Process Biochemistry 35 (2000) 659–664

Production and regeneration of Trichosporon cutaneum protoplasts

Wen Liu a, Wen-Miao Zhu b,*a The State Key Laboratory of Microbial Technology, Shandong Uni6ersity, Jinan 250100, People’s Republic of China

b Department of Microbiology and Immunology, Shandong Medical Uni6ersity, Jinan 250012, People’s Republic of China

Received 7 July 1999; accepted 24 August 1999

Abstract

Trichoderma koningii UC174 produced a number of exoenzymes when grown in liquid medium containing cell walls fromTrichosporon cutaneum SL409 as the sole carbon source. Exoenzymes were isolated from the culture broth and used to prepareprotoplasts from T. cutaneum. An enzyme prepared by bentonite treatment from the original Trichoderma enzyme had lessproteinase activity and protoplasts were fairly stable with this product during incubation for 6 h. Neither Cellulase Onozuka RSnor snail gut enzymes from Helix pomatia were able to generate protoplasts either alone or in combination. Protoplasts wereproduced, however, when commercial chitinase was added to either of these two enzymes. © 2000 Published by Elsevier ScienceLtd. All rights reserved.

Keywords: Protoplast; Regeneration; Lytic enzyme; Trichosporon cutaneum ; Trichoderma koningii

www.elsevier.com/locate/procbio

1. Introduction

A variety of organisms have been described assources of enzymes capable of degrading fungal cell-walls [1]. Protoplasts of fungi and yeast are suitable forthe isolation of organelles such as vacuoles or mito-chondria under mild conditions. In addition, their usein transformation experiments with plasmid DNA orfor protoplasts fusion crosses has widened the interestin improving methods for obtaining protoplasts from avariety of fungi or yeast of biotechnological interest [2].Fusion and transformation systems depend on theavailability of protoplasts in large numbers. Anothermajor aspect requiring consideration relates to thespeed of protoplast production [3,4]. In the past, proto-plasts were isolated using enzymes from the snail Helixpomatia [5]. Microbial enzymes are now used to digestthe cell wall and are available commercially. However,enzyme treatment for fungi and yeast is not routine[6,7]. These protoplasts do not regenerate well, and

efforts have been made to improve the conditions forregeneration. Factors in the enzyme treatment mayreduce the rate of regeneration; for example proteinasein cell-wall-lytic enzyme preparations may be detrimen-tal. Recently a Deuteromycotina yeast, Trichosporoncutaneum SL409, isolated from decaying beet residuewas shown to produce a xylanase that is highly cellu-lase-free when grown on xylan or wheat bran as themajor substrate [8]. Mycelia were formed during theactive growth of T. cutaneum SL409. Trichosporon spp.lack sexuality and to have full knowledge of theirgenetics and regulatory mechanisms, recombinantstrains can be produced by somatic hybridizationthrough protoplast fusion, for which a suitable methodfor protoplast generation is required. This study firstdealt with the use of the lytic enzyme prepared fromTrichoderma koningii UC174. Cell wall lysis by theenzyme was studied by the production of protoplastsfrom T. cutaneum SL409, and compared with thatachieved with combinations of several commercial en-zymes. A detrimental factor in the enzyme preparationwas also investigated and an enzyme system with lessproteinase was prepared to maintain the liberated pro-toplasts intact and improve the rate of regeneration.

* Corresponding author.E-mail address: liuwen@life.sdu.edu.cn (W.-M. Zhu)

0032-9592/00/$ - see front matter © 2000 Published by Elsevier Science Ltd. All rights reserved.

PII: S 0 0 3 2 -9592 (99 )00115 -6

W. Liu, W.-M. Zhu / Process Biochemistry 35 (2000) 659–664660

2. Materials and methods

2.1. Chemicals and enzymes

The cell-wall-lytic enzyme used for most of the studywas prepared by ammonium sulphate precipitation of aculture filtrate of T. koningii UC174 followed by dialy-sis and lyophilization. An enzyme preparation with lessproteinase activity was made by treating the originalenzyme with bentonite. Other enzymes used were Cellu-lase Onozuka RS (Yakult Honsha, Tokyo, Japan),Novozyme 234 (Novo Alle, Denmark), chitinase(Sigma, St Louis, MO), and snail gut enzymes from H.pomatia (Sigma, St Louis, MO). Chitin, laminarin, nig-eran, carboxymethyl cellulose, and p-nitrophenyl-b-D-glucopyranoside were from Sigma (St Louis, MO).Other chemicals used were reagent grade.

2.2. Microorganisms

The microorganisms used in this work were T. cuta-neum SL409 previously isolated from decaying beetresidue in this laboratory [8] and T. koningii UC174from the Institute of Microbiology, Chinese Academyof Science.

2.3. Isolation of cell walls of T. cutaneum

Malt extract agar medium was used for T. cutaneumpropagation. The fungus was grown in liquid culture ina medium containing (g l−1): yeast extract, 5;(NH4)2SO4, 4; KH2PO4, 1; CaCl2, 0.02; MgSO4·7H2O,0.89; sucrose, 10. Aliquots (100 ml) of the medium wereplaced in 500-ml Erlenmeyer flasks, inoculated with2×104 spores per ml and incubated at 35°C on arotary shaker (160 rev. min−1) for 10 h. Cultures wereharvested by suction filtration, washed with 0.5 M KCland then freeze-dried. The lyophilisate was ground in amortar and then suspended in 50 ml per g of 50 mMphosphate buffer, pH 7.4, containing 0.1% (w/v)sodium dodecyl sulphate. The suspension was homoge-nized in a Waring blender at room temperature andcentrifuged at 20 000×g for 20 min. The supernatantwas discarded and the pellet resuspended in the samebuffer. This procedure was repeated several times untilthere was no more protein detectable in the supernatantusing the method of Lowry et al. [9]. Finally the cellwall preparation was washed twice with distilled water,resuspended in 20 ml water per g wet wt and autoclavedat 121°C for 15 min.

2.4. Liquid culture of T. koningii

Aliquots (100 ml) of a salt solution (0.5 g K2HPO4,0.2 g MgSO4·7H2O, 1.0 g NaCl, 2.0 g (NH4)2SO4, 1litre of tap water) containing 2% (v/v) of cell wall

preparation were placed in 500-ml Erlenmeyer flasks.Agar media were prepared by adding 1.5% agar to thebroth media. In control experiments, the cell walls werereplaced by 1% (w/v) glucose. T. koningii UC174 (108

spores) was spread on a cell wall-containing agar andincubated for 48 h at 32°C. Mycelia were harvested andsuspended in 20 ml sterile saline and used for inocula-tion of liquid cultures. Each flask was given a 1% (v/v)inoculum of the suspension of the Trichoderma myceliaand incubated for 72 h at 28°C on a rotary shaker (160rev. min−1).

2.5. Isolation of cell-wall-lytic enzyme

After 72 h of incubation, 1 litre of liquid culture of T.koningii UC174 grown on cell wall medium was cen-trifuged at 8000×g for 10 min at 4°C in order toremove the T. koningii cells. Solid ammonium sulphatewas slowly added to the filtrate to give 80% saturation.After standing at 4°C overnight the precipitate wascollected by centrifugation (20 000×g) for 25 min. Thepellet containing lytic enzyme was dissolved in 5 ml of50 mM phosphate buffer, pH 6.8, and the solutiondialyzed against the same buffer and lyophilized. Lyticactivity was tested by placing 80 mg of the enzymepreparation (dissolved in 50 ml of 20 mM acetate buffer,pH 5.4) into wells (3 mm diameter) in cell wall-contain-ing agar plates and incubating overnight at 30°C.

2.6. Enzyme assay

2.6.1. b-Glucanase (EC 3.2.1.6)This enzyme was assayed using a modification of the

method of Huotari et al. [10]. Suitably diluted enzymesolution (0.5 ml) was mixed with 0.5 ml substratesolution (0.1% w/v laminarin) in 50 mM sodium citratebuffer, pH 5.8, and the mixture incubated for 15 min at37°C.

2.6.2. a-Glucanase (EC 3.2.1.61)To 0.5 ml of suitably diluted enzyme solution, 0.5 ml

substrate solution containing 0.2% (w/v, in sodiumcitrate buffer, pH 5.8) nigeran was added and themixture was incubated for 30 min at 37°C.

2.6.3. Endo-b-1,4-glucanase (EC 3.2.1.4)The enzyme was estimated by incubating tubes (at

37°C for 30 min) containing appropriate dilutions of0.5 ml of crude enzyme and 0.5 ml of 1% (w/v) CMCsolution in 0.1 M acetate buffer at pH 5.0 [11].

2.6.4. Exo-b-1,4-glucanase (EC 3.2.1.91)The appropriate dilutions of enzyme samples were

incubated at 50°C for 1 h with 50 mg of WhatmanNo.1 filter paper (1×6 cm strip) in a final volume of 1ml made with 0.1 M acetate buffer at pH 5.0 [11].

W. Liu, W.-M. Zhu / Process Biochemistry 35 (2000) 659–664 661

The amount of reducing sugar in each of the abovereactions was estimated by the dinitrosalicylic acid(DNS) reagent method [12]. According to this method,2 ml of DNS reagent was added to each tube andcolour was developed by heating in a boiling water bathfor 10 min; 4 ml of water was added to it after coolingand the absorbance was read at 580 nm.

2.6.5. Chitinase (EC 3.2.1.14)To suitably diluted 0.5 ml of enzyme sample, 0.5 ml

of 0.1% (w/v) suspension of colloidal chitin prepared in50 mM citrate buffer (pH 5.8) was added and themixture was incubated for 2 h at 37°C in a shakingwater bath. Then, 100 ml of 0.8 M potassium tetrabo-rate, pH 9.1, was added and the mixture was heated ina boiling water bath for 3 min. After cooling under tapwater, 3 ml of p-dimethylaminobenzaldehyde wasadded and incubated at 37°C for 20 min. Absorbancewas read immediately at 585 nm [13].

2.6.6. b-1,4-glucosidase (EC 3.2.1.21)To 0.5 ml of appropriate dilution of enzyme sample,

0.5 ml of 1 mM p-nitrophenyl-b-D-glucopyranoside(pH 5.0) was added and the mixture incubated at 37°Cfor 30 min. The reaction was terminated by adding 4 mlof 0.2 M NaOH/glycine buffer (pH 10.6). The yellow-coloured p-nitrophenol liberated was determined bymeasuring absorbance at 420 nm [11].

2.6.7. ProteinaseProteinase activity was assayed by the method of

McDonald and Chen [14] with some modifications,with casein as the substrate at pH 5.5. The concentra-tion of tyrosine liberated in the supernatant was mea-sured by the method of Lowry et al. [9].

2.7. Enzyme units

Enzyme activity is expressed in units (U) as theamount of the enzyme needed to release 1 mmol ofglucose, p-nitrophenol, tyrosine, N-acetylglucosamine,or their equivalent per ml per min.

2.8. Protein estimation

The soluble protein contents were estimated accord-ing to the method of Lowry et al. [9].

2.9. Production of protoplasts of T. cutaneum

A 150-ml portion of the medium (20 g sucrose, 10 gmalt extract, 1 g yeast extract, 1 g KH2PO4, 0.3 gMgSO4·7H2O, 0.1 g CaCl2·2H2O, 1 litre of distilledwater, pH 6.5) was placed in a 500-ml Erlenmeyer flaskand inoculated with 2×104 spores per ml of T. cuta-neum. After 10 h at 35°C on a rotary shaker (160 rev.

min−1) mycelia were harvested by centrifugation(1000×g, 5 min) and washed twice with distilled water.Protoplasting was performed using 50 mg wet myceliain 5.0 ml of 50 mM citrate–phosphate buffer, pH 5.4.Lytic enzyme preparation of T. koningii was added atdifferent concentrations and with different osmotic sta-bilizers; after that mycelium suspensions were incubatedat 37°C on a rotary shaker (120 rev. min−1).Novozyme 234, Cellulase Onozuka RS, and snail gutenzymes from H. pomatia were also tested for theircapability of releasing protoplasts from T. cutaneum.Progress of protoplast formation was examined by mi-croscopy and counted using a haemocytometer.

2.10. Protoplast regeneration

The crude protoplast suspension was filtered througha G2 sintered glass funnel and centrifuged at 1000×gfor 5 min. The pellet, resuspended in stabilizer solution,was diluted and plated onto regeneration medium(0.7% agar). The mixture was then overlaid on the samemedium (2% agar) that had hardened in a Petri dish.The regeneration medium (pH 6.0) was composed of(w/v) 0.15% peptone, 1% malt extract, 0.1% KH2PO4,and 0.1% yeast extract. The additional supplementsincluded osmotic stabilizers and 0.1% Triton X-100 ascolony restrictor. The cultures were incubated at 32°Cuntil colonies became visible.

2.11. Data presentation

All results reported are the average of at least threedeterminations.

3. Results and discussion

Cell walls of T. cutaneum SL409 were isolated byhomogenization of lyophilized mycelium in a buffercontaining sodium dodecyl sulphate, subsequent cen-trifugation and thorough washing of the pellet to freethe cell walls from any soluble proteins. This prepara-tion was used as substrate to test the ability of T.koningii to produce lytic exoenzymes for cell wall degra-dation. T. koningii was grown in liquid culture in amedium containing the cell walls of T. cutaneum SL409as the sole carbon source. Total lysis of added cell wallswas observed within 72 h. The growth of T. koningii onthe cell wall material of T. cutaneum produced variouslytic enzymes, viz b-1,4-glucosidase, b-1,3-glucanase,a-1,3-glucanase, exo-b-1,4-glucanase, endo-b-1,4-glu-canase, chitinase and proteinase. b-1,4-Glucosidase, a-1,3-glucanase, and b-1,3-glucanase showed an earlypeak on the third day and proteinase on the secondday. Endo-b-1,4-glucanase, exo-b-1,4-glucanase andchitinase yields were maximum on the fourth day (datanot shown).

W. Liu, W.-M. Zhu / Process Biochemistry 35 (2000) 659–664662

For isolation of the lytic activity from the liquidculture of T. koningii UC174, total protein was precipi-tated from the culture filtrate with ammonium sulphateand lyophilized. From 1 litre of culture, about 20 mg of

protein could be isolated. To test the lytic activity ofthis preparation aliquots were filled into wells in a cellwall containing agar plate. Within 72 h of incubationthe agar was cleared around the wells indicating cellwall degradation. In order to test whether the lyticactivity of T. koningii was induced by growth on thecell walls of T. cutaneum, the organism was grown inliquid culture containing glucose instead of the cellwalls and exoenzymes were isolated as described above.This preparation, however, was not capable of cell walllysis. Therefore, the lytic activity seems to be inducibleby the presence of its substrate.

There are a number of important factors that influ-ence protoplast isolation, i.e. organisms, lytic enzymes,osmotic stabilizers and others. In this sense, the condi-tions for protoplast formation and regeneration of T.cutaneum were established.

The preparation of lytic enzyme of T. koningii wasused to prepare protoplasts from T. cutaneum. Theorganism was grown in liquid culture at 35°C andharvested after 10 h. The mycelia were washed andresuspended in 0.7 M MgSO4 and exoenzyme prepara-tion was added to a final concentration of 1.0 mg ml−1.During 2 h a titre of 1.5×107 protoplasts per ml wasattained (Fig. 1). Besides MgSO4, various other osmoticstabilizers were tested, all at 0.7 M concentration. InKCl and sucrose, good results of protoplast formationwere observed. In mannitol and NH4Cl, formation ofprotoplasts with a lower yield (B4×106 ml−1) wasobserved, while in glucose and NaCl, protoplast forma-tion was nearly zero (Fig. 1). The efficiency of proto-plast formation strongly decreased with the age of theTrichosporon cultures. When cells were older than 18 hno protoplast release was observed regardless whichkind of osmotic stabilizer was used.

Fig. 2 shows the patterns of protoplast formationduring incubation with T. koningii enzyme at concen-trations of 0.2–2.0 mg ml−1. The initial rate of proto-plast formation and the yields of protoplasts from T.cutaneum were increased by the addition of a highconcentration of the enzyme, but were not improved byadditions over 1.0 mg ml−1. The maximum yields ofprotoplasts were reached after 2–3 h incubation, butthe number decreased above 3 h because some proto-plasts burst. The bursting of protoplasts might becaused by injury to membranes by the proteinase in theT. koningii enzyme preparation. Some authors havereported that proteolytic activity present in lytic en-zyme systems affects protoplast yield and regenerationcapacity [15,16]. To prevent such injury, the enzymewas treated with bentonite. The stability of the proto-plasts of T. cutaneum produced by the treated enzymewas examined and compared with those produced bythe original enzyme. The bentonite treatment reducedthe proteinase activity of the enzyme without muchdecrease in the b-1,3-glucanase, endo-b-1,4-glucanase,

Fig. 1. Effect of osmotic stabilizers on protoplast release from T.cutaneum by the lytic enzyme from T. koningii. Osmotic stabilizers(0.7 M) used were: 1, MgSO4; 2, KCl; 3, Sucrose; 4, Mannitol; 5,NH4Cl; 6, Glucose; 7, NaCl. The incubation time was 2 h.

Fig. 2. Course of protoplast formation of T. cutaneum by the lyticenzyme from T. koningii. Concentrations of the enzyme were (w/v)0.2% (�), 0.5% (), 1.0% (�). 1.2% (�), 1.5% ( ), and 2.0% ().

Table 1Effects of bentonite treatment on the protoplast-forming ability ofcrude T. koningii enzyme from T. cutaneum

Trichoderma enzyme (1 mg ml−1) Original Bentonite-treated

b-1,3-Glucanase activity (U ml−1) 1.38 1.12Endo-1,4-b-glucanase activity (U 1.671.96

ml−1)Chitinase activity (U ml−1) 1.391.52

0.35 0.028Proteinase activity (U ml−1)Protoplasts formation from T. cuta-

neum (×106 ml−1)2 h 14.90 11.24

4.174 h 11.631.816 h 11.87

W. Liu, W.-M. Zhu / Process Biochemistry 35 (2000) 659–664 663

Table 2Frequency of regeneration of T. cutaneum protoplasts produced bythe original and treated preparations of the Trichoderma enzyme

Trichoderma enzyme (1 mg ml−1) Regeneration (%)a

47.4OriginalProteinase-reduced 70.6

a Regeneration rates were measured by counting colonies formedand number of protoplasts plated.

injury might reduce the regeneration frequency of pro-toplasts, hence the removal of the factor contained inthe cell-wall-lytic enzyme that caused bursting (i.e.proteinase) should improve the regeneration of proto-plasts. This was examined with protoplasts of T. cuta-neum produced by the original and treated enzymes.The rate of regeneration measured by colony formationwas increased some 1.5 times by the proteinase-reducedenzyme (Table 2).

Among the different osmotic stabilizers used in theregeneration medium, T. cutaneum exhibited good re-sults with KCl and NH4Cl (Fig. 3). T. cutaneum proto-plasts did not seem to regenerate in the presence ofglucose or NaCl, while in mannitol and sucrose regen-eration rose to 12 and 19%, respectively, but still quitelow. The effect of T. koningii enzyme digestion time onthe regeneration of T. cutaneum is shown in Fig. 4.Lengthy treatment with T. koningii enzyme decreasedthe ability of the protoplasts to regenerate. It has beenobserved that protoplasts obtained in shorter exposuretimes to lytic enzymes have greater capacity to regener-ate than those which have been in contact with theseenzymes for longer periods, since the membrane isliable to be damaged [17]. For this reason, the digestiontime was kept to a minimum with T. cutaneum. Regen-eration of T. cutaneum with spread plates was com-pleted in 60 h. The use of soft-agar overlays did notsignificantly increase the regeneration frequencies of T.cutaneum. However, regeneration was faster with theoverlays, as regeneration was complete after 36 h.

Finally, a number of commercial preparations oflytic enzymes known to induce the formation of proto-plasts from many organisms were tested with T. cuta-neum. Novozyme 234, Cellulase Onozuka RS, and snailgut enzymes from H. pomatia were tried in differentcombinations to produce protoplasts from T. cutaneum(Table 3). Novozyme 234, the lytic enzyme mixtureproduced by Trichoderma harzianum, gave good yieldsof T. cutaneum protoplasts in the presence of 0.7 MMgSO4. However, the addition of the above lytic en-zymes to Novozyme 234 proved detrimental. NeitherCellulase Onozuka RS nor snail gut enzymes from H.pomatia were able to generate protoplasts either aloneor in combination. However, protoplasts were pro-duced when commercial chitinase was added to Cellu-lase Onozuka RS or snail gut enzymes (Table 3). Theaddition of chitinase to the lytic enzyme systems is notuncommon and has been shown to be necessary forhydrolysis of the cell walls of many organisms [18,19].

In summary, the present study established the condi-tions by which T. cutaneum can be protoplasted and bywhich the resulting protoplasts can be regenerated. Italso gives some insights into the factors that control thesuccess in forming and regenerating protoplasts of T.cutaneum. These methods should be widely applicableto Trichosporon strains and produce a basis for proto-plast fusion experiments with these organisms.

Fig. 3. Effect of osmotic stabilizers on regeneration frequency ofprotoplasts from T. cutaneum. Protoplasts were regenerated on regen-eration medium at 32°C. 1, KCl; 2, Sucrose; 3, Mannitol; 4, NH4Cl;5, Glucose; 6, NaCl.

Fig. 4. Effect of enzyme digestion time on regeneration frequency ofprotoplasts from T. cutaneum. The osmotic stabilizer used is 0.7 MMgSO4. Protoplasts were regenerated on regeneration medium at32°C. �, protoplast formation; , regeneration frequency of proto-plasts.

and chitinase activities (Table 1). The initial formationof protoplasts from T. cutaneum was somewhat loweredby the bentonite-treated enzyme. However, after 6 hincubation, the number of protoplasts had decreased byonly 12% in the original enzyme, while it was stillincreasing with the treated enzyme. Hence, no signifi-cant difference in protoplast number was observed be-tween the two enzymes. The after-effects of membrane

W. Liu, W.-M. Zhu / Process Biochemistry 35 (2000) 659–664664

Table 3Effect of different lytic enzymes on the formation of T. cutaneumprotoplasts

Lytic enzymea Concentration Protoplasts(mg ml−1) released

(×105 ml−1)

5Novozyme 234 63.5Cellulase Onozuka RS B0.0015

B0.001Snail gut enzymes 55+5Cellulase Onozuka RS+ B0.003

snail gut enzymes5+5 3.16Novozyme 234+Cellulase

Onozuka RS5+5Novozyme 234+snail gut 2.08

enzymesCellulase Onozuka RS+ 5+1 19.2

chitinase5+1 28.6Snail gut enzymes+

chitinase

a The osmotic stabilizer used was 0.7 M MgSO4. The incubationtime was 2 h.

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Acknowledgements

The assistance of Drs W Xu and Q Wu in this workis appreciated. This work was supported by a grantfrom the Natural National Foundation of China.

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