Enzymatic Lysis of Yeast Cell Walls

D. KNORR,” K. J . SHETTY, and J . E. KINSELLA, Department of Food Science, Cornell University, Ithaca, New

York 14853

Summary Lysis of yeast cell walls using zymolase and lysozyme was studied. During a

coupled zymolase-lysozyme treatment, nearly three times more reducing sugars were released from the yeast cells compared to controls. Enzyme treatment followed by extraction at pH 9 resulted in a yield of more than 80% of the total nitrogen of the yeast cell. Protein degradation occurred during enzyme treatment. The precipi- tation of proteins was significantly increased by succinylation after enzyme treat- ment. This also reduced the nucleic acid content of the yeast proteins to less than 2% and enhanced the extractability of nitrogenous material.

INTRODUCTION

Current problems associated with the use of single cell proteins (SCPs) as a human food include the undesirably high levels of nucleic acids and the questionable nutritional value of whole dried cells. The nucleic acids (which if consumed in large amounts cause uricemia and gout’) can be markedly reduced by succinylation dur- ing extraction of the protein from y e a ~ t . ~ , ~ . The limited nutritional value of intact cells can be improved by separation of the protein from the cell-wall material.4-5 The thick, tough, and rigid cell walls of the whole yeast cells resist intestinal digestion thereby limiting the availability of intracellular protein.6 Consequently, it is neces- sary to remove or break the cell wall to facilitate digestion of intracellular proteins.

Numerous methods such as autolysis (plasmolysis), chemical, and mechanical treatments, have been used for cell disruption and the release of cell components. The major disadvantages of autolysis are the long reaction time required, the low yield of protein, and

* Present address: Department of Food Science and Human Nutrition, University of Delaware, Newark, Delaware 19711.

Biotechnology and Bioengineering, Vol. XXI, Pp. 201 1-2021 (1979) @ 1979 John Wiley & Sons, Inc. OOO6-3592/79/Oo2 1-20 1 1$0 I .OO

2012 KNORR, SHETTY, AND KINSELLA

high cost^.^'^ The principal problems with chemical treatments such as urea or butanol are recovery of chemicals used, potential toxicity, and costs.8-11 Protein denaturation, restriction to laboratory scale, apparatus inefficiency, and high cost are commonly reported prob- lems of mechanical disruption methods such as homogenization, pressure release, sonic oscillation, grinding, stone milling, freeze- thaw, etc.12-17 Proteolytic enzymes have been used for the release of intracellular components of the microbial cells but little specific information is available. 18*19 There have been report^^^-^^ showing that various microorganisms accumulate enzymes with lytic activ- ities against yeast cell walls in culture media. Some of these en- zymes digest yeast cell walls effectively only after the yeast has been subjected to heat treatment.

Enzymes secreted into cultures of Bacillus cirulans caused com- plete lysis of the cell walls of bakers’ yeast.25 In contrast to the crude culture fluid, two P-glucanases, purified from the media, assumed to be responsible for the cell-wall lysis, showed very lim- ited hydrolytic action on yeast cell walls.26

Arthrobacter luteus secretes enzymes with very strong lytic ac- tivity on yeast cell walls into their culture f l ~ i d . ~ ~ , ~ ~ These enzymes have been isolated from A . luteus and were thoroughly studied by Kitamura et al.28-31 and Kaneko et al.32 Studies on the hydrolysis of p-( 1+3)-glucans (i.e., yeast glucans) showed that this enzyme zymolase, is an endo-p-( 1 +3)-glucanase, which specifically releases laminaripentose as the product.

This paper describes studies on the enzymatic lysis of cell walls of brewer’s yeast using zymolase and lysozyme combinations and the recovery of the yeast protein. This is part of ongoing studies to develop practical methods for the extraction of intact, undenatured protein, with low nucleic acid content from yeast cells.

MATERIALS AND METHODS

Materials

Batches of brewer’s yeast in the stationary growth phase (ob- tained from Genesee Brewing Co., NY) were washed in distilled water (ratio 1 : 10) and centrifuged at 12100 g . The supernatant was discarded and the yeast cells resuspended in distilled water. This procedure was repeated three times. The yeast cells were freeze- dried and stored at 3 3 ° C . Zymolase-5000 (The Research Labora- tories, Kirin Brewery Co., Ltd., Takasaki, Japan) and lysozyme

YEAST LYSIS 2013

(“Muramidase”) from egg white (Sigma Chemical Co., St. Louis, MO) were used for the enzyme treatments.

Methods

Total carbohydrate was determined with the phenol-sulfuric acid method.33 D-Glucose was used for establishing the calibration curve, and the carbohydrate content was expressed in glucose equivalents. Nitrogen was ascertained by the Kjeldahl procedure. For the de- termination of protein and nucleic acid concentrations, the methods of Lowry et al.34 and Herbert et al.33 were used, respectively. Succinylation was carried out according to the method described by Shetty and K i n ~ e l l a . ~ A Braun mechanical cell homogenizer (Braun-Melsung, West Germany) was used to disrupt the yeast cells .35

Lysis Procedure

Freeze-dried yeast was suspended (2.5% w/v) in phosphate buffer (50mM, pH 7 . 3 , the slurry was stirred for 5 min at room temper- ature, and then the buffer solutions containing the enzyme were added. The incubation was carried out in a shaking incubator at 37°C. The samples were cooled and centrifuged at 4°C at 12100 g for 30 min. The supernatant was used for the determination of total carbohydrates released by the treatments. For the nitrogen and protein release studies, after initial incubation the samples were stirred at pH 7.5, or at pH 9.0 for an additional 30 or 45 min and then centrifuged. All experiments were repeated four times.

RESULTS

Effect of Enzyme Concentration on Yeast Cell Wall

The integrity of yeast cell is mainly determined by the structured carbohydrate-protein complex of the cell waL6 The release of re- ducing sugars following the incubation of cells with zymolase or lysozyme was monitored to determine the effect of these enzymes on cell-wall structure. Freeze-dried cells were suspended in phos- phate buffer (50mM, pH 7.5) and incubated with various enzyme concentrations (16 to 4000 pg enzyme/g yeast or an enzyme: yeast ratio ranging from 1:250 to 1:64000) for 2 hr at 37°C. After incu- bation, the suspension was centrifuged, and the carbohydrates in the supernatant were estimated (Fig. 1). The controls without en-

2014 KNORR, SHETTY, A N D KINSELLA

0 230 - W v) Q W I --It u r n = a

Y w > + 4 - L L O D t O I.. 0 0 W E L L - Q 100 - u

0

I I I I 1000 2000 3000 4000

ENZVME CONCENTRATION ( V G / G OF Y E A S T )

Fig. 1 . Influence of enzyme concentration on the release of carbohydrates from yeast cell followed by incubation at 37°C for 2 hr. (0) Zymolase; (0) lysozyme.

zymes resulted in a carbohydrate release of 44.3 * 5.6 mg/g dry yeast. The lysozyme treatment had no significant effect on the release of carbohydrate; however, zymolase progressively en- hanced the release of carbohydrates at increasing concentrations of enzyme.

Because of their differing specificities, mixtures of zymolase and lysozyme in varying concentrations were studied (Table I). A com- bination of both enzymes enhanced the release of carbohydrates from yeast cell walls. By using a combination of zymolase and lysozyme, both at an enzyme : yeast ratio of 1 : 500 the release of carbohydrates was 44% more compared to zymolase alone at the same ratios. A zymolase-lysozyme combination using enzyme yeast ratios of 1 : 500/1: 250 resulted in an increase (58%) in release of carbohydrates. For further studies a zymolase-lysozyme combi- nation was used at yeast to enzyme ratio of 1 : 500.

Effect of Incubation Time on Yeast Cell Wall

The effects of the zymolase and the zymolase-lysozyme treat- ments on release of carbohydrate from yeast cell during 120 min incubation are shown in Figure 2 . After 90 min of incubation the zymolase-lysozyme treatment gave a 35% higher carbohydrate re-

YEAST LYSIS 2015

TABLE I Effect of Zymolyse-Lysozyme Treatmenta on Release of Carbohydrates from

Yeast Cells

Carbohydrates Zymolase : yeast Lysozyme : yeast released

ratio ratio (mdg yeast, DM)

1 : 1000 1 : 1000 1 : 1000 1 : 1000

1 : 500 1 : 500 1 : 500 1 : 500

- 1 : 1000 1:500 1 : 250

-

1 : 1000 1 : 500 1 : 250

77.83 k 10.86 77.54 ? 1.29 98.30 ? 22.64 98.33 ? 22.70

95.40 ? 3.86 135.90 * 12.23 135.74 2 12.11 151.22 k 45.26

a Two hr at 3 7 T , pH 7.5.

lease than the zymolase treatment alone. This was 3.3 times more when compared with the control (yeast dispersed in buffer). Maxi- mum release occurred after 90 min of incubation.

Extractability of Nitrogenous Materials and Protein from Enzyme- Treated Yeast Cells

Because we are interested in recovering the protein from yeast cells, we examined the extractability of nitrogen from enzyme- treated cells. The amount of nitrogen recovered from yeast cells after extraction of the enzyme-treated cells for 30 min at pH 7.5 or 9.0 is shown in Figure 3 . By increasing the pH of extraction to 9.0, the release of nitrogen after zymolase and zymolase-lysozyme treatment increased to 75% of the total nitrogen content of the yeast cells compared to 3% from the control and lysozyme-treated cells. Fifty-two to 6% of the nitrogen released was precipitable at pH 4.5.

The amount of total nitrogen and protein released from enzyme- treated yeast cells following extraction for 45 min at pH 9.0 is shown in Figure 4. Extension of the extraction to 45 min resulted in an increased extraction of nitrogen to 83.8%. However, only 33.5% of this was intact protein, the remainder being hydrolyzed by endogenous proteases. The release of protein from the non- enzyme-treated samples showed similar trends as the release of total nitrogen. However, the protein recovery did not increase after 60 min although the overall nitrogen yield was increased. Enzyme

2016

INCUBATION TIME (MIN)

Fig. 2. Influence of incubation time on the release of carbohydrates from yeast cells using various enzyme systems. (0) Untreated yeast; (0) zymolase-treated yeast (zymolase : yeast ratio = I : 500); (A) lysozyme-treated yeast (lysozyme: yeast ratio = 1 : 500); (0) zymolase-lysozyme-treated yeast (zymolase : lysozyme: yeast ratio = 1:1:500).

treatment of cells with zymolase-lysozyme increased the extracta- bility of nitrogen and protein almost twofold without the need for any mechanical treatment of cells.

The decrease in protein recovery while nitrogen yield continued to increase (Fig. 4) is presumably caused by proteolysis during lysis of the cell wa1L6 However, succinylation of the enzyme-treated yeast homogenate markedly increased the yield of precipitable pro- tein at pH 4.5 (Fig. 5 ) . Mechanical disruption gave comparable yields, but the nucleic acid levels in the succinylated sample were reduced to less than 2% (on protein basis). The yield of protein was significantly greater in mechanically disrupted cells (Table 11).

DISCUSSION

Treatment of yeast cells with zymolase resulted in an increase of carbohydrate release which was almost 2.5 times higher compared to the control cells without enzyme. Incubation of yeast cells with lysozyme alone had no effect on the release of reducing sugars,

YEAST LYSIS

H

2017

-

m

J w u

c 4 Y t

r a Y

c1 Y + " a + w z w 0 0 a + - - I_

c z Y " a w a

A B C D

PI1 7 . 5 PH 9.0

Fig. 3. Extractability of total cellular nitrogen from yeast cells following treat- ment with lytic enzymes. A, untreated yeast; B, zymolase-treated yeast; C, lyso- zyme-treated yeast: D, zymolase-lysozyme-treated yeast: E, untreated yeast; F, zymolase-treated yeast; G, lysozyme-treated yeast: H, zymolase-lysozyme-treated yeast.

whereas when zymolase was coupled with lysozyme the release of reducing sugars was significantly enhanced being nearly 1.5 times more than that of zymolase alone and three times more than the

TABLE I1 Influence of Various Treatments on the Amount of Precipitable Protein Released

from Yeast Cells

Precipitable protein Treatment (5%)

Enzyme treatment 39.99 + extraction at pH 9

Enzyme treatment + succinylation

Mechanical disruption

48.70

72.59

2018

z 0

r U

= c x w

t z Y U a Y

a

-

KNORR, SHETTY, AND KINSELLA

I

I * - 0 30 60 90

I N C U B A T I O N T I M E ( M I N )

Fig. 4. Effect of duration of extraction on the recovery of total nitrogen and protein from enzyme treated yeast cells. Extraction was at pH 9 and protein was precipitated at pH 4.5. (0) Total nitrogen extracted from untreated cells; (m) total nitrogen extracted from zymolase-lysozyme-treated cells; (0) protein recovery from nontreaterl cells; (0) protein extracted from zymolase-lysozyme-treated cells.

control. This observation indicates that lysozyme may hydrolyze the glucosidic bonds of mannan-protein and glucan-protein complexes of the cell wall after partial disorganization of the cell wall which is caused by the partial hydrolysis of the glucan and mannan com- ponents of the carbohydrate-protein complex of the cell wall, by zymolase. It is also possible that zymolase hydrolyzes these car- bohydrates more easily following the disruption of the carbohy- drate-protein complexes by lysozyme.

This study indicates that partial softening and weakening of the yeast cell walls caused by the enzyme treatment may facilitate mechanical cell rupture of yeast cells for the recovery of the cellular protein as suggested by Phaff.6 Although nitrogen release was equal for zymolase and zymolase-lysozyme treatments, the higher release of reducing sugars by the combination of these enzymes indicated a more effective weakening of the cell walls. This could increase

YEAST LYSIS 2019

the effectiveness of mechanical cell disruption for protein extrac- tion.

Extraction at pH 9.0 for 45 min was effective in recovering more than 80% of the total nitrogen of the yeast cells. Since protein degradation by endogenous proteases occurred during enzyme treatment and because the nucleic acid concentration of the precip- itated protein was 26%, the influence of succinylation on the re- duction of nucleic acid and on the yield of precipitable protein following enzyme treatment was studied. Succinylation significantly increased the precipitation of proteins at isoelectric point and hence increased protein recovery. Succinylation also decreased the nu- cleic acid concentration of the precipitated protein to less than 2%. These observations are in agreement with earlier studies by Shetty and Kin~e l l a .~

Fig. 5 . Influence of various treatments on extractability of nitrogen from yeast cells. A, untreated yeast cell: B , yeast cells treated with zymolase-lysozyme for 90 rnin and extracted for 45 min at pH 9.0: C, yeast cells treated with zymolase- lysozyme and then succinylated: D, mechanical disrupted yeast cell extracted at pH 9.0 for 45 min.

2020 KNORR, SHETTY, AND KINSELLA

In summary, enzyme treatment of yeast cells with zymolase and lysozyme followed by extraction at pH 9.0 results in a nitrogen release comparable to those obtained by conventional mechanical disruption and extraction methods. The advantages of this treatment are low cost, negligible protein denaturation, and application to large-scale operations.

Further research on this treatment should be directed to large- scale application, optimization of the process, and further inhibition of proteolytic enzyme activity.

This research was supported in part by NSF Grant No. Eng 75-17273.

References 1. C. I. Waslien, D. Calloway, S . Margen, and F. Costa, Food Technol., 35, 294

2. J . E. Kinsella. K. J . Shetty, and M. Friedman, Eds., ACS Publication (1978). 3. K. J . Shetty and J . E. Kinsella, Biotechnol. Bioeng., 21, 329 (1979). 4. J . T. Worgen, Plant FoodsJbr Man (Newman, London, 1975). 5 . P. Kihlberg, A m . Re) , . Microbiol.. 26, 427 (1972). 6. H. J . Phaff, in Food Proteins: Improvement Through Chemicals and Enzy-

matic Modification (Advances in Chemistry Series 160) R. E. Feeney and J . R. Whitaker, Eds. (American Chemical Society, Washington, D.C., 1977).

7. J . C. Johnson, Yeast f o r Food and Other Purposes (Noyes Data Corp., Park Ridge, NJ , 1977).

8. S. R. Tannenbaum, Single-Cell Protein, R. I. Mateles and S. R. Tannenbaum, Eds. (M.I.T. Press, Cambridge, MA, 1968).

9. M. Follows, J . S . Hetherington, P. Dunhill, and M. D. Lilly, Biotechnol. Bioeng., 13, 549 (1971).

10. H. Mitsuada, Book, J . M. Leitch, Ed. (Gordon & Breach, New York, 1965), Vol. 2, p. 529.

I I . H. Mitsuada, K. Yasurnoto, and H. Nakamura, paper presented at Engineering of Unconventional Protein Production Conference, Santa Barbara, CA, 1967.

12. S. D. Cunningham, C. M. Cater, and K. F. Mattil, J . Food Sci., 40, 732 (1975).

13. D. Fraser, Nature, 167, 33 (1951). 14. I . Hayakawa, N. D. Khai, and D. Nomura, Sci . Bull. Fac. Agri . , Kyuslu

15. W. J . Nickerson, Bacteriol. Rev . , 27(3), 305 (1963). 16. S . R. Tannenbaum, R. 1. Mateles, and G. R. Capeo, in World Protein Re-

sources (Advances in Chemistry Series No. 57). R. F. Gould, Ed. (American Chem- ical Society, Washington, D.C., 1966), p. 254.

17. P. Vananuvat and J . E. Kinsella, J . Agric . Food Chem. , 23, 613 (1976). 18. E. A. Weaver, U.S. patent 3,088,879 (1963). 19. E. A. Weaver, U.S. patent 3,178,359 (1965). 20. A. Furuya and Y . Ikeda, J . Grn. Appl . Microbiol., 6, 40 (1960). 21. K. Horikoshi and K. Sakaguchi, J . Gen. Appl . Microbiol., 4, I (1958). 22. C. G. Mendoza and J . R. Villanueva, Nature (Lond.), 195, 1326 (1962).

( 1970).

Univ., 26, 528 (1972).

YEAST LYSIS 202 I

23. M. R. J. Salton, J . Gen. Microbiol., 12, 25 (1955). 24. H. Sugimoto,Agri. Biol. Chem., 31, 1111 (1967). 25. H. Tanaka and H. J. Phaff, J . Bacteriol., 89, 1570 (1965). 26. G. H. Fleet and H. Phaff, J . Bacteriol., 119, 207 (1974). 27. K. Kitamura, T. Kaneko, and Y. Yamamoto,Arch. Biocheirr. Biophys., 145,

28. K. Kitamura, T. Kaneko, Y. Yamamoto, and Y. Kuroiwa, U.S. patent 3,-

29. K. Kitamura, T. Kaneko, and Y. Yamamoto,.!. Gen. Appl. Microbiol., 18, 57

30. K . Kitamura and Y. Yarnamoto,Arch. Biochein. Biophys., 153, 403 (1972). 31 . K. Kitamura, T. Kaneko, and Y . Yamamoto, J . Gen. Appl. Microbiol., 20,

32. T. Kaneko, K. Kitamura, and Y. Yamamoto, Agri. Bid. Chem., 37, 2295

33. D. Herbert, P. J. Phipps, and R. E. Strange, Methods in Microbiology, J. R.

34. 0. H. Lowry, N. J . Rosebrough, A. L. Farr, and R. J . Randall, J . Biol.

35. K. J . Shetty and J . E. Kinsella, Biotechnol. Bioeng., 20, 755 (1978).

402 (1971).

716,452 (1973).

(1972).

323 (1974).

( 1973).

Morns and D. W. Robbons, Eds. (Academic, London, NY, 1971), Vol. 5B.

Chem., 193, 265 (1951).

Accepted for Publication January 28, 1979