Enhanced secretion of peroxidase from carrot hairy roots using polyethylene glycol

JOURNALOP FERMENTATION AND BIOENOINEBRINO Vol. 83, No. 4, 397-400. 1997

Enhanced Secretion of Peroxidase from Carrot Hairy Roots Using Polyethylene Glycol YONG HWAN KIM, JI HYEON KIM, Department

AND

YOUNG JE YOO*

of Chemical Engineering and the Institute of Genetics and Molecular Biology, Seoul National University, Seoul 151-742, Korea Received 18 September 199YAccepted 4 July 1996

Effects of various organic solvents and polyethylene glycol (PEG) on the secretion of peroxidase from carrot hairy roots were investigated. The peroxidase activity in culture broth was signifkantiy enhanced without adverse effects on root growth when PEG was employed while other organic permeabilixing agents exerted harmful effects on root growth. PEG with a molecular weight of over 6,OtMlg/mol enhanced the secretion of peroxldase dramatically. Since PEG did not induce the formation of pores in the root cell membrane as contlrmed by stalnlng using Neutral Red, it was thought that the mechanism of secretion enhancement of peroxidase by PEG is dMerent from that of other organic solvents which mahe the cell membrane permeable. Since PEG does not adversely affect the root growth, PEG can be applied to the repeated use of root cells for production of

peroxidase. IKey words: Daucus carota, peroxidase, hairy roots, polyethylene glycol, secretion] Peroxidase [EC 1.11.1.71 has been widely used as an important component of reagents for clinical diagnosis and in various laboratory experiments (1). The major limitation in continuous production of peroxidase using plant cells is that the desirable product is synthesized intracellularly and most of it is stored in the cells. Various methods have been studied in efforts to overcome these difficulties and to enhance the secretion of the product into the medium. Several techniques involving using of organic solvents and surfactants (2) either alter or permeabilize the cell membranes. Very few techniques were reported to enhance the secretion of proteins from plant cells. Kato et al. (3) enhanced the secretion of peroxidase from horseradish hairy roots by using light and NaCl. But the final cell mass was smaller than that in the control experiment. Therefore it is important to develop a method which enhances the secretion of peroxidase from the cells without inhibiting cell growth. We describe here a new method involving use of polyethylene glycol (PEG) to enhance the secretion of peroxidase from carrot hairy roots (4) without adverse effects on cell growth and discuss the possibility of adaptation of this technique for a repeated batch production system. Hairy roots of Daucus carota were induced as described elsewhere (5) and maintained by regular subculture in the dark at 3 week intervals, on hormone-free Murashige-Skoog (MS) medium supplemented with 3% (w/v) sucrose. For the culture experiments, the roots were grown for 14 d at 27°C and then transferred aseptically into 250ml Erlenmeyer flasks containing lOOm1 aliquots of the required medium. The temperature of medium during the cultivation was kept at 27°C. Fresh root mass was determined after vacuum filtration on filter paper. For the determination of extracellular peroxidase activity, the supernatant of the broth was obtained by centrifugation at 13,000 rpm for 5 min at 4°C. Peroxidase activity was determined at 30°C with a spectrophotometer (UVIKON 930, Kontron Co., USA) following the formation of tetraguaiacol (OD,,=470nm, E

(extinction coefficient)=26.6 mM-lcm-l) in 3 ml of reaction mixture containing 1 ml of 0.1 M phosphate buffer (pH 6.0), 1 ml of 15 mM 2-methoxyphenol (guaiacol), 1 ml of 3 mM HzOz and 50 1-11of enzyme extract. One unit (U) of peroxidase activity represents the amount of enzyme catalyzing the oxidation of 1 ,umol of guaiacol in 1 min. All the chemicals used in the experiments were of analytical grade. PEGS of various molecular weights were purchased from Sigma Co. (USA). Permeabilizing agents sterilized by microfiltration were added to the culture broth at 12 d after inoculation of hairy roots. Peroxidase activity in the culture broth was measured periodically after addition of permeabilizing agents. Enhancement of peroxidase secretion: The hairy roots cultivated in MS media secreted peroxidase into the broth to some extent (e.g. 0.1 U/mol). The activity of peroxidase in the broth was much lower than that of the peroxidase in the root cells (e.g. 5.OU/g fresh root mass) (4). To develop an effective process for producing peroxidase, alternative means of stimulating both the production and the secretion of peroxidase from carrot hairy roots, was investigated. Various organic solvents (2, 6) used as permeabilizing agents for release of secondary metabolites from carrot hairy roots were tested for their ability to permeabilize the cell membrane of the hairy roots. Also, PEG (M.W. 6,OOOg/mol) was added to stabilize the peroxidase secreted from the cells. One day after various agents were added to the broth, the peroxidase activity in the culture broth was analyzed. As shown in Table 1, the peroxidase activity in the broth was elevated when various organic solvents were added. When diethyl ether (0.1 g/l) was added to the culture broth, the peroxidase activity in the culture broth approached about 0.86U/ml. Also addition of the other organic solvents enhanced the peroxidase activity in the culture broth. However, the growth of the hairy roots was severely inhibited even in the case of dimethylsulfoxide (DMSO) which has been reported to be nontoxic to the cells (2). Addition of PEG (M.W. 6,OOOg/mol) enhanced the peroxidase activity in the medium and the peroxidase activity approached about 0.51 U/ml. When

* Corresponding author. 397

398

KIM ET AL. TABLE 1.

J. FERMENT. BIOENG.,

Peroxidase activity in the broth for various permeabilizing agents

Permeabilizing agent*

Peroxidase activity (U/ml) at Id 20d

Control Toluene (0.01 g/[) Diethyl ether (0.1 g/c) DMSO (0.1 g/f) Triton X-100 (l.Og/[) Tween 80 (1 .O g/l) PEG (l.Og/f) PEG (0.5 g/f) + toluene (0.005 g/l) PEG (0.5 g/f)+ diethyl ether (0.05 g/[)

Fresh root mass (g/0 at Id 20d

0.1 0.6 0.86 0.75 0.78 0.55 0.51 0.38

0.34 0.11 0.21 0.26 0.13 0.21 2.23 1.63

110 85 88 96 78 87 106 101

340 80 75 190 85 115 325 300

0.71

1.69

99

290

* Each permeabilizing agent was added into 250ml Erlenmeyer flasks containing 100 ml of culture medium 12 d after inoculation.

PEG was added, the intracellular peroxidase activity in the finally harvested roots (1.0 g/l of PEG 6,000 was added) was 5.0 U/g fresh root mass, which was a normal value of intracellular peroxidase activity (4). Addition of PEG only did not result in inhibition of the root growth, and the final root mass in this case was comparable to that in the control experiment. Also, addition of PEG together with another organic solvent such as toluene or ether did not result in inhibition of the root growth to as great an extent as the organic solvent alone. While the elevation of the peroxidase activity in the broth was thought to be due to the bursting of the root cells in the case of an organic solvent, PEG was thought to have mechanisms of action different from those of the other organic solvents. To test this assumption, we analyzed the time course of peroxidase activity in the broth. Each permeabilizing agents was added 12 d after inoculation as shown in Fig. 1. Addition of an organic solvent without PEG resulted in an increase in peroxidase activity in the broth only for one day after their addition to the broth and peroxidase activity reached about 0.7 U/ml. However, these organic

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solvents could not maintain the enhanced peroxidase activity and the activity gradually decreased toward the control level near the end of cultivation. This implies that these organic solvents make pores in the cell membrane and that the root cells cannot live any more after all. Since the organic solvents inhibited the cell growth and the peroxidase activity in the broth did not increase consequently, these organic solvents do not seem to be appropriate permeabilizing agents for the production of peroxidase. The presence of PEG 6,000 continuously enhanced the peroxidase activity in the broth; the activity almost approached 2.0 U/ml at the end of cultivation as shown in Fig. 1. This value is about 7 times higher than the control value. When a mixture of PEG and another organic solvent such as toluene or ether was added to the broth, an increase in peroxidase activity in the broth was also observed. The intracellular peroxidase activity in the finally harvested roots was 5.OU/g fresh root mass when 1.0 g/l of PEG 6,000 was added. This means that the level of intracellular peroxidase activity remained constant even after the secretion of peroxidase. Based on this result, we propose following two ways in which addition of PEG to the culture broth resulted in an increase in peroxidase activity in the broth. First, while the amount of secreted peroxidase is almost the same as that of the control experiment when PEG is added, PEG stabilizes the peroxidase in the broth. Second, PEG enhances the secretion of peroxidase into the broth. Since to our knowledge there is no report that PEG can be used as a permeabilizing agent or to enhance the secretion of peroxidase from carrot hairy root, it was necessary to elucidate the effect of PEG on the secretion of peroxidase from carrot hairy root. Effects of PEG on the secretion of peroxidase: Since the effect of PEG on the stability of enzymes has been reported (7), the effect of PEG on peroxidase storage stability was tested. The results together with the effect of toluene on the storage stability of peroxidase are shown in Fig. 2. Addition of toluene resulted in a decrease of the storage stability of peroxidase and the relative peroxidase activity dropped to 20% of the initial activity. The peroxidase activity decreased to about 30%

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Time (d)

FIG. 1. Time courses of activity of peroxidase secreted from the hairy roots cultured in the presence of various permeabilizing agents (addition of permeabilizing agents at 12 d after inoculation). Symbols: 0, control; ??, toluene (0.01 g/l); A, ether (0.1 g/l); 7, PEG (6,000 1.0 g/[); +, PEG (6,000 1.0 g/f)+toluene (0.005 g/l); ?? , PEG (6,000 1.0 g/l)+ether (0.005 g/l).

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FIG. 2. Effects of organic solvent and PEG addition on the peroxidase storage stability. Experiments were performed using medium (pH 5.8) as incubation solution at 25°C. Symbols: 0, control; ??, toluene (0.01 g/l); A, PEG (6,000 1.0 g/[).

NOTES

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FIG. 3. Effects of molecular weights of PEGS on the peroxidase activity. 1.Og/l of PEG was added at 12 d after inoculation. Symbols: 0, control; V, PEG (M.W. 6600); 0, PEG (M.W. 35660); 0, PEG (M.W. 206); v, PEG (M.W. 8000).

the basis of the initial activity even when PEG was added and the extent of decrease for peroxidase activity was almost same as that of the control. Therefore, the effect of PEG on peroxidase storage stability was not considerable. So it can be postulated that PEG in fact enhances the secretion of peroxidase into the broth during the cultivation. While PEG did not inhibit the growth of the hairy roots, the organic solvents used as permeabilizing agents were severely toxic to the cells as shown in Table 1. A study of peroxidase localization in the onion root revealed that peroxidase seems to be present in the cell wall, in the plasma lemma, in the Golgi apparatus cisternae, and in the endoplasmic reticulum (3). It appears that the peroxidase which was secreted in the presence of PEG in the present study may have been localized in the cell wall of the carrot hairy roots. To investigate the effect of the molecular weight of PEG in enhancing the secretion of peroxidase from carrot hairy root, several PEGS having different molecular

FIG. 5. Repeated use of the hairy roots for peroxidase production. The total broth volume was replaced with fresh culture medium containing 1.0 g/l PEG (M.W. 6,060 g/mol) 12, 20 and 31 d after inoculation. Symbols: 0, fresh root mass; 0, peroxidase activity in broth.

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Time (d) FIG. 4. Effects of Red release from cells PEG 6066 and toluene 0, control; 0, PEG;

organic solvent and PEG addition on Neutral in 3% sucrose solution. The concentrations of were 1.O and 0.01 g/l, respectively. Symbols: V, toluene.

weights were tested. As shown in Fig. 3, PEGS with a molecular weight of over 6,000 g/mol enhanced the secretion of peroxidase from carrot hairy root PEG with a lower molecular weight (M.W. 2OOg/mol) did not enhance the secretion of peroxidase from carrot hairy root. The differences between organic solvent and PEG effects on the cell membrane were investigated using a Neutral Red staining method. While the optical density of the Neutral Red released from the root cells in the control experiment was almost the same as that the case of the PEG addition, the optical density of the Neutral Red released from the cells in the case of toluene addition was much higher than that in the case of PEG addition as shown in Fig. 4. This suggests that PEG itself did not permeabilize the cell membrane like an organic solvent and has a different mechanism of action for the enhancement of peroxidase secretion. However, the mechanism of the PEG action on the cell membrane is still unknown. Because PEG does not inhibit the growth of the cells, repeated use of hairy roots for peroxidase production was performed as shown in Fig. 5. On days 12, 20 and 31 after inoculation, the broth was exchanged with fresh culture medium containing 1 g/l PEG 6,000. Since the hairy roots grew like an entagled ball of thread (4), it was easy to exchange the medium without use of sophisticated equipment. Within 2 d after exchange of the medium, the peroxidase activity increased gradually and approached about 6OOU/I and the fresh root mass increased gradually and approached about 6OOg/l of liquid volume. It was possible to enhance the secretion of peroxidase repeatedly when PEG was employed. Since in general hairy roots grow slower than microbial cells, it will be beneficial to use hairy roots repeatedly to save time for the cultivation of hairy roots. At present, we are trying to adapt this method to a large-scale fermentor for the production of peroxidase. Although the exact mechanism of PEG in enhancing secretion was not elucidated, this technique could be applied to a continuous culture system for increasing peroxidase productivity.

400

J. FERMENT. BIOENG.,

KIMET AL.

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hairy root cell culture. Enzyme Microbial Technol., 18, 531 (1996). Hwang, B., Cho, D. Y., and Hong, S. S.: Physiological studies on the formation of hairy root by the Agrobacterium rhizogenes. Korean J. Bot., 29, 275 (1986). Felix, H.: Permeabilized cells. Anal. Biochem., 120, 211 (1982). Inada, Y., Furukawa, M., Sasaki, H., Kodera, Y., Hiroto, M., Nisbimura, H., and Matsuhima, A.: Biomedical and biotechnological application of PEG- and PM-modified proteins. TIBTECH., 13, 86 (1995). Delmer, D. P.: Dimethylsulfoxide as a potential tool for analysis of compartmentation in living plant cell. Plant Physiol., 64, 623 (1979).