigma (London) Chemical Co. Ltd., London S. W. 6, U. K), as standard. The fumarase activity was determined by the rate of formation of fumarate from L-malate in the spectrophotometric method described by Racker4 except that tris buffer was used instead of phosphate. The assay of invertase was based on the method of Gascon and L a m ~ e n . ~ The glucose formed was determined using Fermocotest (Hughes & Hughes Enzymes Ltd., Brentwood, Essex, U. K.).

552 FOLLOWS ET AL.

TABLE I Approximate Reaction Conditions for Automated Assay of Enzymes

Final concentration Reaction Enzyme of reactants conditions

ADH

GPGDH

GGPDH

Acid phosphatase

Alkaline phosphatase

0.2M ethanol 0.21 x 10-aM NAD

0.24 x 10-3M 6PG 0.20 x 1 0 - 3 ~ NADP

as for GPGDH and 0.27 x 10-3M G6P

0.06M PNPP

0.06M PNPP

O.1M tris pH 8.5, 7 mM semicarbaeide; assayed at 340 nm

0.07M MgCl, 0.1M tris, pH 7.4; assayed

at 340 nm

as for 6PGDH

0.08M sodium citrate, pH 4.8, reaction stopped by 2 vols of 0.5M NaOH; as- sayed at 420 nm

0.4M tris, pH 8.0 reaction stopped by 2 vols of 0.5M NaOH; assayed at 420 nm

All enzyme assays were carried out at 30°C and activities are expressed as pmoles of substrate utilized/min. The values for enzyme release, E , (pmoles/min/g yeast) and protein release, R (mg protein/g yeast), were calculated from

where CE and CB are the enzyme activity/ml and protein concentra- tion for the clarified disrupted yeast, C , was the starting yeast con- centration (g/ml), and F is the fraction of aqueous phase in the disrupted yeast suspension. Values for F have been reported pre- v iou~ ly .~

RESULTS

Fjl'ect of Disruption Time The aim of the work reported in this paper was t o investigate the

relationship between the rate of release of particular enzymes and BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII, ISSUE 4

DISRUPTION OF YEAST FOR ENZYME RELEASE 553

the rate of release of soluble protein. Since the rate of release of soluble protein was time-dependent, i t was necessary to examine the effect of disruption time on the activities of the released enzymes.

In preliminary experiments done over the range 2-55"C, significant losses of activity of the enzymes with time were observed a t tempera- tures over 35°C. All further experiments were done a t 5 or 30°C.

At high operating pressures over 95y0 of the extractable protein was released in 30 min. During the next 60 min for experiments both at 5 and 30°C, there was no decrease in the enzyme activities in the extracts, confirming that during the period of these experiments enzyme denaturation was insignificant. Only in two isolated cases, once for fumarase and once for invertase (see Figs. 7 and S), was there an apparent decline in activity during the disruption process.

Except for these two cases, as no losses of enzymic activity were observed during the experiments at 5 and 30"C, the relationship between enzyme release and protein release was independent of the actual rates of release of protein. Thus i t was possible to present all the results for each enzyme in the form of plots of enzyme release versus protein release. Readings obtained after disruption was al- most complete have not been included, since these points would all lie very close to each other on such graphs.

Most disruption experiments lasted for 90 min.

Reproducibility of Results

Experiments were done over many months. To determine the variation in enzyme content and release between batches, several replicate experiments were done at intervals. The results of two such experiments are shown in Figure 1. From these and other experiments we concluded that the variation in enzyme content between batches of yeast was within the experimental error.

Effect of Valve Seat Design

Also shown in Figure 1, are the results of an experiment in which t!he knife-edge valve seat was replaced by the standard flat-valve seat. Although the rate of protein release with the latter valve seat was found to be lower,3 the ratio of enzyme to protein release is unaffected.

554 FOLLOWS ET AL.

E

1

20 a 60 m 100

R

Fig. 1. Enzyme release vs. protein release curves for three experiments using 750 g/liter yeast suspension at 30°C and 460 kgf/cm* operating pressure (open symbols for two identical experiments using a knife-edge valve seat, closed symbols for an experiment with a flat valve seat): (0, 0) acid phosphatase ( X 0.8); (0, +)invertase ( X ( A , A ) G6PDH ( X 0.1); (a, m) fumarase ( x 0.02).

Effect of Storage Bakers’ yeast was supplied at weekly intervals, stored at 4°C and

used within 2 days. After storage of a batch for 7 days the yo changes in enzyme activity released/g yeast were : acid phosphatase, - 16; alkaline phosphatase, -9; invertase and ADH, -4; GPGDH, +4; GGPDH, +S; fumarase, + 14. There was no detectable change in rate of release of these enzymes after the yeast had been stored for 7 days. Thus the changes in the enzyme activities of the yeast during the two days within which experiments were done were small.

Effect of Operating Variables The results for 7 experiments done at two yeast concentrations

(450 and 750 g/liter), two temperatures (5 and 30°C) and three pressures (100, 270, 460 kgf/cm2) are given in Figures 2 through 8. For each enzyme a single curve can be fitted to the results. The rates of release of the enzymes relative to protein release seem, within experimental error, to be independent of the operating conditions.

BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIII, ISSUE 4

DISRUPTION OF YEAST FOR ENZYME RELEASE 555

3

2 E

1

20 40 60 80 100

R

Fig. 2. Acid phosphatase release vs. protein release for yeast concentrations of 450 g/liter (closed symbols) and 750 g/liter (open symbols) at the following operating pressures (kgr/cm*) and temperatures ("C) : (0 ) 460, 30°C, (0) 460, 5°C; (A) 270, 30°C; (+) 100, 30°C (450 g/liter only).

3

E 2

1

20 40 60 80 100

R Fig. 3. Alkaline phosphatase release vs. protein release. Symbols as in Figure 2.

556

30t FOLLOWS ET AL.

?

2o t E !

20 &O 60 60 1M)

R

Fig. 4. ADH release vs. protein release. Symbols as in Figure 2.

For comparison the curves from Figures 2 through 8 have been These results are expressed as % release replotted in Figure 9.

where 100% release corresponds to E when R = 96 mg/g yeast.

DISCUSSION

The results presented show that the Manton-Gaulin/APV homog- enizer may be used for the release of a variety of enzymes from bakers’ yeast. Except in 2 isolated cases, release of the enzymes

Fig. 5.

20 40 60 80 1M)

R GGPDH release vs. protein release. Symbols as in Figure 2.

BIOTECHNOLOGY BND BIOENGINEERING, VOL. XIII, ISSUE 4

DISRUPTION OF YEAST FOR ENZYME RELEASE 557

20 40 60 80 100

R Fig, 6. GPGDH release vs. protein release. Symbols as in Figure 2.

was completed without apparent loss of enzyme activity even when disruption was done at 30°C. The release of an enzyme relative to the overall protein release was independent of the disruption pressure, the temperature, and initial yeast concentration.

The enzymes investigated were released at different rates (Fig. 9). Acid phosphatase and invertase are predominantly outside the cell

Although an intracellular invertase is present in yeast, it is only a small proportion of the total invertase when the

30

20

E

10

/

/: 20 LO R 60 80 100 J

Fig. 7. Fumarase release vs. protein release. Symbols as in Figure 2.

558 FOLLOWS ET AL.

2 0 0

20 LO 60 80 100

R Fig. 8. Invertase release vs. protein release. Symbols as in Figure 2.

invertase activity of the yeast is high.* Thus acid phosphatase and invertase are released faster than the rest of the protein and intra- cellular enzymes. Matile et d9 state that in yeast, alcohol dehy- drogenase, enzymes of glycolytic pathway and of the oxidative pentose phosphate cycle are soluble enzymes. Rodgers and Hughedo found that alcohol dehydrogenase release from yeast by ultrasonics

2 0 4 0 6 0 8 0 K x )

PROTEIN RELEASE (*Id

Fig. 9. Enzyme release vs. protein release for: 1, acid phosphatase; 2, invertase; 3, G6PDH and 6PGDH; 4, ADH; 5, alkaline phosphatase; and 6, fumarase.

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DISRUPTION OF YEAST FOR ENZYME RELEASE 559

was faster than overall protein release and similar results were obtained by Rosett."

Alcohol dehydrogenase, G6PDH and 6PGDH were released at the same rate or slightly faster than the overall protein release. Hughes'O found that fumarase was released very slowly. Nossall* reported that fumarase was mainly present in the mitochondria and was only solubilized after further shaking with glass beads. A similar effect, was observed in our experiments where fumarase release was slower than overall protein release. The exact location of alkaline phospha- tase in the yeast cell is not certain but Suomalainen et aL16 reported that most of the alkaline phosphatase was in the plasma membrane. The slow release of this enzyme in our experiments is compatible with this location. Thus the rates of release of the enzymes we have investigated are in good agreement with their reported locations in the yeast cell. The differences between these rates however are not sufficient to allow fractionation of the enzymes into groups a t this disruption stage.

ADH G6P GGPDH NAD NADP 6PG 6PGDH PNPP

C E

C R

Cll E F R R ,

Abbreviations alcohol dehydrogenase glucose-6-phosphate glucose-6-phosphate dehydrogenase nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate 6-phosphogluconate 6-phosphogluconate dehydrogenase p-nitrophenyl phosphate

Symbols enzyme activity per ml, pmoles/min/ml protein concentration, mg/ml yeast concentration, g packed yeast/ml enzyme activity release, pmoles/min/g packed yeast fraction of aqueous phsse in disrupted yeast suspension protein release, mg/g packed yeast maximum protein release, 96 mg/g packed yeast

The authors wish to thank Whatman Biochemicals Ltd., for their support of this work and Mr. G. Buch and Mr. F. Farky for their invaluable technical assistance.

560 FOLLOWS ET AL.

References 1. J. W. T. Wimpenny, Process Biochem., 2(7), 41 (1967). 2. L. Edebo, in Fermentation Advances, D. Perlman, Ed., Academic Press,

3. P. J. Hetherington, M. Follows, P. Dunnill, and M. D. Lilly, Trans.

4. E. Racker, Biochim. Biophys. Acta, 4, 211 (1950). 5. S. Gascon and J. 0. Lampen, J. Biol. Chem., 243, 1567 (1968). 6. H. Suomalainen, T. Nurminen, and E. Oura, Arch. Bwchem. Biophys.,

7. W. L. McLellan and J. 0. Lampen, Biochim. Biophys. Acta, 67,324 (1963). 8. M. Burger, E. E. Bacon, J. S. D. Bacon, and J. W. Millbank, Nature,

9. P. Matile, H. Moor, and C. F. Robinow, in The Yeasts, Vol. l., Academic

New York, 1969, p. 249.

Inst. Chem. Eng., 49, 142 (1971).

118, 219, (1967).

205, 622 (1965).

Press, New York, 1969, ch. 6. 10. D. E. Hughes, J. Biochem., Microbiol., Technol. Eng., 3, 405 (1961). 11. T. Rosett, Appl. Microbiol., 13, 254 (1965). 12. P. M. Nossal, Biochem. J . , 57,62 (1954).

Accepted for Publication May 10, 1971