Enzymatic lysis and disruption of microbial cells

TIBTECH - OCTOBER 1987 [Vol. 5]

EI BDDDB// Enzymatic lysis and disruption of microbial cells B. A. Andrews and J. A. Asenjo The development of expression systems for large recombinant proteins which cannot be secreted by the host microbial cells necessitates the development of novel techniques for cell disruption. The use of enzyme systems which provide biological specificity to the process of cell lysis and disruption shows tremendous promise as a method of controlled lysis and selective product release. Enzymatic lysis and disruption of microbial cells has found a number of important applications (Fig. 1). These include the production of specialty wall polysaccharides 1, production of intracellular, membrane bound or wall enzymes 2, production of yeast autolysates and products from subcellular structures 3 and the recovery of recombinant DNA products such as surface antigen particles manufactured in yeast 4. At present, the only industrial scale microbial cell breakage equipment is the high pressure homogenizer: other equipment, such as bead mills are used mainly at bench and pilot plant scale. However, these mechanical methods have drawbacks. They have no biological specificity, they generate high temperatures in the cell suspensions which impose great demands on cooling equipment, they generate high shear stresses which can harm the molecules to be recovered, and they necessitate a large capital investment in specialized equipment. Enzymes have none of these disadvantages. They can be used on their own for the release of intracellular soluble proteins, particulate inclusions and for wall and membrane associated materials. Or they can be used in conjunction with

B. A. Andrewsand J. A. Asenjo are at the Biochemical Engineering Laboratory, University of Reading, PO Box 226, Reading RG6 2AP, UK.

mechanical disruption to increase the selectivity of product release, to increase the rate and yield of extraction, and to minimize product damage. The industrial use of enzymes for the release of specific cell proteins is coming of age with the development of expression systems for large recombinant proteins that cannot be secreted by the cell. By choosing an appropriate enzyme system, selective and sequential product release may be obtained. This review summarizes the latest developments and trends in the production and utilization of enzyme systems which lyse microbial cells.

The substrates for lysis- the cell walls The cell walls of yeast and bacteria are distinctly different, hence, in general, lytic systems are specific for particular groups of microorganisms. Yeast cell walls (Fig. 2) have

two main layers, an outer layer of protein-mannan complex and an inner glucan layer 5. Enzyme systems for yeast cell lysis are, therefore, usually a mixture of several different enzymes which include one or more [3(1-3)glucanase, protease, [5(1-6)glucanase, mannanase or chitinase. They act synergistically in the lysis of the cell wall but only two are essential for whole cell breakage; a specific wall-lytic protease to degrade the outer layer of protein-mannan and a lytic [~(13)glucanase to degrade the inner glucan layer. In both Gram-positive bacteria and Gram-negative bacteria, a peptidoglycan component is responsible for the strength of the wall. In Grampositive bacteria peptidoglycans are the major wall polymers and are associated with teichoic acids and polysaccharides. Gram-negative bacteria have a two layer wall structure: the inner, rigid peptidoglycan component is covered by an outer layer or outer membrane composed of proteins, phospholpids, lipoproteins and lipopolysaccharides 6. Consequently, a single enzyme can lyse Gram-positive bacteria but pretreatment with a detergent (e.g. Triton X-100) is usually necessary to remove the outer membrane of Gramnegative cells. Three types of bacteriolytic enzyme have been isolated; glycosidases which split polysaccharide chains, acetylmuramyl-Lalanineamidases which cleave the junction between polysaccharides and peptides; and endopeptidases which split polypeptide chains.

Sources and properties of cell lyric enzymes A large number of microorganisms

-Fig. 1

Recombinant proteins Antibiotics

Lysed cell

Specialized lipids Enzyme attack

~

Wall polysaccharides Pigments Enzymes

Productsthatcanbeisolated ~ ~'~-'~ after enzymatic lysis of microbial cells.

~

Intracellular polymers

~) 1987, Elsevier Publications, Cambridge 0166- 9430•87/$02.00

TIBTECH --Fig. 2 Opening in mannoprotein layer

Openinginglucanlayer

Exposed glucanlayer have been found to produce microbial lytic enzyme systems 7-25 (Table 1). These organisms exhibit predatory activity against yeast and other microbial cells and have been isolated from such diverse habitats as decaying plant material (e.g. rice7), brewery effluents 8, sewage plants 9, estuaries 9, soil 1° and the human mouth 11. Virtually all microorganisms used for production of lytic enzymes are safe (class 1 classification in ATCC) and non-pathogenic. The only strains that do not fall into this category are some strains of Staphylococcus (class 2 in ATCC) and they are considered only mildly pathogenic. Conditions for use of microbial cell-lytic enzyme systems have been investigated by different authors 12. The pH and temperature optima vary considerably in the range pH 6-11 and 35-60°C (Table 1). For all the lytic systems, there is considerable variation between the molecular weights of the different systems and their component enzymes. However, most are relatively small (in the range of 10 to 30 kDa), a property which is important in that it makes them relatively easy to separate from large intracellular proteins after cell lysis. There is little information available on the effect of enzyme and substrate concentration on microbial cell-lyric enzymes. However, one study 28 showed that the rate of protein release from whole yeast cells in the presence of lytic enzymes was a linear function of enzyme concentration and was used with substrate concentrations up to 110 gl -~ dry weight of yeast cells.

Lysis of yeast The yeast-lytic enzymes produced by different Arthrobacter sp. have been extensively studied in batch culture and found to be inducible (by whole yeast cells and cell walls) and subject to catabolite repression by g l u c o s e 1 5 ' 2 6 ' 2 7 : [~(1-3)glucanase, protease and mannanase activities have been detected. The cell wall degrading enzymes from different strains of Oerskovia xanthineolytica have also *Jeffries, T. W. (1976)PhD Dissertation, Rutgers University, New Jersey, USA.

wall ofOuter yeast cell

OCTOBER1987[Vol.5]

f ~

" ~

Lysing yeast cell showing wall structure.

been purified and characterized 13'*. Jeffries* found four different fi(1-3)glucanases with distinct action patterns in batch culture with autolysed yeast cells as inducer: Scott and Schekman z3, using a different strain, found two synergistic activities in batch culture with yeast glucan as the carbon source; an endo-lytic glucanase and an alkaline protease. In general, in the enzyme systems which lyse yeast, the optimum pH values for the constituent enzymes are markedly different, for glucanase it is usually neutral whereas for protease it is alkaline in most cases.

Lysis of bacteria Bacteriolytic enzymes tend to have pH optima around 6 or 7. Optimum temperature ranges from 30 to 60°C with most between 35 and 40°C. At temperatures 5-10°C above optimum temperature, the stability of the enzyme is usually low and the rate of denaturation is high. Most of the enzymes are active, not on whole, live cells but on pretreated cells (e.g. lyophilised or heat killed) or on isolated cell walls. Many enzymes are specific for Gram-positive bacteria and few are active on Gramnegative cells. However, the lytic protease of Micromonospora was active against lyophilised cells of Serratia marcescens, Pseudomonas aeruginosa, E. coli and Bacillus subtilis 23 and the enzyme produced by Streptomyces coelicolor lyses cells of both Gram-positive and Gram-negative bacteria 25. The lytic protease from Bacillus subtilis can lyse cells of E. coli apparently without the need for pretreatment 24. The enzyme system from Cytophaga sp. has been used for lysis of E. coli cells in the presence of detergent (unpublished results from the authors' laboratory).

Plasmamembrane surface

(TakenfromHunterandAsenjo5.)

Methods of production The production of bacteriolytic enzymes has been studied mainly for possible use in food preservation and investigation of bacterial cell wall structures. Almost all of the work has been done in batch culture and early studies indicated that enzyme synthesis was non-inducible 22'29. However, in these studies a complex nitrogen and carbon source was used (bean cake extract), a component of which might have induced bacteriolyric activity. The production of enzymes able to lyse whole yeast cells has been studied in batch and continuous culture. The synthesis of the lyric enzymes of Cytophaga 9497 in batch culture appeared to be constitutive 3°. Recent work on the regulation of cell lytic enzyme synthesis in Cytophaga NCIB 9497 and Oerskovia xanthineoIflica included both batch and continuous culture studies 19'*. For both strains, the synthesis of lytic enzyme systems is inducible and subject to catabolite repression by glucose. At low dilution rates in carbon limited continuous culture, high ~(1-3)glucanase activities and a high glucanase/protease ratio are obtained in both strains, at high dilution rates all enzyme activities are similar to batch values. For both systems, continuous culture provides a several fold increase in enzyme concentration and productivity over batch culture. This has meant that a process for the production of protein using yeast lyric enzymes can be designed in which an enzyme production fermenter of only 1.5-2 m 3 is required to lyse the yeast cells produced in a 100 m 3 continuous culture fermenter. The medium required for enzyme production

~Andrews, B. A. (1985) PhD Thesis, University of London, UK.

TIBTECH- OCTOBER1987 [Vol. 5]

--Table 1 M i c r o b i a l lytic enzyme systems

Source

Optimum Optimum temperature pH (°C) Substrate

Oerskovia xanthineolytica/ Arthrobacter luteus A rth ro bacter GJ M - 1 (Zymolyase/Lyticase) g lucanase protease (alkaline) whole yeast cell activity Oerskovia CK glucanase with proteolytic activity whole yeast cell activity Rhizoctonia sp.

glucanase protease whole yeast cell activity Cytophaga NCIB 9497

Ref.

Saccharomyces, 13-15 Candida, Hansenula, Pichia and other yeasts

5-6.5 9-10 7.5

45-50 35 30-35

S. cerevisiae

16

9.0

35-40 60 35-40

5.5 6.5 6.0 9.0

55-60 40 40 45-55

Candida,

18

Saccharomyces, Hansenula S. cerevisiae,

19

Bacillus, Corynebacteria, E. coli

Lysozyme (hen egg-white)

6-7

35

E. coli M. lysodecticus and other

9.5

50

Staphilococcus

a

6-7

37

21

6.5

50

M~rococcus luteus M. lysodecficus

bacteria Cytophaga B-30 (Lysopeptase) Staphylococcus sp. Streptomyces globisporius (N-acetylmuramidase) (Mutanolysin) Micromonospora sp. Nr. 152 (lytic protease)

Bacillus subtilis 797 (lytic protease)

22

Streptococcus 11

7.8-8.5

60

E. coil

23

30

S. marcescens P. aeroginosa B. subtilis E. coil

24

aMiles Technical Information (1984)lysopeptidase.

in this process is only 0.25% of the medium necessary for yeast production 31. Cloning the genes for the lytic enzymes could result in a several fold increase in activity of the enzyme systems in the producer strains. H o w are lyric enzymes used? The cells to be lysed are usually harvested from the fermenter by centrifugation, ultrafiltration or microporous filtration. They are mixed in a lysis reactor with the lytic enzymes and buffer. Typically, the enzyme will be added as a crude supernatant at a concentration of 3 30% v/v (which corresponds to 0.121.2 g protein 1-1 in the enzyme reaction) or as low as 0.4% v/v if the enzyme is produced in continuous culture 15. These values are for the

lysis of B. subtilis cells at a concentration of 9-13 g 1-1 using the enzymes produced by Cytophaga sp. With yeast cells, concentrations of up to 110 g 1-1 dry weight have been used in disruption reactors. With lysozyme it was estimated that using 4000-5000 U 1-1, good protein extraction could be obtained from bacterial cells in one hour. Commercial enzyme preparations contain 14 000-22 000 U g-1. The only lytic enzyme available on a commercial scale for the industrial disruption of cells is lysozyme (active only on bacterial cells and specific activity as stated above); other bacteriolytic and yeast lytic enzymes (e.g. Zymolyase) are only available as laboratory reagents, so with present data it is not possible to

make an accurate cost comparison with non-enzymic breakage methods on an industrial scale. The enzyme system from Cytophaga sp. has been efficiently used for the lysis of bacterial cells. The activity of the Cytophaga sp. system was approximately one order of magnitude higher than that of other bacteriolytic strains. Process design calculations similar to those carried out for yeast cell lysis have shown that the enzyme production fermenter w o u l d be 1.5-2% of the volume of the cell production vessel in batch enzyme production and if continuous culture is used, only 0.4-0.5%; in other words, a 42 1 fermenter could produce sufficient enzyme for a cell production unit of 10 m 3 (Ref. 20). Products of lysis Ideally, the cell debris would be particulate and the protein product to be recovered would be larger than the lytic enzyme protein. Then cell debris separation could be achieved by using a centrifuge or microporous membrane filter and product purification and separation from the lytic enzyme would be achieved by a 'classic' chromatography sequence (e.g. ion exchange followed by gel filtration). Some lytic enzymes have a strong affinity for yeast glucan and wall debris 2a, which w o u l d allow a substantial fraction of the lytic enzymes to be separated along with the cell debris. Process design Tailoring enzyme systems for a particular use and manipulating process conditions introduces a considerable degree of specificity to cell disruption and product release. Since the pH and temperature optima of the crucial enzymes needed for cell breakage can be very different, pH or temperature changes could provide, for example, high protease activity in the early part of the lysis reaction and high glucanase and low protease activities at a later stage 32. Protease inhibitors (e.g. mannan) could also be used to control the process35~ Eventually, it might be possible to improve control by using genetic manipulation to develop an organism to produc e its own inducible lytic

TIBTECH- OCTOBER1987[Vol. 5] - Fig. 3 1

M

enzyme system: this option is being explored. Yeast cells have a double layer wall and by choosing an enzyme system with a higher glucanase or higher lytic protease or a combination of different glucanases (producing glucan oligomers of different sizes), the rate of release of final product and byproducts as well as its quality and composition can be engineered. Examples of this are the release of invertase from cell walls, the production of protein-flee glucan and the production of glucan oligomers with pre-specified characteristics 12. If the enzymes of the yeast lytic systems are purified it should be possible to first treat cells with lytic protease then remove or inactivate the protease and degrade the wall with lytic glucanase 12. In osmotic solution the intracellular osmotic pressure would not break the periplasmic membrane thus allowing the degradation and depolymerization of glucan only. Gentle agitation, or another means of protoplast breakage, would then allow release of intracellular material (Fig. 3) 12. It has been found that in most cases wall lytic proteases are very specific with little or no activity on intracellular proteins 33.

Applications Bacteriolytic enzymes are already used commercially on a large scale for the release of intracellular and membrane bound enzymes and antibiotics 12. Some of the applications of microbial lytic enzyme systems are

]

Lytic enzymes

E

~-'~ ,~,Cellsr~ ,~, I-erm' ~ /~ ~ ~ 1

Organelle product Protein particle

Ex.Prod.

Wall enzymes

Cytoplasmic enzymes

Process for the lysis of microbial cells including sequential disruption for selective product release. A, wall lysis in osmotic support; B, protoplast disruption; C, lysis of organelles or protein particles. RM, raw materials; I, inoculu[;n; Ferm, fermenter; Ex. Prod., extracellular product; E, enzyme of reagent used to lyse organelle or renaturation of protein. (Taken from Asenjo and Andrews12).

shown in Table 2. These include a several fold increase in yield in preparation of a soluble glucan polysaccharide (12-15 fold with bakers yeast) 1, the extraction of lipids a4 and the extraction of human serum albumin made by genetically engineered yeast cells 33. Other important applications of cell lytic enzyme systems are cell killing for microbial containment and the selective lysis of mixed microbial populations which cannot be achieved by mechanical means.

Conclusions Enzymatic methods of cell lysis can be highly specific in terms of the microorganism lysed and the product released. These two variables will determine the activity profile of a lytic enzyme system to be used in a particular application. Mechanical techniques for cell disruption are effective but highly non specific. The concept of a biochemical cell refinery where enzymatic, physico-chemical and/or genetic techniques are used to

--Table 2

Present and potential applications o f microbial lyric enzymes Ref.

Preparation of protoplasts, cell fusion and transformation of yeast Production of intracellular enzymes Pretreatment to increase yeast digestibility Preparation of soluble glucan polysaccharide Alkali extraction of yeast protein Pretreatment for Dyno-mill mechanical breakage of cells Extraction of specialized lipids from yeast Production of yeast extracts Food preservation Extraction of pigments from red yeast Release of recombinant proteins e.g. human serum albumin Ethanol recovery from spent brewers yeast a Lysis of caries inducing microorganisms aKrauss (1985) pers. commun.

r ~ - [ ,~, r~ ,~, ~ ~ , 1 . ) - ~ 1 ~-~ ~

26 2 18 1 18 18 34 18 35 17 33 36

selectively release products from the cell is important. It appears that continuous culture is highly advantageous for the production of lytic enzyme systems both to overcome catabolite repression and to design systems with desired enzyme composition: enzyme concentrations can be increased many fold and the profile of enzyme components can be manipulated. Lytic systems with extremely high activities can thus be obtained making large scale application of this technology feasible in the near future. Specific enzyme activities of lytic systems are high and thus should mean that the cost of large scale enzymatic lysis is very reasonable. Cloning of the genes for lytic enzymes in producer strains should further decrease the cost of this technology. Lytic enzyme technology shows great promise for the isolation of high value subcellular fractions of some large intracellular recombinant proteins that cannot be secreted by the cell, and for other specialized applications. New and future developments should focus on understanding the mechanism by which whole microbial cells and subcellular fractions are enzymatically cleaved. This will allow the optimization of product extraction particularly in those cases where protein secretion cannot be obtained. It will also result in the design of new processes for the specific extraction of intracellular proteins.

References 1 Jamas, S., Rha, C. K. and Sinskey, A. J. (1986) Biotechnol. Bioeng. 28, 769784 2 Er-A1, Z., Klein, D., Buttat, E. and

TIBTECH - O C T O B E R 1987 [Vol. 5]

3 4

5

6 7 8 9

10

11

12 13 14 15 16 17 18 19 20 21 22 23

Zorner, E. (1984) Third Eur. Cong. Biotechnol. (Vol. I), 1-629 D'Souza, S. F. (1983) Biotechnol. Bioeng. 25, 1661-1664 Valenzuela, P., Colt, D., MedinaSelby, M. A., Kuo, C. H., Van Nest, G., Burke, R. L., Bull, P., Urdea, M. S. and Graves, P. V. (1985) Bio/Technology, 3, 323-326 Hunter, J. B. and Asenjo, J. A. (1986) in Separation, Recovery and Purification in Biotechnology: Recent Advances and Mathematical Modelling, (Asenjo, J. A. and Hong, J., eds), pp. 931, American Chemical Society Schnaitman, C. A. (1971) J. Bacteriol. 108, 553-563 Kobayashi, R., Miwa, T., Yamamoto, S. and Nagasaki, S. (1980) J. Ferment. Technol. 58, 311-317 Kaneko, T., Kitamura, K. and Yamamoto, Y. (1969) J. Gen. Microbiol. 15,317-326 Reichenbach, H. and Dworkin, M. (1981) in The Prokaryotes (Starr, M. P., Stolp, H., Truper, H. G., Balows, A. and Schlegel, H. G., eds), pp. 356379, Springer-Verlag Lechevalier, H. A. and Lechevalier, M. P. (1981)in TheProkaryotes (Starr, M. P., Stolp, H., Truper, H. G., Balows, A. and Schlegel, H. G., eds), pp. 21202123, Springer-Verlag Newman, M. G., Socransky, S. S., Savitt, E. D., Propas, D. A. and Crawford, A. (1976) J. Periodontology, 47, 373-379 Asenjo, J. A. and Andrews, B. A. Adv. Bioehem. Eng./Biotechnol. (in press) Scott, J. H. and Schekman, R. (1980) J. Bacteriol. 142, 414-423 Kitamura, K. (1982) Agric. Biol. Chem. 46, 963-969 Vrsanska, M., Kratky, Z. and Biely, P. (1977) Z. Allg. Mikrobiol. 17,391--402 Obata, T., Iwata, H. and Namba, Y. (1977) Agric. Biol. Chem. 41, 23872394 Okagbue, R. N. and Lewis, M. J. (1983) Biotech. Lett. 5,731-736 Kobayashi, R., Miwa, T., Yamamoto, S. and Nagasaki, S. (1982) Eur. J. Appl. Microbiol. Biotechnol. 15, 14-19 Andrews, B. A. and Asenjo, J. A. (1986) Biotechnol. Bioeng. 28, 13661375 LeCorre, S., Andrews, B. A. and Asenjo, J. A. (1985) Enzyme Microb. Technol. 7, 73-77 Valisena, S., Varaldo, P. E. and Satta, G. (1982) J. Bacteriol. 51,636-647 Hayashi, K., Kasumi, T., Kubo, N. and Tsumura, N. (1981) Agric. Biol. Chem. 45, 2289-2300 Suzuki, K., Uyeda, M. and Shibata, M. (1985) Agric. Biol. Chem. 49, 17191727

24 Borovikova, V. P., Arsenovskaya, V. E., Laurenova, G. I., Kislukhina, O.V., Kalunyants, K. A. and Strepanov, V.M. (1980) Biokhimiya, 45, 1524-1533 25 Wohner, G., Voeiskow, H., Prave, P., Luck, E. and von Rymon Lipinski, G. (1986) European Patent Application EPO 181 562 A2 26 Kitamura, K. (1982) J. Ferment. Technol. 60, 253-256 27 Rowley, B. I. and Bull, A. T. (1977) Biotechnol. Bioeng. 19, 879-899 28 Asenjo, J. A. (1981) Advances in Biotechnol., 3, (Vezina, C. and Singh, K., eds), pp. 295-300, Pergamon Press 29 Yoshimoto, T. and Tsuru, S. (1972) J. Biochem. 72,379-390 30 Asenjo, J. A., Dnnnill, P. and Lilly, M.D. (1981) Biotechnol. Bioeng. 23, 97-109 31 Asenjo, J. A., Andrews, B. A., Hunter, J.B. and LeCorre, S. (1985) Process

[]

[]

[]

[]

[]

[]

Riochem. 20, 158-164 32 Liu, L. C., Prokopakis, G. J. and Asenjo, J.A. (1986) AIChE 1986 Annual Meeting, T203, FL, USA 33 Asenjo, J. A., Andrews, B. A. and Pitts, J. M. (1987) Proc. 4th European Congress of Biotechnology, Vol. 2, (Neijssel, O. M., van der Meet, B. R. and Lyben, K. Ch. A. M., eds), p. 497, Elsevier 34 Hammond, E. G., Glatz, B. A., Choi, Y. and Teasdale, M.T. (1981) in New Sources of Fats and Oils (Pryde, E. H., Princess, L. H. and Mukherjee, K. D., eds), pp. 171-187, American Oil Chemistry Society 35 Scott, H., Hammer, F. E. and Szalkucki, T.J. (1987) in Food Biotechnology (Knorr, D., ed.), pp. 413-440, Marcel Dekker 36 Johnson, J. C. (1977) in Industrial Enzymes, Recent Advances, p. 203, Noyes Data Corporation []

[]

[]

[]

[]

Expression, glycosylation and secretion of fungal hydrolases in yeast Hajirne Yoshizumi and Toshihiko Ashikari Although there are now some systems for transfer and expression of fungal genes, none gives expression at a level useful for production. There are useful expression systems in yeast, however, and these have been used to express genes coding for fungal extracellular hydrolases. This review examines how properties of the genes and gene product affect production and secretion of the enzyme in yeast. Similar considerations apply to expression of other heterologous genes in yeast and in other hosts. Various h y d r o l a s e s used in i n d u s t r y p r o d u c e d b y filamentous fungi offer attractive targets for industrial gene technologists. R e c o m b i n a n t DNA t e c h n o l o g y is e x p e c t e d to i m p r o v e the p r o d u c t i v i t y , stability and substrate specificity of such enzymes. A l t h o u g h the m o l e c u l a r genetics of

H. Yoshizumi and T. Ashikari are at the Laboratories of Applied Microbiology, Research Center, Suntory Ltd., 1-1-1 Wakayama-dai, Shimamoto-eho, Mishima-gun, Osaka, Japan.

f i l a m e n t o u s fungi has r e c e n t l y progressed and some gene transfer systems for filamentous fungi have b e e n r e p o r t e d 1-3. These have not yet r e a c h e d the stage of practical applications. On the other h a n d m a n y foreign proteins of various origins have already b e e n e x p r e s s e d in the yeast, Saccharomyces cerevisiae, some of t h e m at a high level 4-7. Genes of fungal extracellular proteins have, therefore, b e e n i n t r o d u c e d into yeast and their e x p r e s s i o n and secretion studied. In this article, w e r e v i e w the

(~) 1987, Elsevier Publications, Cambridge 0166-9430/87/$02.00