Autolysis: A tool for protoplast production from Aspergillus nidulans

Trans. Br. mycol. Soc. 78 (3) 389-394 (1982)

Printed in Great Britain

AUTOLYSIS: A TOOL FOR PROTOPLAST PRODUCTION FROM ASPERGILLUS NIDULANS By SUSAN ISAAC AND A. V. GOKHALE Department of Botany, University of Liverpool, P.O. Box 147, Liverpool L69 3BX

The rate and degree of autolysis of Aspergillus nidulans was investigated during a 16 day incubation period. Changes in the activity of chitinase, s-glucanase, a-glucanase, invertase, esterase, acid and alkaline phosphatase and protease liberated into the culture fluid were monitored during growth and autolysis. Protoplasts were released from A. nidulans mycelium by lytic enzymes present in autolytic-phase culture filtrates of the same organism. Maximum protoplast-liberating activity corresponded to both the point of maximum autolysis and maximum lytic enzyme activity. The method described represents a highly efficient and reliable technique for protoplast production from Aspergillus nidulans.

Filamentous fungi excrete enzymes into the culture medium during growth and autolysis. Events associated with the high levels of autolysis which can be achieved by Penicillium oxalicum, Neurospora crassa and Aspergillus niger have been described (Lahoz, Reyes & Perez Leblic, 1976) and the release of degradative enzymes during autolysis, in response to variations in culture conditions, has also been investigated (Gomez, Reyes & Lahoz, 1977; Lahoz & Miralles, 1970). There are few reports concerning the action of autolytic enzymes on the cell walls of those organisms concerned (Lahoz, Rubio-Huertos & Martinez de Ibarreta, 1970; Po lacheck & Rosenberger, 1975). However, enzymes from various microbial origins active in cell wall lysing ability have been used extensively in the liberation of protoplasts from filamentous fungi (Villanueva & Garcia Acha, 1971; Peberdy, 1976, 1979). The isolation of protoplasts from an organism using autolytic enzyme from a culture of the same organism has been reported for Streptococcus faecalis (Joseph & Shockman, 1974) and Saccharomyces cerevisiae (Necas, 1956). Reyes & Lahoz (1976) described the production of 'autoplasts' in Neurospora crassa although such a method has not been widely adopted. This paper investigates the time course of autolysis and lytic enzyme liberation in Aspergillus nidulans. An efficient and reliable method for protoplast liberation using autolytic enzymes from A. nidulans cultures is described.

MATERIALS AND METHODS

Organism Aspergillus nidulans BDUN 33 (supplied by Dr J. F. Peberdy) was maintained on 1 % (w/v) malt extract agar (Oxoid). Similar cultures were used for the preparation of conidial suspensions. Lytic enzyme production Mycelium was grown in mineral salts medium (Vogel, 1964) supplemented with glucose (10 g 1-1). Erlenmeyer flasks (2 I) containing 500 ml medium were incubated at 30°C with shaking (200 r.p.m.) for up to 16 days. Conidia were harvested in 10 ml 0'1 % (vlv) Tween 80 and washed twice with distilled water prior to inoculation at a final concentration of 2 x 106 / m !. Samples (10 ml) were taken at intervals and centrifuged (1500 g for 10 min). Supernatants were retained for analysis. Enzyme assays Chitinase (poly[ 1,4 -,8-(z-acetamido-z-deoxy- Dglucoside)] glycanohydrolase, E. C. 3.2.1 . 14) assay mixtures were composed of 2 ml enzyme solution, at a suitable concentration, and 2 ml of a 0'1 % (w Iv) suspension of colloidal chitin, prepared from ball-milled crustacean chitin according to the method of Berger & Reynolds (1958), in 0'05 Msodium maleate buffer pH 5'8. Incubation was for 2 h at 37°C in a shaking water bath. Assay for

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Protoplasts from Aspergillus

x-acetyl-n-glucosaminc in the supernatant was by the method of Reissig, Strominger & Leloir (1955), fJ-glucanase (fJ- 1,3(4) glucan glucanohydrolase, E , C . 3 . 2. 1 .6) was assayed using a modification of the method of Huotari, Nelson, Smith & Kirkwood (1968). Enzyme solution (0'1 rnl) was incubated with substrate solution (0'9 ml) containing 0'05 % (w/ v) laminarin (Koch-L ight) in 0'05 M-sodium maleate buffer pH 5,8 . a-Glucanase (a- 1,3(4) glucan glucanohydrolase, E. C , 3.2.1 ,61 ) was assayed by the same method using 0'1 % (w/ v) nigeran as substrate, Incubation was for 15 min at 37°, The reaction was stopped by placing in a boiling water bath for 5 min , The concentration of reducing sugar in the supernatant was estimated by the 3,5-dinitrosalicylic acid method of Sumner & Somers (1949) with glucose as standard. Proteolytic activity was determined by measuring the hydrolysis of 0'1 % (wI v) Hammarsten casein, denatured by boiling for 15 min, in 0'05 M-sodium maleate buffer pH 5'8 . One ml substrate was incubated with an equal volume enzyme solution at 32° for 30 min. The reaction was stopped by the addition of 2 ml 10 % (w/ v) trichloroacetic acid, and the absorbance of the supernatant was measured at 280 nm in a Pye Unicam SP 600 spectrophotometer. .. Acid phosphatase (ort hophosphoric-monoester phosphohydrolase (acidoptimum),E . C , 3 .1,3 ,2 ) and alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), E , C . 3 . 1 . 3 . 1) activities were measured using a modification of the method of Brightwell & Tappel (1968), Enzyme solution (0' 1 rnl) was incubated with 0"4 ml 15 mxt-a-nitrophenyl phosphate in either 0'5 M-sodium acetate buffer pH 5'0 or 0'5 M-tris/HCI buffer pH 9'0 for 15 min at 32°, The reaction was stopped by the addition of 2 ml 0'5 M-NaOH and the concentration of 4-nitrophenol estimated by measuring absorbance at 410 nm, using a Cecil C.E.373 spectrophotometer. Invertase (fJ-fructofuranoside fructohydrolase, E. C , 3 ,2. 1 .26) activity was measured using a modified method of Arnold (1969). Enzyme solution (0'1 ml) was incubated with 0"4 ml 5 mM-sucrose in 0'1 M-phosphate buffer pH 5'0 at 32° for 15 min. The reaction was stopped by boiling and free reducing sugar estimated using 3,5dinitrosalicylic acid reagent (Sumner & Somers, 1949), Esterase (carboxylic-ester hydrolase, E. C . 3 ,1 .1 . 1) activity was measured using a modification of the method of Huggins & Lapide (1947). 4-nitrophenyl acetate was dissolved in a small volume of methanol and diluted with 0'1 M-phosphate buffer pH 7'0 to give a concentration of 1 mM. This substrate (2 ml) was then

incubated with enzyme solution (1 ml ) at 32°, Absorbance at 410 nm was measured after 30 min , Protein determination Concentration of protein in culture filtrates was measured using the method of Lowry, Rosebrough, Farr & Randall (1951). Dry weight determination Samples were filtered through paper discs (Whatman, 542), thoroughly washed with water and dried to constant weight (36 h at 80 0). Quadruple determinations were made in each case. Protoplast isolation

Protoplasts were prepared by the method of Peberdy & Isaac (1976) from 18 h mycelium of A. nidulans (0'02 g fresh weight /rnl ), using 0'1 Mphosphate buffer pH 5,8 plus 0·6 M-KCI (final concentration in digestion mixture) as buffer/stabilizer system. An equal volume of culture filtrate from autolysing cultures of A . nidulans was used as lytic enzyme after centrifugation (1500 g for 10 min ) to remove debris. Protoplasts were harvested from the lytic mixtures by filtration through sintered glass filters (porosity 1), to remove hyphal debris, and centrifugation at 800 g for 10 min . Yields were determined, after washing preparations three times with buffer/ stabiliser solution, by counting in a haemocytometer. RESULTS

The change in dry weight of mycelium of Aspergillus nidulans in batch culture over 18 days is given in Fig. 1. Under the conditions described maximum yield was reached after 48 h incubation. Thereafter dry weight fell due to autolysis which reached a maximum of 85 % after 8 days. Culture medium pH dropped to 4'9 during rapid grewth of mycelium but rose steadily after 24 h reaching a stable value of 7'0 at the end of autolysis (Fig. 2). Variation in the concentration of protein in the culture medium is shown in Fig . 3, No free reducing sugar was present in the medium after 48 h incubation. Increases in the activities of lytic enzymes in the culture medium were measured during autolysis. Activities of chitinasc and fJ-glucanase increased during the autolytic phase, reaching maximum values at the end of the incubation period (F ig. 4a, b). Activities of a-glucanase and protease followed a similar pattern (F ig. 4C, d ) reaching

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maximum activity slightly earlier during autolysis (6 days incubation). Activities of acid and alkaline phosphatase, invertase and esterase also rose during autolysis with maximum levels being reached at, or slightly after, the end of autolysis (Fig. 5 a-d). Culture filtrate samples were used as lytic enzyme preparations in the isolation of protoplasts from 18 h A. nidulans mycelium. Culture filtrate samples from different stages of incubation were tested for their ability to liberate protoplasts (Fig. 6). Those samples taken towards the end of autolysis showed greatest protoplast-liberating

The time course and general pattern of autolysis of Aspergillus nidulans was similar to that found in cultures of A. niger (Gomez, Reyes & Lahoz, 1977). The final level of autolysis obtained here with A. nidulans (85 %) was higher than that obtained with A. niger (- 50%) in cultures containing similar initial glucose concentrations. The appearance of wall-degrading lytic enzymes within the culture medium followed a very similar pattern in both species. The cell wall of A. nidulans is composed predominantly of chitin and p-linked glucan (Bull, 1970).Zonneveld(1973)detectedsomea-l,3-glucan in older hyphae. Enzymes active in the degradation of these polymers have been shown to be essential in the liberation of protoplasts from A. nidulans mycelium (Isaac, 1978; Isaac & Peberdy, 1979). In this paper it has been shown that culture filtrates from autolysing cultures possess p-glucanase, chitinase and a-glucanase activity and are highly effective in releasing protoplasts from exponentialphase mycelium of the same organism. Those filtrates showing maximum enzyme activity liberated the maximum number of protoplasts. Good protoplast yields have previously been obtained from A. nidulans using enzymes from various microbial origins. The most successful

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REFERENCES

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enzyme mixtures have been produced by Streptomycesvenezuelus (Peberdy & Gibson, 1971; Peberdy & Buckley, 1973) and Trichoderma harzianum (Peberdy & Isaac, 1976; Isaac & Peberdy, 1979). Higher numbers of protoplasts were produced using Trichoderma enzyme (2'5 x 10 8 protoplasts from 50 mg mycelium) than with Streptomyces enzyme although the method has proved to be unreliable (Isaac, 1978) and requires that the resulting filtrate be concentrated re-fold by rotary evaporation. More recently Van den Broek, Stunnenberg & Wennekes (1979) have reported high levels of protoplast liberation from A. nidulans using an enzyme combination consisting of a mixture of enzyme extracted from A. nidulans mycelium at a cleistothecium-producing stage and extra-cellular enzyme from Oerskovia xanthineolytica grown on A. nidulans wall material (2'5 x 10 8 protoplasts from 40 mg mycelium). Expensive commercial enzyme from Cytophaga sp. (Lytic enzyme Ll, BDH) has also been used successfully in protoplast production in this laboratory (2 x 10' protoplasts from 40 mg mycelium; A. Gokhale, unpub!.). The method reported here claims several advantages over other systems currently in use. Filtrates from A. nidulans autolytic cultures are high in enzyme activity and can be used directly for protoplast production. There is no requirement for the mixture to be concentrated or to mix enzymes produced from various origins. No complex medium is required. Debris may be removed from the culture filtrate by centrifugation in a bench

ARNOLD, W. N. (1969). Heat activation kinetics of yeast p-fructofuranosidase. Biochimica et Biophysica Acta 178,347-53. BERGER, L. R. & REYNOLDS, D. M. (1958). The chitinase system of a strain of Streptomyces griseus. Biochimica et Biophysica Acta 29, 522-34. BRIGHTWELL, R. & TAPPEL, A. L. (1968). Lysosomal acid pyrophosphatase and acid phosphatase. Archives of Biochemistry Biophysics 124, 333-343. BULL, A. T. (1970). Chemical composition of wild type and mutant Aspergillus nidulans cell walls: The nature of polysaccharide and melanin constituents. Journal of General Microbiology 63, 75--94. GOMEZ, P., REYES, F. & LAHOZ, R. (1977). Effect of the level of the carbon source on the activity of some lytic enzymes released during autolysis of Aspergillus niger. Mycopathologia 62, 23-30. HUGGINS,H. & LAPIDE,J. (1947). Chromogenic substrates. IV. Acyl esters of p-nitrophenol as substrates for the colorimetric determination of esterase. Journal of Biological Chemistry 170,467-482. HUOTARI, F. L., NELSON, T. E., SMITH, F. & KIRKWOOD, S. (1968). Purification of an exo-p-D(1,3)-glucanase from Basidiomycete species QM806. Journal of Biological Chemistry 243, 952--956. ISAAC, S. (1978). Biochemical properties of protoplasts from Aspergillus nidulans. Ph.D. Thesis, University of Nottingham. ISAAC, S. & PEBERDY, J. F. (1979). Production of mycolytic enzymes. In Protoplasts - Applications in Microbial Genetics (ed. J. F. Peberdy), pp. 1'2-16. University of Nottingham Press. JOSEPH, R. & SHOCKMAN, G. D. (1974). Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis 9790: release of cell wall, autolysin, and formation of stable autoplasts. Journal of Bacteriology 118, 735-746. LAHOZ, R. & MIRALLES, M. (1970). Influence of the level of carbon source on the autolysis of Aspergillus niger. Journal of General Microbiology 62, 271-276. LAHOZ, R., REYES, F. & PEREZ LEBLIC, M. I. (1976). Lytic enzymes in the autolysis of filamentous fungi. Mycopathologia 60, 45-49. LAHOZ, R., RUBIO-HuERTOS, M. & MARTINEZ DE IBARRETA, B. (1970). Stability of the cell walls of Neurospora crassa during autolysis. Annals of Botany 34, 625- 629. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265- 275. NECAS, O. (1956). Regeneration of yeast cells from naked protoplasts. Nature, London 177,898-899. PEBERDY, J. F. (1976). Isolation and properties of protoplasts from filamentous fungi. In Microbial and Plant Protoplasts (4th International Symposium on

394

Pro top lasts from Aspergillus (ed. J. F. Peberdy, REYEs, F. & LAHOZ, R.

Yeasts and other protoplasts) A. H. Rose, H. J. Rogers & E. C. Cocking), pp. 39-50. London & New York: Academic Press. PEBERDY, J. F. (1979). Fungal protoplasts: Isolation, reversion and fusion. Annual Reviews of Microbiology 33,21-39. PEBERDY, J. F. & BUCKLEY, C. E. (1973). Adsorption of fluorescent brighteners by regenerating protoplasts of Aspergillus nidulans. Journal of General Microbiology 74, 281-288. PEBERDY, J. F. & GIBSON, R. K. (1971). Regeneration of Aspergillus nidulans protoplasts. Journal of General Microbiology 69, 325-330. PEBERDY, J. F. & ISAAC, S. (1976). An improved procedure for protoplast isolation from Aspergillus nidulans. Microbios Letters 3, 7-9. POLACHECK, Y. & ROSENBERGER, R. F. (1975). Autolytic enzymes in hyphae of Aspergillus nidulans: Their action on old and newly formed walls. Journal of Bacteriology 121, 332-337. REISSIG, J. L., STROMINGER, J. L. & LELOIR, L. F. (1955). A colorimetric method for the estimation of Nacetyl amino sugars. Journal of Biological Chemistry 217, 959-9 66.

(1976). Liberation of protop lasts from mycelium of Neurospora crassa by means of enzymes obtained from autolysed cultures of this fungus. Antonie van Leeuwenhoek 42, 457-460. SUMNER, J. B. & SOMERS, G. F. (1949). Dinitrosalicylic method for determination of glucose. In Laboratory Experiments in Biological Chemistry p. 38. New York: Academic Press. VAN DEN BROEK, W. J., STUNNENBERG, H. G. & WENNEKES, L. M. J. (1979). Protoplasts from Aspergillus nidulans. Microbios 26, 115-128. VILLANUEVA, J. R. & GARCIA ACHA, I. (1971). Production and use of fungal protoplasts. In Methods in Microbiology, vol. 4 (ed. C. Booth), pp. 665-718. London: Academic Press. VOGEL, H. J. (1964). Distribution of lysine pathways among fungi; evolutionary implications. American Naturalist 98, 435-46. ZONNEVELD, B. J. M. (1973). Biochemical analysis of the cell wall of Aspergillus nidulans. Biochimica et Biophysica Acta 249, 506-514.

(Received for publication 30 June 198 I)