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Efficient production of chitinase by Wasabia japonica protoplasts immobilized in double-layered gel fibers
JOURNALOF FERMENTATION AND BIOENGINEERING Vol. 81, No. 5, 394-399. 1996
Efficient Production of Chitinase by Wasabia japonica Protoplasts Immobilized in Double-Layered Gel Fibers HIDE0
TANAKA,‘*
TAKANORI
YAMASHITA,’ HIDEKI AOYAGI,’ AND YUKIO FUKUNAGA*
YOSHINARI
YAMAMOT0,2
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 3OS and Gakken Co. Ltd., Outa-ku, Tokyo 145,* Japan Received 13 November 199UAccepted
29 January 1996
Double-layered gel fibers consisting of an inner layer in which Wasabiajaponica protoplasts were immobilized and an outer layer of calcium alginate was prepared and used for chitinase production in Erlenmeyer flasks. Immobilized protoplasts could be cultivated in shake cultures at low osmotic pressure without disruption. The chitinase productivity by the protoplasts immobilized in double-layered gel fiber (2.0 U/ml at 5 d) was much higher than that of the immobilized cells (0.36 U/ml at 5 d). On the third day of cultivation, cell wall regeneration in the immobilized protoplasts was detected under tight microscope. This implies that prevention of cell wall regeneration is a pre-requisite for long term process with protoplasts. When 2,6-dichlorobenzonitrile was added to the broth, cellulose cell wall synthesis was inhibited, and active protoplasts were maintained for a long time without cell wall regeneration. The chitinase productivity increased significantly when the alginate-immobilized protoplasts were cultivated under pure oxygen aeration. Similar results were also obtained in a bubble column reactor. At high chitinase concentrations, product inhibition was observed. The chitinase production in the batch culture of the immobilized protoplasts could be represented by the following equation, dP/dt = ti - (1 - P/P,,J”.29, where P= chitinase concentration (U-enzyme/l-broth), P,,,= limit chitinase concentration (U-enzyme/Z-broth) and rip= maximum chitinase production rate (U-enzyme/Z-broth-d). During chitinase production by the immobilized protoplasts, product inhibition was observed from the start of the culture and in order to maintain the chitinase productivity above 80% of the maximum productivity, it was necessary to keep the chitinase concentration in the broth lower than 1.8 U/ml. A system for continuous production with simultaneous recovery of chitinase was therefore developed. A production column containing W. japonica protoplasts immobilized in double-layered gel fibers was coupled to a chitin column and by circulating the fermentation broth between the two columns, continuous production with simultaneous recovery of chitinase was successfully carried out. With this system, it was possible to maintain high and stable chitinase production for 25 d and a high amount of chitinases (26,000 U) was obtained. [Key words: protoplast, maiography,
pioduci
Wmzbia juponica, chitinase,
double-layered
gel fibers,
chitin
affinity
chro-
inhibition]
is located in the cell membrane (14-19). Therefore, we considered that exposure of the cell membrane by removing the cell walls would enhance the degree of elicitation, and developed a novel production system consisting of isolated protoplasts. However, protoplasts were very fragile and could not be used for a long term production process, and active protoplasts easily regenerated cell wall during cultivation. In this study, the use of alginate as both an elicitor and immobilization carrier (providing stability to the protoplasts) for efficient production of chitinase by W. japonica protoplasts was investigated. The productivity was further enhanced by simultaneous chitinase recovery from the broth.
Most of the useful metabolites biosynthesized by plant cells are stored within the cells, thus making their efficient and continuous production very difficult. Moreover, after the cultivation, the cells have to be disintegrated in order to extract and purify the desired product. These add both to the complexity of the process and production cost. The amounts and the rates of production of these metabolites by plant cell cultures are still very low. Various methods for releasing useful products into the culture broth have been investigated. These include, pH cycling (l), use of permeabilizing agents (2), high ionic strength (3) and electroporation (4). Although these methods do enhance product release, they are usually detrimental to cell viability and therefore not suitable for long term processes. It has been reported with some plant cells that immobilization in calcium alginate gel results in increased specific productivity and/or induces the release of the intracellular useful metabolites into the culture broth (5-11). We also observed the promotion effect of immobilization of Wasabia japonica cells in alginate gel fiber on chitinase production (12) and discussed that alginate acts as a kind of elicitor (13). Many researchers have reported the possibility that a receptor for elicitor (oligosaccharides)
MATERIALS
AND METHODS
Plant cell and culture conditions W. japonica cells used in all the experiments secrete high concentrations of chitinase into the culture broth (20). Cell suspension cultures were maintained and subcultured every 14 d in Murashige-Skoog (MS) medium (21) supplemented with glucose (30 g/f) and 2,4-dichlorophenoxy acetic acid (2,4D, 0.1 mg/Z) while the pH was adjusted to 5.8. The cultivations were done in 200 ml Erlenmeyer flasks containing 45 ml of the medium on a rotary shaker (120 rpm) at
* Corresponding author. 394
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25°C. Five ml (packed cell volume [PCV]) of the cells were used to inoculate each flask, giving an inoculum ratio of 10%. Preparation of the enzyme Protoplast isolation solution used for isolating protoplasts was carried out as follows: Cellulase Onozuka RS (Yakult Honsha Co. Ltd., Tokyo [l.O% (w/v)]) and 0.1% (w/v) of Pectolyase Y23 (Seikagakukogyo Co. Ltd., Tokyo) were dissolved in CPW solution (21) containing 13% (w/v) mannitol, 0.5% (w/v) potassium dextran sulfate (Meito Sangyo Co. Ltd., Nagoya) and 0.06% (w/v) 2-N-morpholino ethane sulfonic acid (MES). The pH of the solution was adjusted to 5.6. Cultured II’. japonica cells at 8 d were incubated with the enzyme solution for 1 h. Immobilization of W. japonica protoplasts in doublelayered gel fibers and cultivation in Erlenmeyer flasks Double-layered gel fibers were prepared according to the method described previously (12, 23, 24) with a minor modification. The internal diameters of the inner and outer nozzles were 1 mm and 2 mm, respectively. Sodium alginate solution (2.5% [w/v]) containing 8% glucose (final volume =33 ml) was used for the outer layer while the inner layer was prepared with the 2.5% (w/v) Naalginate (final volume= 12 ml) containing 8% glucose and protoplasts isolated from 5 ml (PCV) of W. japonica cells. The gelling agent was a 0.1 M CaCl, solution containing 8% glucose and 3 mM MES (PH 5.6). After the preparation, the spiral double-layered gel fibers were stabilized by maintaining them in the CaCI, solution containing 8% glucose and 3 mM MES (pH 5.6) for 30 min. The CaC& solution was then removed and the gel fibers were washed with MS medium. After the washing, the immobilized protoplasts were cultivated in a 200mlErlenmeyer flask containing 45 ml of the medium on a rotary shaker (120 rpm) at 25°C in the dark. In order to inhibit cellulose cell wall synthesis in the protoplasts, 2,6dichlorobenzonitrile (2,6-D, Wako Pure Chemical Industries, Osaka) was added to the medium. The protoplasts imCultivation in an aerated-flask mobilized in double-layered gel fibers were cultivated in 200ml aerated-flask (12, 25) on a rotary shaker at 120 rpm. Pure oxygen was used for aeration at 1.O vvm. Cultivation in a bubble column reactor Sodium
BY IMMOBILIZED
B’. JAPONICA
PROTOPLASTS
395
alginate solution (2.5% [w/v]) containing 8% glucose (final volume= 123 ml) was used for the outer layer while the inner layer was prepared with the 2.5% (w/v) Naalginate (final volume=43 ml) containing 8% glucose and protoplasts isolated from 18.5 ml (PCV) of W. japonica cells. The double-layered gel fibers were prepared directly inside a bubble column reactor (#=40 mm, h =4OOmm, Fig. 1A). After stabilizing and washing the fibers as described above, 266 ml of the MS medium was added to the column and the cultivation was done at 25°C in the dark. Pure oxygen was used for aeration at 1.0 vvm. Production of chitinase coupled with its continuous Commercially available recovery by chitin column cheap Chitin EX [purified shrimp chitin powder, mesh size 70 (Katokichi Co. Ltd., Tokyo)] could be used as an affinity adsorbent to adsorb the chitinase in the culture broth (12, 20). The immobilized protoplasts were cultivated in a bubble column reactor which was connected to another column ($=30mm, h=250 mm, Fig. 1B) in which chitin powder (20g) was packed for chitinase recovery. By considering the optimum conditions for both chitinase production and adsorption, a modified MS medium [five times diluted MS medium supplemented with 2,4-D (0.1 mg/Z), glucose (log/l), 2,6-D (2.0mg/l) and MES (2g/l)] was used and the pH was adjusted to 5.8. The elution of chitinase from the chitin column was done as described previously (12, 20). Analytical method The characteristics of the chitinase and the method for measurement of chitinase activity have been described in our previous paper (20). The glucose concentration was measured enzymatically with glucose oxidase and peroxidase. All the experiments were performed in triplicate and the results were expressed as the mean values. There were no significant differences within the triplicate values. RESULTS AND DISCUSSION Production of chitinase by protoplasts immobilized in It is difficult to cultivate susdouble-layered gel fibers pended protoplasts in shake cultures because of their weakness to hydrodynamic stress. Furthermore, protoplasts are easily disrupted under low osmotic pressure
(4
FIG. 1. Experimental set-up for chitinase production by immobilized W’.japonica cells in column-type reactor (A) and chitinase recovery system (B). 0 Fresh medium supply, @medium circulation, @ thermostat water inlet, @ thermostat water outlet, @ thermostat water inlet, @ thermostat water outlet, (3 sampling nozzle.
396
TANAKA ET AL.
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condition. Protoplasts immobilized in double-layered gel fibers could be cultivated in shake cultures and under low osmotic pressure without disruption. Alginate gel gives an artificial mechanical support to the protoplasts and thus enables them to withstand osmotic pressure changes and hydrodynamic stress. Figure 2 shows the time courses of chitinase activity in the broth during the cultivation of immobilized W. juponica cells and protoplasts in 200ml Erlenmeyer flasks. The chitinase production by the immobilized protoplasts increased rapidly, reaching about 2.0 U/ml in 5 d and then levelled off. The chitinase production by the protoplasts immobilized in double-layered gel fiber was much higher than that produced by immobilized cells. Cell wall regeneration in the immobilized protoplasts was detected under light microscope on the third day of cultivation. Prevention of cell wall regeneration is thus a pre-requisite for long term process with protoplasts. In this study, 2,6-D was used as an inhibitor of cellulose cell wall synthesis (26). When 2.0mg// of 2,6-D (molecular weight= 172.02) was added into the broth, it diffused easily into the alginate gel fiber, and active protoplasts were maintained for 15 d without cell wall regeneration. Chitinase activity in the broth increased during the cultivation and reached about 3.0 U/ml at 8 d and then levelled off (Fig. 2). In the subsequent experiments, 2.0mg/l of 2,6-D was therefore added to the broth during the cultivation of the immobilized protoplasts in double-layered gel fibers. Since in the W. juponica immobilized cell culture, more than 82% of the chitinase was secreted into the medium, the high productivity obtained with immobilized protoplasts is not only due to the release of the stored chitinase but the protoplasts themselves actively produced chitinase. In our previous paper (12), the chitinase activity produced by immobilized W. japonica cells was highly increased when the dissolved oxygen concentration was raised by aeration with pure oxygen gas. The protoplasts immobilized in double-layered gel fibers were therefore cultivated in an aerated-flask with pure oxygen at l.Ovvm. As shown in Fig. 3, chitinase production was stimulated, reaching about 3.0U/ml after 4d of cultivation. The rate of chitinase production by the immobilized protoplasts aerated with pure oxygen gas was about four times higher than that of immobi-
5
10
15
Cultivation time (d)
FIG. 3. Effect of oxygen gas supply on chitinase production by immobilized W. juponica cells and protoplasts. Symbols: A, immobilized cells without aeration; A, immobilized cells with pure oxygen gas at 1.Ovvm; 0 , immobilized protoplasts without aeration; 0, immobilized protoplasts with pure oxygen gas at 1.Ovvm.
lized cells. The results demonstrated that the W. juponicu protoplasts immobilized in alginate double-layered gel fibers are very effective in chitinase production. The mechanism of the promotive effect of oxygen on chitinase production by W. japonica cells and protoplasts is currently being investigated. Cultivation in a bubble column reactor As shown in Fig. 4, the same level of chitinase productivity was obtained even when the process was scaled up from the aerated-flask to a bubble column reactor. The results of a repeated-batch culture in the bubble column reactor with protoplasts immobilized in double-layered gel fibers are shown in Fig. 5. The repeated-batch culture was performed by replacing the broth with a fresh MS medium every 4 d. During five repeated batches lasting for 20 d, the active protoplast was maintained without cell wall regeneration and there was no decrease in the chitinase production during each batch culture. However, even though there was enough nutrients left at the end of each batch culture, the maximum chitinase activity levelled off at 3.2U/ml. This was also observed during the batch culture (Fig. 4). In our previous paper, the
’
Cultivation time (d)
FIG. 2. Effect of 2,6-dichlorobenzonitrile (2,6-D) on chitinase production by immobilized W. juponica protoplasts. Symbols: A, immobilized cells; 0, immobilized protoplasts; n , immobilized protoplasts with 2,6-D.
10 5 Cultivation time (d)
15
‘0
FIG. 4. Comparison of chitinase production and glucose consumption by immobilized W. japonica protoplasts in column-type reactor (0 , n ) and aerated-flask (0, 0 ).
CHITINASE PRODUCTION
VOL. 81, 1996
5
10
15
20
BY IMMOBILIZED
-
397
O!--TTrK
Cultivation time (d)
Repeated-batch production of chitinase by immobilized W. juponicu protoplasts in column-type reactor.
Cultivation time (d)
FIG. 5.
same phenomenon was observed in the immobilized W. japonica cell culture and it was due to product chitinase inhibition (12). Although the cessation of chitinase production in the immobilized protoplast culture, when excess nutrient was still remaining in the broth, was attributable to product inhibition, it was not clear if it was caused by the produced chitinase or by other metabolites. This was investigated by adding purified chitinase to the broth. No further chitinase was produced in a medium to which 3.2 U/ml of purified chitinase (20) was added at the beginning of the immobilized protoplast culture; while the final chitinase activity in the culture to which 1.6 U/ml of chitinase was added was 3.2 U/ml. These imply that the inhibition was caused by the produced chitinase. The above results have shown that in the immobilized protoplast culture, the cessation of chitinase production was due to chitinase inhibition. In our previous paper, a production column containing W. japonica cells immobilized in double-layered gel fibers was coupled to a chitin column (+= 12 mm, h = 140 mm, 2g of chitin powder was packed) and, by circulating the fermentation broth between the two columns, continuous production with simultaneous recovery of chitinase was successfully carried out. In order to adapt this system to the chitinase production by the immobilized protoplasts, we tried to construct a kinetic model for the batch chitinase production by the immobilized W. japonica protoplasts. The limit chitinase activity (P,) was 3.2 U/ml and by assuming that there is no substrate inhibition, the chitinase specific production rate by the immobilized protoplasts could be described by Eq. 1 (12). dP/dt = vp. (1 -P/P,)
W. JAPONICA PROTOPLASTS
FIG. 6. Estimation of the inhibition constant (n) during chitinase production by immobilized W. juponicu protoplasts in column-type reactor. Symbol and Line: 0, experimental chitinase activity; -, simulation curve from Eq. 1 using Runge-Kutta-Gill method.
The results show that during chitinase production, product inhibition is observed from the beginning of the culture [when the product (chitinase) concentration is still very low]. From Fig. 7, it is seen that in order to maintain the production rate above 80% of the maximum value (1,600 [U-enzyme/l-broth. d], it is necessary to keep the chitinase concentration in the broth below 1.8 U/ml. As the chitinase production rate by the immobilized protoplasts was about four times higher than that of the immobilized cells (Fig. 7), for efficient chitinase removal from the immobilized protoplast culture, the capacity of chitin column must be increased. Production of chitinase coupled with its continuous recovery by chitin column The results of fed-batch production coupled with simultaneous chitinase removal from the broth by using chitin column (Fig. 1B) are shown in Fig. 8. The chitin column was replaced with a new column whenever chitinase activity in the broth reached 1.8 U/ml. Also, by supplying five times diluted fresh MS medium each time the column was changed, (X107
I 100
(1)
where P= chitinase concentration (U-enzyme//-broth), Pm = limit chitinase concentration (U-enzyme//-broth), vp= maximum chitinase production rate (U-enzyme/l-broth. d) and n=inhibition constant (-). In the case of the batch cultivation of immobilized protoplasts, 7jp was calculated to be 2000 (U-enzyme/l-broth. d) from the maximum slope of a chitinase concentration vs time curve during batch cultures. The inhibition constant (n) was estimated from Eq. 1 using Runge-Kutta-Gill method to be 0.29 (Fig. 6) [in the case of immobilized cells, ~~=487.5, n=0.24]. The relationship between P and dP/dt as calculated from Eq. 1, is shown in Fig. 7.
FIG. 7. Relationship between chitinase activity (P) and chitinase production rate (dP/dt) by immobilized W. juponicu cells and protoplasts. The percentage production rate was calculated as (dP/dr)/ or x 100. Lines: -, immobilized protoplasts; --mm,immobilized cells.
398
TANAKA ET AL.
J.
FERMENT. BIOENG.,
1
1 1
0
15
0
20
25
Cuttlvation time (d) FIG.
8.
Fed-batch production of chitinase in modified MS medium coupled with it’s continuous removal by chitin column
the glucose concentration in the broth was maintained at about 6g/l. The result shows that the chitinase was efficiently adsorbed to the adsorption column and chitinase activity in the broth was maintained below 1.8 U/ml. As a result of this, a high rate of chitinase production was maintained over a long period of time and as high as 26,000U of chitinase was obtained in 25 d. Table 1 shows a comparison of the results of ordinary batch cultivation with those of batch and fed-batch cultivation coupled with simultaneous chitinase recovery and pH control. In immobilized protoplast culture, by controlling the pH and simultaneously removing the chitinase from the broth, the production rate increased from 0.6 to 2.90U1ml.d in batch. In fed-batch production coupled with simultaneous chitinase removal from the broth, a high production rate (3.0 U/ml.d) could be maintained for 25 d. This is ten and twenty times higher than those of the immobilized cell culture and free cell efficient production of culture, respectively. Thus, chitinase can be achieved with protoplasts immobilized in double-layered gel fibers if product inhibition is prevented by circulating the culture broth through a chitin column. This method of using protoplasts (exposure of cell membrane by removing cell walls) is potentially useful for production of many metabolites where the receptors for the elicitors are located in the cell membrane. Aside from facilitating elicitation, the products are released freely into the broth with the double conseTABLE 1. Comparison of chitinase productivity by free cells (suspension culture), immobilized cells and immobilized protoplasts Type of cultivation
Cultivation time (d)
Suspension culture Free cell without chitin column O-16 Immobilized cells Fed-batch with chitin column and pH controla Immobilized protoplasts Batch without chitin column Batch with chitin column and pH control Fed-batch with chitin column and pH control a From Ref. 12.
O-40 O-8 &8 o-25
Total activity (U)
Production rate (U/ml. d)
540
0.13
3,211
0.29
864
0.60
8,761
2.90
26,000
3.00
quences of increasing the overall productivity and facilitating downstream processing. Furthermore, since many high molecular weight substances (including elicitors) which otherwise are not accessable to the cells (due to the presence of cell walls) are able to access directly to the cell membrane, the composition and concentration of the metabolites produced by protoplasts may vary from those of cells. REFERENCES 1. Renaudio, J. P.: Uptake and accumulation of an indole alkaloid, [I%] tabernanthine by cell suspension cultures of Catharanthus roseus (L.) G. Don and Acer pseudoplatanus L. Plant Sci. Lett., 22, 59-69 (1981). 2. Brodelius, P. and Nilsson, K.: Permeabilization of plant cells resulting in release of intracellular stored products with preserved cell’s viability. Eur. J. Appl. Microbial. Biotechnol., 17, 275-280 (1983). 3. Tanaka, H., Hirao, C., Semba, H., Tozawa, Y., and Ohmomo, S.: Release of intracellulary stored 5’-phosphodiesterase with preserved plant cell viability. Biotechnol. Bioeng., 27, 89G 893 (1985). 4. Brodelius, P.: Permeabilization of plant cells for release of intracellularly stored products: viability studies. Appl. Microbiol. Biotechnol., 27, 561-566 (1988). 5. Brodelius, P., Deus, B., Mosbach, L., and Zenk, M. H.: Immobilized plant cells for the production and transformation of natural product. FEBS Lett., 103, 93-97 (1979). 6. Brodelius, P. and Nilsson, K.: Entrapment of plant cells in different matrixes. FEBS Lett., 122, 312-316 (1980). 7. Ayabe, S., Iida, K., and Furuya, T.: Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells. Plant Cell Rep., 3, 186-189 (1986). 8. Haldimann, D. and Brodelius, P.: Redirecting cellular metabolism by immobilization of cultured plant cells: a method study with CoJka arabica. Phytochem., 26, 1431-1434 (1987). 9. Asada, M. and Shuler, M. L.: Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ, elicitors and alginate immobilization. Appl. Microbial. Biotechnol., 30, 475-481 (1989). 10. Kim, D. J. and Chang, H. N.: Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization. Biotechnol. Bioeng., 36, 460466 (1990). 11. Ramakrishna, S. V., Reddy, G. R., Curtis, W. R., and Humphrey, A. E.: Production of solavetivon by immobilized cells of Hyoscyamus muticus. Biotechnol. Lett., 15, 301-306 (1993). 12. Tanaka, H., Kaneko, Y., Aoyagi, H., Yamamoto, Y., and Fukunaga, Y.: Efficient production of chitinase by immobilized Wasabia japonica cells in double-layered gel fibers. J. Ferment.
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Bioeng., 81, 220-225 (1996). 13. Aoyagi, H., Okada, M., Akimoto, C., Katsuyama, K., Yosbida, S., Kusakabe, I., and Tanaka, H.: Promotion effect of alginate on chitinase production Lett. (1996). in press
by Wasobia japonica cells. Biotechnol.
14. Yoshikawa, M., Keen, N. T., and Wang, M. C.: A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation. Plant Physiol., 73, 497-506 (1983). 15. Schmit, W. E. and Ebel, J.: Specific binding of fungal glucan phytoalexin elicitor to membrane from soybean Glycine max. Proc. Natl. Acad. Sci. USA, 84, 4117-4121 (1987). 16. Mark, A. H., Peter, F. H., and Philip, S. L.: Receptor-mediated endocytosis in plant cells. The Plant Cell, 1, 1003-1009 (1989). 17. Cheong, J. J. and Hahn, M. G.: A specific, high-affinity binding site for the hepta-p-glucoside elicitor exists in soybean membrane. Plant Cell, 3, 137-147 (1991). 18. Cosio, E.G., Frey, T., and Ebel, J.: Identification of a highaffinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean. Eur. J. Biochem., 204, 1115-1123 (1992). 19. Sbibuya, N., Kaku, H., Kuchitsu, K., and Maliarik, M. J.: Identification of a novel high-affinity binding site for N-acetylchitooligosaccharide elicitor in the membrane fraction from suspension-cultured rice cells. FEBS Lett., 329, 75-78 (1993). 20. Yamamoto, Y., Fukunaga, Y., Aoyagi, H., and Tanaka, H.:
21.
22.
23.
24.
25.
26.
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Purification and characteristics of secreted chitinase by cultured Wusabia japonicu cells. J. Ferment. Bioeng., 80, 148-152 (1995). Murashige, T. and Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15, 473-479 (1963). XII, Z. H., Davey, M. R., and Cocking, E. C.: Isolation and sustained division of Phaseolus aweus (Mung Bean) root protoplasts. Z. Pflanzenphysiol. Bd., 104, 289-298 (1981). Tanaka, H., Irie, S., and Ochi, H.: A novel immobilization method for preventation of cell leakage from the gel matrix. .I. Ferment. Bioeng., 68, 216-219 (1989). Ochi, H., Takahashi, M., Kaneko, T., Suzuki, H., and Tanaka, H.: Diacetyl production by co-immobilized citrate-positive Lactococcus iactis subsp. lactis 3022 and homogenized bovin liver in alginate fibers with double gel layers. Biotechnol. Lett., 13, 505-510 (1991). Tanaka, H., Ishikawa, H., Nobayashi, H., and Takagi, Y.: A new scale-up method based on the effect of ventilation on aerated fermentation system. J. Ferment. Bioeng., 72, 204-209 (1991). David, W. G. and Barbara, A. S.: The effect of inhibitors of cell wall synthesis on tobacco protoplast development. Physiol. Plant., 55, 25-30 (1982).
Efficient Production of Chitinase by Wasabia japonica Protoplasts Immobilized in Double-Layered Gel Fibers HIDE0
TANAKA,‘*
TAKANORI
YAMASHITA,’ HIDEKI AOYAGI,’ AND YUKIO FUKUNAGA*
YOSHINARI
YAMAMOT0,2
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 3OS and Gakken Co. Ltd., Outa-ku, Tokyo 145,* Japan Received 13 November 199UAccepted
29 January 1996
Double-layered gel fibers consisting of an inner layer in which Wasabiajaponica protoplasts were immobilized and an outer layer of calcium alginate was prepared and used for chitinase production in Erlenmeyer flasks. Immobilized protoplasts could be cultivated in shake cultures at low osmotic pressure without disruption. The chitinase productivity by the protoplasts immobilized in double-layered gel fiber (2.0 U/ml at 5 d) was much higher than that of the immobilized cells (0.36 U/ml at 5 d). On the third day of cultivation, cell wall regeneration in the immobilized protoplasts was detected under tight microscope. This implies that prevention of cell wall regeneration is a pre-requisite for long term process with protoplasts. When 2,6-dichlorobenzonitrile was added to the broth, cellulose cell wall synthesis was inhibited, and active protoplasts were maintained for a long time without cell wall regeneration. The chitinase productivity increased significantly when the alginate-immobilized protoplasts were cultivated under pure oxygen aeration. Similar results were also obtained in a bubble column reactor. At high chitinase concentrations, product inhibition was observed. The chitinase production in the batch culture of the immobilized protoplasts could be represented by the following equation, dP/dt = ti - (1 - P/P,,J”.29, where P= chitinase concentration (U-enzyme/l-broth), P,,,= limit chitinase concentration (U-enzyme/Z-broth) and rip= maximum chitinase production rate (U-enzyme/Z-broth-d). During chitinase production by the immobilized protoplasts, product inhibition was observed from the start of the culture and in order to maintain the chitinase productivity above 80% of the maximum productivity, it was necessary to keep the chitinase concentration in the broth lower than 1.8 U/ml. A system for continuous production with simultaneous recovery of chitinase was therefore developed. A production column containing W. japonica protoplasts immobilized in double-layered gel fibers was coupled to a chitin column and by circulating the fermentation broth between the two columns, continuous production with simultaneous recovery of chitinase was successfully carried out. With this system, it was possible to maintain high and stable chitinase production for 25 d and a high amount of chitinases (26,000 U) was obtained. [Key words: protoplast, maiography,
pioduci
Wmzbia juponica, chitinase,
double-layered
gel fibers,
chitin
affinity
chro-
inhibition]
is located in the cell membrane (14-19). Therefore, we considered that exposure of the cell membrane by removing the cell walls would enhance the degree of elicitation, and developed a novel production system consisting of isolated protoplasts. However, protoplasts were very fragile and could not be used for a long term production process, and active protoplasts easily regenerated cell wall during cultivation. In this study, the use of alginate as both an elicitor and immobilization carrier (providing stability to the protoplasts) for efficient production of chitinase by W. japonica protoplasts was investigated. The productivity was further enhanced by simultaneous chitinase recovery from the broth.
Most of the useful metabolites biosynthesized by plant cells are stored within the cells, thus making their efficient and continuous production very difficult. Moreover, after the cultivation, the cells have to be disintegrated in order to extract and purify the desired product. These add both to the complexity of the process and production cost. The amounts and the rates of production of these metabolites by plant cell cultures are still very low. Various methods for releasing useful products into the culture broth have been investigated. These include, pH cycling (l), use of permeabilizing agents (2), high ionic strength (3) and electroporation (4). Although these methods do enhance product release, they are usually detrimental to cell viability and therefore not suitable for long term processes. It has been reported with some plant cells that immobilization in calcium alginate gel results in increased specific productivity and/or induces the release of the intracellular useful metabolites into the culture broth (5-11). We also observed the promotion effect of immobilization of Wasabia japonica cells in alginate gel fiber on chitinase production (12) and discussed that alginate acts as a kind of elicitor (13). Many researchers have reported the possibility that a receptor for elicitor (oligosaccharides)
MATERIALS
AND METHODS
Plant cell and culture conditions W. japonica cells used in all the experiments secrete high concentrations of chitinase into the culture broth (20). Cell suspension cultures were maintained and subcultured every 14 d in Murashige-Skoog (MS) medium (21) supplemented with glucose (30 g/f) and 2,4-dichlorophenoxy acetic acid (2,4D, 0.1 mg/Z) while the pH was adjusted to 5.8. The cultivations were done in 200 ml Erlenmeyer flasks containing 45 ml of the medium on a rotary shaker (120 rpm) at
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25°C. Five ml (packed cell volume [PCV]) of the cells were used to inoculate each flask, giving an inoculum ratio of 10%. Preparation of the enzyme Protoplast isolation solution used for isolating protoplasts was carried out as follows: Cellulase Onozuka RS (Yakult Honsha Co. Ltd., Tokyo [l.O% (w/v)]) and 0.1% (w/v) of Pectolyase Y23 (Seikagakukogyo Co. Ltd., Tokyo) were dissolved in CPW solution (21) containing 13% (w/v) mannitol, 0.5% (w/v) potassium dextran sulfate (Meito Sangyo Co. Ltd., Nagoya) and 0.06% (w/v) 2-N-morpholino ethane sulfonic acid (MES). The pH of the solution was adjusted to 5.6. Cultured II’. japonica cells at 8 d were incubated with the enzyme solution for 1 h. Immobilization of W. japonica protoplasts in doublelayered gel fibers and cultivation in Erlenmeyer flasks Double-layered gel fibers were prepared according to the method described previously (12, 23, 24) with a minor modification. The internal diameters of the inner and outer nozzles were 1 mm and 2 mm, respectively. Sodium alginate solution (2.5% [w/v]) containing 8% glucose (final volume =33 ml) was used for the outer layer while the inner layer was prepared with the 2.5% (w/v) Naalginate (final volume= 12 ml) containing 8% glucose and protoplasts isolated from 5 ml (PCV) of W. japonica cells. The gelling agent was a 0.1 M CaCl, solution containing 8% glucose and 3 mM MES (PH 5.6). After the preparation, the spiral double-layered gel fibers were stabilized by maintaining them in the CaCI, solution containing 8% glucose and 3 mM MES (pH 5.6) for 30 min. The CaC& solution was then removed and the gel fibers were washed with MS medium. After the washing, the immobilized protoplasts were cultivated in a 200mlErlenmeyer flask containing 45 ml of the medium on a rotary shaker (120 rpm) at 25°C in the dark. In order to inhibit cellulose cell wall synthesis in the protoplasts, 2,6dichlorobenzonitrile (2,6-D, Wako Pure Chemical Industries, Osaka) was added to the medium. The protoplasts imCultivation in an aerated-flask mobilized in double-layered gel fibers were cultivated in 200ml aerated-flask (12, 25) on a rotary shaker at 120 rpm. Pure oxygen was used for aeration at 1.O vvm. Cultivation in a bubble column reactor Sodium
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alginate solution (2.5% [w/v]) containing 8% glucose (final volume= 123 ml) was used for the outer layer while the inner layer was prepared with the 2.5% (w/v) Naalginate (final volume=43 ml) containing 8% glucose and protoplasts isolated from 18.5 ml (PCV) of W. japonica cells. The double-layered gel fibers were prepared directly inside a bubble column reactor (#=40 mm, h =4OOmm, Fig. 1A). After stabilizing and washing the fibers as described above, 266 ml of the MS medium was added to the column and the cultivation was done at 25°C in the dark. Pure oxygen was used for aeration at 1.0 vvm. Production of chitinase coupled with its continuous Commercially available recovery by chitin column cheap Chitin EX [purified shrimp chitin powder, mesh size 70 (Katokichi Co. Ltd., Tokyo)] could be used as an affinity adsorbent to adsorb the chitinase in the culture broth (12, 20). The immobilized protoplasts were cultivated in a bubble column reactor which was connected to another column ($=30mm, h=250 mm, Fig. 1B) in which chitin powder (20g) was packed for chitinase recovery. By considering the optimum conditions for both chitinase production and adsorption, a modified MS medium [five times diluted MS medium supplemented with 2,4-D (0.1 mg/Z), glucose (log/l), 2,6-D (2.0mg/l) and MES (2g/l)] was used and the pH was adjusted to 5.8. The elution of chitinase from the chitin column was done as described previously (12, 20). Analytical method The characteristics of the chitinase and the method for measurement of chitinase activity have been described in our previous paper (20). The glucose concentration was measured enzymatically with glucose oxidase and peroxidase. All the experiments were performed in triplicate and the results were expressed as the mean values. There were no significant differences within the triplicate values. RESULTS AND DISCUSSION Production of chitinase by protoplasts immobilized in It is difficult to cultivate susdouble-layered gel fibers pended protoplasts in shake cultures because of their weakness to hydrodynamic stress. Furthermore, protoplasts are easily disrupted under low osmotic pressure
(4
FIG. 1. Experimental set-up for chitinase production by immobilized W’.japonica cells in column-type reactor (A) and chitinase recovery system (B). 0 Fresh medium supply, @medium circulation, @ thermostat water inlet, @ thermostat water outlet, @ thermostat water inlet, @ thermostat water outlet, (3 sampling nozzle.
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condition. Protoplasts immobilized in double-layered gel fibers could be cultivated in shake cultures and under low osmotic pressure without disruption. Alginate gel gives an artificial mechanical support to the protoplasts and thus enables them to withstand osmotic pressure changes and hydrodynamic stress. Figure 2 shows the time courses of chitinase activity in the broth during the cultivation of immobilized W. juponica cells and protoplasts in 200ml Erlenmeyer flasks. The chitinase production by the immobilized protoplasts increased rapidly, reaching about 2.0 U/ml in 5 d and then levelled off. The chitinase production by the protoplasts immobilized in double-layered gel fiber was much higher than that produced by immobilized cells. Cell wall regeneration in the immobilized protoplasts was detected under light microscope on the third day of cultivation. Prevention of cell wall regeneration is thus a pre-requisite for long term process with protoplasts. In this study, 2,6-D was used as an inhibitor of cellulose cell wall synthesis (26). When 2.0mg// of 2,6-D (molecular weight= 172.02) was added into the broth, it diffused easily into the alginate gel fiber, and active protoplasts were maintained for 15 d without cell wall regeneration. Chitinase activity in the broth increased during the cultivation and reached about 3.0 U/ml at 8 d and then levelled off (Fig. 2). In the subsequent experiments, 2.0mg/l of 2,6-D was therefore added to the broth during the cultivation of the immobilized protoplasts in double-layered gel fibers. Since in the W. juponica immobilized cell culture, more than 82% of the chitinase was secreted into the medium, the high productivity obtained with immobilized protoplasts is not only due to the release of the stored chitinase but the protoplasts themselves actively produced chitinase. In our previous paper (12), the chitinase activity produced by immobilized W. japonica cells was highly increased when the dissolved oxygen concentration was raised by aeration with pure oxygen gas. The protoplasts immobilized in double-layered gel fibers were therefore cultivated in an aerated-flask with pure oxygen at l.Ovvm. As shown in Fig. 3, chitinase production was stimulated, reaching about 3.0U/ml after 4d of cultivation. The rate of chitinase production by the immobilized protoplasts aerated with pure oxygen gas was about four times higher than that of immobi-
5
10
15
Cultivation time (d)
FIG. 3. Effect of oxygen gas supply on chitinase production by immobilized W. juponica cells and protoplasts. Symbols: A, immobilized cells without aeration; A, immobilized cells with pure oxygen gas at 1.Ovvm; 0 , immobilized protoplasts without aeration; 0, immobilized protoplasts with pure oxygen gas at 1.Ovvm.
lized cells. The results demonstrated that the W. juponicu protoplasts immobilized in alginate double-layered gel fibers are very effective in chitinase production. The mechanism of the promotive effect of oxygen on chitinase production by W. japonica cells and protoplasts is currently being investigated. Cultivation in a bubble column reactor As shown in Fig. 4, the same level of chitinase productivity was obtained even when the process was scaled up from the aerated-flask to a bubble column reactor. The results of a repeated-batch culture in the bubble column reactor with protoplasts immobilized in double-layered gel fibers are shown in Fig. 5. The repeated-batch culture was performed by replacing the broth with a fresh MS medium every 4 d. During five repeated batches lasting for 20 d, the active protoplast was maintained without cell wall regeneration and there was no decrease in the chitinase production during each batch culture. However, even though there was enough nutrients left at the end of each batch culture, the maximum chitinase activity levelled off at 3.2U/ml. This was also observed during the batch culture (Fig. 4). In our previous paper, the
’
Cultivation time (d)
FIG. 2. Effect of 2,6-dichlorobenzonitrile (2,6-D) on chitinase production by immobilized W. juponica protoplasts. Symbols: A, immobilized cells; 0, immobilized protoplasts; n , immobilized protoplasts with 2,6-D.
10 5 Cultivation time (d)
15
‘0
FIG. 4. Comparison of chitinase production and glucose consumption by immobilized W. japonica protoplasts in column-type reactor (0 , n ) and aerated-flask (0, 0 ).
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O!--TTrK
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Repeated-batch production of chitinase by immobilized W. juponicu protoplasts in column-type reactor.
Cultivation time (d)
FIG. 5.
same phenomenon was observed in the immobilized W. japonica cell culture and it was due to product chitinase inhibition (12). Although the cessation of chitinase production in the immobilized protoplast culture, when excess nutrient was still remaining in the broth, was attributable to product inhibition, it was not clear if it was caused by the produced chitinase or by other metabolites. This was investigated by adding purified chitinase to the broth. No further chitinase was produced in a medium to which 3.2 U/ml of purified chitinase (20) was added at the beginning of the immobilized protoplast culture; while the final chitinase activity in the culture to which 1.6 U/ml of chitinase was added was 3.2 U/ml. These imply that the inhibition was caused by the produced chitinase. The above results have shown that in the immobilized protoplast culture, the cessation of chitinase production was due to chitinase inhibition. In our previous paper, a production column containing W. japonica cells immobilized in double-layered gel fibers was coupled to a chitin column (+= 12 mm, h = 140 mm, 2g of chitin powder was packed) and, by circulating the fermentation broth between the two columns, continuous production with simultaneous recovery of chitinase was successfully carried out. In order to adapt this system to the chitinase production by the immobilized protoplasts, we tried to construct a kinetic model for the batch chitinase production by the immobilized W. japonica protoplasts. The limit chitinase activity (P,) was 3.2 U/ml and by assuming that there is no substrate inhibition, the chitinase specific production rate by the immobilized protoplasts could be described by Eq. 1 (12). dP/dt = vp. (1 -P/P,)
W. JAPONICA PROTOPLASTS
FIG. 6. Estimation of the inhibition constant (n) during chitinase production by immobilized W. juponicu protoplasts in column-type reactor. Symbol and Line: 0, experimental chitinase activity; -, simulation curve from Eq. 1 using Runge-Kutta-Gill method.
The results show that during chitinase production, product inhibition is observed from the beginning of the culture [when the product (chitinase) concentration is still very low]. From Fig. 7, it is seen that in order to maintain the production rate above 80% of the maximum value (1,600 [U-enzyme/l-broth. d], it is necessary to keep the chitinase concentration in the broth below 1.8 U/ml. As the chitinase production rate by the immobilized protoplasts was about four times higher than that of the immobilized cells (Fig. 7), for efficient chitinase removal from the immobilized protoplast culture, the capacity of chitin column must be increased. Production of chitinase coupled with its continuous recovery by chitin column The results of fed-batch production coupled with simultaneous chitinase removal from the broth by using chitin column (Fig. 1B) are shown in Fig. 8. The chitin column was replaced with a new column whenever chitinase activity in the broth reached 1.8 U/ml. Also, by supplying five times diluted fresh MS medium each time the column was changed, (X107
I 100
(1)
where P= chitinase concentration (U-enzyme//-broth), Pm = limit chitinase concentration (U-enzyme//-broth), vp= maximum chitinase production rate (U-enzyme/l-broth. d) and n=inhibition constant (-). In the case of the batch cultivation of immobilized protoplasts, 7jp was calculated to be 2000 (U-enzyme/l-broth. d) from the maximum slope of a chitinase concentration vs time curve during batch cultures. The inhibition constant (n) was estimated from Eq. 1 using Runge-Kutta-Gill method to be 0.29 (Fig. 6) [in the case of immobilized cells, ~~=487.5, n=0.24]. The relationship between P and dP/dt as calculated from Eq. 1, is shown in Fig. 7.
FIG. 7. Relationship between chitinase activity (P) and chitinase production rate (dP/dt) by immobilized W. juponicu cells and protoplasts. The percentage production rate was calculated as (dP/dr)/ or x 100. Lines: -, immobilized protoplasts; --mm,immobilized cells.
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1
1 1
0
15
0
20
25
Cuttlvation time (d) FIG.
8.
Fed-batch production of chitinase in modified MS medium coupled with it’s continuous removal by chitin column
the glucose concentration in the broth was maintained at about 6g/l. The result shows that the chitinase was efficiently adsorbed to the adsorption column and chitinase activity in the broth was maintained below 1.8 U/ml. As a result of this, a high rate of chitinase production was maintained over a long period of time and as high as 26,000U of chitinase was obtained in 25 d. Table 1 shows a comparison of the results of ordinary batch cultivation with those of batch and fed-batch cultivation coupled with simultaneous chitinase recovery and pH control. In immobilized protoplast culture, by controlling the pH and simultaneously removing the chitinase from the broth, the production rate increased from 0.6 to 2.90U1ml.d in batch. In fed-batch production coupled with simultaneous chitinase removal from the broth, a high production rate (3.0 U/ml.d) could be maintained for 25 d. This is ten and twenty times higher than those of the immobilized cell culture and free cell efficient production of culture, respectively. Thus, chitinase can be achieved with protoplasts immobilized in double-layered gel fibers if product inhibition is prevented by circulating the culture broth through a chitin column. This method of using protoplasts (exposure of cell membrane by removing cell walls) is potentially useful for production of many metabolites where the receptors for the elicitors are located in the cell membrane. Aside from facilitating elicitation, the products are released freely into the broth with the double conseTABLE 1. Comparison of chitinase productivity by free cells (suspension culture), immobilized cells and immobilized protoplasts Type of cultivation
Cultivation time (d)
Suspension culture Free cell without chitin column O-16 Immobilized cells Fed-batch with chitin column and pH controla Immobilized protoplasts Batch without chitin column Batch with chitin column and pH control Fed-batch with chitin column and pH control a From Ref. 12.
O-40 O-8 &8 o-25
Total activity (U)
Production rate (U/ml. d)
540
0.13
3,211
0.29
864
0.60
8,761
2.90
26,000
3.00
quences of increasing the overall productivity and facilitating downstream processing. Furthermore, since many high molecular weight substances (including elicitors) which otherwise are not accessable to the cells (due to the presence of cell walls) are able to access directly to the cell membrane, the composition and concentration of the metabolites produced by protoplasts may vary from those of cells. REFERENCES 1. Renaudio, J. P.: Uptake and accumulation of an indole alkaloid, [I%] tabernanthine by cell suspension cultures of Catharanthus roseus (L.) G. Don and Acer pseudoplatanus L. Plant Sci. Lett., 22, 59-69 (1981). 2. Brodelius, P. and Nilsson, K.: Permeabilization of plant cells resulting in release of intracellular stored products with preserved cell’s viability. Eur. J. Appl. Microbial. Biotechnol., 17, 275-280 (1983). 3. Tanaka, H., Hirao, C., Semba, H., Tozawa, Y., and Ohmomo, S.: Release of intracellulary stored 5’-phosphodiesterase with preserved plant cell viability. Biotechnol. Bioeng., 27, 89G 893 (1985). 4. Brodelius, P.: Permeabilization of plant cells for release of intracellularly stored products: viability studies. Appl. Microbiol. Biotechnol., 27, 561-566 (1988). 5. Brodelius, P., Deus, B., Mosbach, L., and Zenk, M. H.: Immobilized plant cells for the production and transformation of natural product. FEBS Lett., 103, 93-97 (1979). 6. Brodelius, P. and Nilsson, K.: Entrapment of plant cells in different matrixes. FEBS Lett., 122, 312-316 (1980). 7. Ayabe, S., Iida, K., and Furuya, T.: Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells. Plant Cell Rep., 3, 186-189 (1986). 8. Haldimann, D. and Brodelius, P.: Redirecting cellular metabolism by immobilization of cultured plant cells: a method study with CoJka arabica. Phytochem., 26, 1431-1434 (1987). 9. Asada, M. and Shuler, M. L.: Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ, elicitors and alginate immobilization. Appl. Microbial. Biotechnol., 30, 475-481 (1989). 10. Kim, D. J. and Chang, H. N.: Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization. Biotechnol. Bioeng., 36, 460466 (1990). 11. Ramakrishna, S. V., Reddy, G. R., Curtis, W. R., and Humphrey, A. E.: Production of solavetivon by immobilized cells of Hyoscyamus muticus. Biotechnol. Lett., 15, 301-306 (1993). 12. Tanaka, H., Kaneko, Y., Aoyagi, H., Yamamoto, Y., and Fukunaga, Y.: Efficient production of chitinase by immobilized Wasabia japonica cells in double-layered gel fibers. J. Ferment.
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