Biotechnol. Bioprocess Eng. 1999, 4, 281-286

Kin e t i c Model for B i o t r a n s f o r m a t i o n of D i g i t o x i n in P l a n t Cell S u s p e n s i o n Culture of Digitalis lanata

�9 1~ }~im 1, Hyun-Kyu P a r k 1, W o n Hong Lee 1, and Dong-I1 K i m 2 J e o n g - W o o C h o l ', Y o u n g - K e e

1Department of Chemical Engineering, Sogang University, C.P.O. Box 1142, Seoul 100-611, Korea 2Department of Biological Engineering, Inha University, Inchon 402-751, Korea

Batch suspension cultures of Digitalis lanata plant cell were performed to investigate the biotransformation of digitoxin. Digitalis Ianata K3OHD plant cells were used to biotransform digitoxin into deacetyllanatoside C. A kinetic model was proposed to de- scribe cell growth, substrate consumption, depletion of digitoxin, formation and deple- tion of digoxin and purpureaglycoside A, and formation of deacetyllanatoside C. The di- goxin and purpureaglycoside A are intermediates of deacetyllanatoside C formation from digitoxin. Interactions between extracellular and intracellular compounds were considered. The proposed model could accurately predict cell growth, substrate con- sumption and product synthesis. And it can provide a useful framework for quantita- tive analysis of biotransforrnation in a plant cell culture system.

Key words: kinetic model, Digitalis lanata, biotransformation, digitoxin, digoxin

I N T R O D U C T I O N

Biotransformation by cell culture has been devel- oped as an attractive alternative to modify" func- tional groups of exogenous organic compounds [1,2]. Although microbial cells were initially investigated for biotransformation, plant cells are being used re- cently in biotransformation process. Significant ad- vances have been made in mass cultivating plant cells in a large scale in bioreactors in the last two decades [3,4], but plant cell cultures are considered to be inferior compared to microbial cell cultures because of low productivity and mainly slow growth rate [5]. Therefore, in the case of biological reactions which are restricted to plant cells and would yield high valuable products, biotransformation using plant cells is considered to be an attractive tech- nique [6].

Digoxin is one of the important pharmaceuticals in the treatment of various cardiac diseases and widely used in medicine [7]. Digoxin derivatives come from Digitalis lanata EHRH (Scrophulariaceae), which is the main source of cardenolides, the compounds that can only be extracted from plant sources. Since a cardenolide molecule consists of a steroid nucleus and a sugar side-chain of variable length, cardeno- lides are classified according to the substitution pat- terns of the steroid moieties. The chemical struc- tures of cardenolides are shown in Table 1. Due to its pharmacological properties, digoxin is the most valuable cardenolide in demand [8]. In product syn- thesis of Digitalis lanata plant cell culture, digitoxin is a directly by-product, produced in larger quantity than digoxin. As shown in Table l, the steroid nucleus

* Corresponding author Tel: +82-2-705-8480 Fax: +82-2-711-0439 e-maih jwchoi@ccs.sogang.ac.kr

of digoxin differs from that of digitoxin only at posi- tion C-12~, where the former contains a functional hydroxyl group. In the past few years, the biotrans- formation of digitoxin into digoxin by Digitalis lanata cell had been studied extensively [7-9]. The biotrans- formation mechanism of digitoxin is illustrated in Fig. 1. As shown in Fig. 1, digoxin and purpureagly- coside A are intermediates of deacetyllanatoside C production from digitoxin. In order to investigate the optimal conditions to maximize the synthesis of digoxin, a kinetic model describing cell growth and biotransformation of digitoxin would be useful in the process optimization. The proposed kinetic model also need to be useful in developing operating strate- gies to achieve high digoxin production.

In this study, a kinetic model was developed to de- scribe the biotransformation of digitoxin. Batch sus- pension cultures of Digitalis lanata cells were per- formed to investigate the characteristics of the bio- transformation of digitoxin and to validate the pro- posed kinetic model. The cell line used for this study was Digitalis lanata K3OHD plant cells that can bio- transform digitoxin into deacetyllanatoside C. The mathematical kinetic model had been formulated to describe cell growth, consumption of nutrient, deple- tion of digitoxin, formation and depletion of digoxin and purpureaglycoside A, and formation of deacetyl- lanatoside C. Interactions between extracellular and intracellulm ~ compounds have been considered.

M A T E R I A L S A N D M E T H O D S

C u l t u r e s a n d M e d i a

Digitalis lanata K3OHD plant cell were provided by Dr. Wolfgang Kreis (Friedrich-Alexander-Universit/it Erlangen-Niirnberg, Germany) [10]. Cell suspension

282 Biotechnol. Bioprocess Eng. 1999, Vol. 4, No. 4

R 2 ~ O

CH3 OH CH 3 CHar " T- \

OH CH3 OH

Table 1. Chemical structures of the cardiac glycosides

Compound Name R 1 R z

Digoxin H OH Digitoxin H H Purpureaglycoside A Glucosyl H Deacetyllanatoside C Glucosyl OH

Digitoxin

Hydroxylatio/

Digoxin

Glucosylation~

~ lucosylation

Purpureaglycoside A

ydroxylation

Deacetyllanatoside C

Fig. 1. Biotransformation of digitoxin by Digitalis lanata cells.

cultures have been maintained on modified Mura- shige and Skoog (MS) medium. The medium was prepared with distilled water, and pH of the medium was adjusted to 5.5 before autoclaving. The produc- tion medium of 8% glucose aqueous solution with its pH adjusted to 5.5 was confirmed to be a suitable production medium for biotransformation of digi- toxin.

B i o t r a n s f o r m a t i o n Exper iment s

Biotransformation experiments were carried out in a 100-mL shake flask containing 30 mL of medium. Cells were incubated in growth medium for 7 day and transferred to production medium. Three days after transferring the cells to production medium, digitoxin was added to the cultured cells. In all ex- periments, digitoxin was added in the solution form of dimethyl sulfoxide.

A n a l y t i c a l P r o c e d u r e

Quantitative glucose analysis was performed by an isocratic HPLC system (Model 910, Young-In Scien- tific Co., Seoul, Korea) with a refractive index detec- tor (Model 410 Differential Refractometer, Waters, U.S.A.), under the following conditions; carbohy- drate column (4.6 x 250 mm, Waters, U.S.A.); flow rate, I mL/min; mobile phase, 75% (v/v) MeCN in water. For cardenolide analysis, the total methanolic extract of the suspension culture was obtained by adding the same amount of methanol as that of the culture broth and sonicating for 20 min. Supernat- ent after centrifugation was used for the determina-

DtE Dffe ~E

Fig. 2. Schematic diagram of the biotransformation mechanism.

tion of cardenolides. Samples were injected into HPLC system with UV detector. Curosil G column (4.6 • 250 mm, 6 t~m, Phenomenex Inc., U.S.A.) was used for analysis. The mobile phase was a mixture of acetonitrile and water (35:65, v/v). Flow rate was 1 mL/min, and measuring wavelength was 220 nm. Standard cardenolide for HPLC analysis was pur- chased from Roth (Germany), and the solvent was obtained from Fisher Scientific (Pittsburgh, PA, U.S.A.).

MODEL DEVELOPMENT

To develop model equations for biotransformation of digitoxin into digoxin by cultured Digitalis lanata cells, the biotransformation system was simplified as illustrated in Fig. 2. In Fig. 2, Dt, Dg, A, and C rep- resent the concentrations of digitoxin, digoxin, pur- pureaglycoside A, and deacetyllanatoside C, respec- tively. And G represents the concentration of glucose. The subscripts E and I designate the extracellular concentration and intracellular concentration, re- spectively. Intracellular digitoxin was biotrans- formed into digoxin and purpureaglycoside A, which is turn were biotransformed into deacetyllanatoside C. In order to established model equations, ki, k2, k3, and k 4 were introduced as the biotransformation rate constants.

Interactions between extracellular and intracellular compounds were considered. It is known that the active transport of extracellular compounds is per- formed by carrier molecules [11], which might be considered as transport proteins; thus, the active transport could be considered as the enzyme reaction. The mechanism of the active transport of extracel-

lular digitoxin could be simply described with the following equation.

KDt kDtl MDt + Dt I MDt + Dt E < > MDtDtE < kDt 2 (1)

where MDt is the concentration of digitoxin car- rier molecules; KDt is the equilibrium constant; kDtl and kDt 2 are the rate constants. It is assumed that the latter step controls the overall transport rate, and thus the overall transport rate of digitoxin (RDt) is represented as follows.

Biotechnol. Bioprocess Eng. 1999, Vol. 4, No. 4 283

RDt -

where

KDtlDtE /(Dt2DtI (2) KDt + DtE (I + DtE / KDt )

KDt 1 = MDto]gDt 1

K'Dt 2 = MDtokDt 2

where , MDt o is the total concentration of digitoxin carrier molecules.

For digoxin, the overall transport rate (RDg) could be represented as follows, based on the silmilar mechanism of digitoxin.

MDg + Og E Kog kDgl < > Mo,~gs < > MDg + Dgi ko~ (3)

where MDg is the concentration of digoxin carrier molecules, Kog is the equilibrium constant, and kog~ and kDg2 are the rate constants. It is assumed that the latter step controls the overall transport rate.

RDg - ~CDglDgE tCDg2DgI (4) ( KDg + DgE ) ( I + DgE / KDg )

where

K'Dg 1 = MDgokDg 1

K'Dg 2 = MDgokDg 2

where Mog o is the total concentration of digoxin car- rier molecules.

The mechanism of the active transport of extracel- lular glucose could be described as follows.

Kc kc Mc + GI~ < > MGG~ < > M~ + GI (5)

where Mo is the concentration of glucose carrier molecules, K G is the equilibrium constant, and k C is the transport rate constant. It was assumed that the latter step controls the overall t ransport rate, and thus the overall transport rate of glucose (R G) is rep- resented as follows.

R G - /CAGE (K~ + GE) (6)

where

= MGokG

where MGo is the total concentration of glucose car- rier molecules.

Only glucose was used as the substrate for cell growth in production medium, but glucose was also used as the reactant for biotransformation of digi- toxin as shown in Fig. 2. Therefore, it is assumed that dig/toxin has an inhibitory effect in the cell growth [12]. Hence, the specific growth rate is rep- resented with the following equation.

r

r = (G I + K m )(1 + Dt I / K i) (7)

where gm~ is the maximum specific growth rate, Km is the Monod type constant for glucose, and K i is the inhibition constant for dig/toxin. Mass balance equation for cell is described as

dX - ~ (8)

dt

where X is the cell mass concentration. The mass balance equation for extracellular digi-

toxin can be as following.

dDt_____~_E_ (/CDtLDtE KDt2DtI I X (9) dt ~ KDt + Dt E 1 + Dt E / KDt

Mass balance equation for intracellular digitoxin is described as

dDtI = - I kaDtt + k2DtIGI 1 dt r\ KDtDg + Dt I (gDt A + Dt; )(KGA + V I )

1 dDt E Dt I dX (10)

X dt X dt

where KDtDg, Koa and KGA are the Michaelis-Menten constants.

On the right-hand side of the equation (10), the first term represents the biotransformation of dig/- toxin into digoxin and the second term represents biotransformation of digitoxin into purpureaglyco- side A. Because the biotransformation is performed by enzymes, it can be assumed that the dependence of biotransformation rate on concentration of reac- tant is Michaelis-Menten type. Therefore, the first and the second term are represented as a Michaelis- Menten type formula. The third term represents the dependence of change of intracellular dig/toxin on that of extracellular digitoxin. The fourth term represents the dilution effect of intracellular digi- toxin due to cell growth. In a well-mixed bulk solution, intracellular digoxin

is transported into bulk and extracellular digoxin is transported into cells, simultaneously. Hence, mass balance of extracellular digoxin could be represented with the following equation.

dDg~ dt

K'DglDg E

KDg + DgE -KD#DgI I X

1 + Dg E / KDg )

Mass balance for intracellular digoxin is

(11)

dDgi_ l hlDti k3Dg~Gi ) dt KDtDg + Dt I (KDg c + Dg I)(KGc + G I)

1 dDg E Dg I dX (12) X dt X dt

where KD~ and Koc are the Michaelis-Menten con-

284

stants. On the right-hand side of the equation (12), the

first term represents the biotransformation of digi- toxin into digoxin, and the second term represents biotransformation of digoxin into deacetyllanatoside C. The third term represents the dependence of change of intracellular digoxin on that of extracellu- lar digoxin. The fourth term represents the dilution effect of intracellular digoxin due to cell growth.

The mass balance of intracellular purpureaglyco- side A can be described with the following equation.

I k2DtlGI k4AI -1 dAi _ (KDt A + dt Dt I )(KGA + G I ) KAC + A I

AI dX X dt

(13)

where KAc is the Michaelis-Menten constant. On the right-hand side of the equation (13), the

first term represents the biotransformation of digi- toxin into purpureaglycoside A, and the second term represents biotransformation of purpureaglycoside A into deacetyllanatoside C. The third term represents the dilution effect of intracellular purpureaglycoside A due to cell growth.

Mass balance of deacetyllanatoside C is

dCI - ( k4AI k3DgIGI 1 dt ~Kac +A I ~ (KD~ +Dgi)(K~c +G I) )

(14) C I dX X dt

On the right-hand side of the above equation, the first term represents the biotransformation of pur- pureaglycoside A into deacetyllanatoside C, and the second term represents biotransformation of digoxin into deacetyllanatoside C. The third term represents the dilution effect of intracellular deacetyllanatoside C due to cell growth.

The mass balance of extracellular glucose can be described with the following equation.

dt K G + G E Gimax (15)

where Gtm~ is the maximum intracellular glucose concentration.

Intracellular glucose is used for cell growth, and the synthesis of purpureaglycoside A and deacetyl- lanatoside C. Therefore, the mass balance equation for intracellular glucose is as follows.

( 1 1 k2DtlG I dG I ]~G l-l+ Y2G (KDtA + D~-~KGA +VI)

X d~- = - /+ 1 k3DgtG I

1 dG E G I dX (16) X dt X dt

where Y represents the yield coefficient.

Biotechnol. Bioprocess Eng. 1999, Vol. 4, No. 4

Table 2. Estimated parameter values of the kinetic model

Parameter Value Dimension

k o 1.32 g/g-cell K i 0.0392 mg/g-cell ~6~ 0.183 1/day KDt 8 mg/L ~CDt 1 13 mg/g-eell/day R'Dt 2 1 1/day k 1 25 mg/g-cell/day k 2 3 9 . 8 mg/g-cell/day k 3 1 . 7 2 mg/g-cell/day h 4 2 . 7 4 mg/g-cell/day KDtD~ 8 mg/g-cell KDtA 4.2 mg/g-cell KDm 0.04 g/g-cell KDg 20 mg/L R'Dg 1 5 mg/g-cell/day ~CDg 2 4 mg/g-cell/day KDg c 0.0613 mg/g-cell KDN 0.06 g/g-cell Kac 0.6 mg/g-cell K c 31.1 g/g-cell/day kG 480. g/L

R E S U L T S A N D D I S C U S S I O N

P a r a m e t e r E s t i m a t i o n

The complete model was composed of a set of dif- ferential equations that are shown in the equation (8)-(16). The parameters involved in the kinetic equations were estimated using the nonlinear pa- rameter estimation technique [13,14]. While the pa- rameter estimation was being performed, the model equations were solved simultaneously with numeri- cal integration using the Runge-Kutta-Verner fifth- order method. The experimental data were com- pared to the model predictions by choosing parame- ters that give a best fit of the model to the data. The estimated values of the set of parameters were shown in Table 2.

C o m p a r i s o n o f E x p e r i m e n t a l a n d M o d e l P r e d i c t i o n R e s u l t s

Cell Growth

For the analysis of the cell growth behavior in the suspension culture of Digitalis lanata cells, batch experiments were carried out in shake flasks, and samples were taken every day. Fig. 3 represents the experimental and the model predicted cell growth behavior. A lag phase in the cell growth was ob- served during the early stage of the cultivation. The cell mass was exponentially increased after 1 day and then ceased after 3 days when digitoxin was in- jected into the culture medium. As mentioned previ- ously, this behavior can be explained with the digi- toxin's inhibitory effect in the cell growth. As shown in Fig. 5, the injected digitoxin was completely de- pleted at 7 days of cultivation. At this time, another exponential growth of cell mass was detected as shown in Fig. 3. Consequently, it can be concluded that the experimental data were fairly consistent with

Biotechnol. Bioprocess Eng. 1999, Vol. 4, No. 4 285

Time (days)

Fig. 3. The experimental and kinetic model results of cell growth time course change in Digitalis lanata K3OHD culture. The circle represents experimental results, and the solid line represents model prediction results.

the solution of the model equations with the given initial conditions.

Substrate Uptake

Fig. 4 represents the experimental and the model predicted time course behavior of the extracellular glucose concentration. It can be concluded that the proposed model accurately predicted the experi- mental results. The extracellular glucose concentra- tion decreased rapidly after inoculation until 3 days due to the cell growth. Then, the glucose concentra- tion was maintained at a certain concentration due to the cell growth inhibition by injected dig/toxin, and finally decreased again due to the depletion of dig/toxin. This behavior of extracellular glucose con- centration was consistent with that of cell mass.

Biotransforrnation

Fig. 5 represents the experimental and the model predicted time course behavior of cardiac glycosides. It can be concluded that the proposed model pre- dicted the experimental data fairly well. As men- tioned before, dig/toxin was added 3 days of cultiva- tion into the production medium. Dig/toxin rapidly transformed into digoxin and purpureaglycoside A, completely depleted within about 3 days after injec- tion. At the time of dig/toxin depletion, digoxin and purpureaglycoside A showed their maximum values. And then, the concentrations of two intermediates (digoxin and purpureaglycoside A) decreased con- tinuously until 10 days of incubation. The amount of dig/toxin transformed into purpureaglycoside A was larger than that of digitoxin transformed into di- goxin. The concentration of deacetyllanatoside C continuously increased as digoxin and purpureag]y- coside A transformed into deacetyllanatoside C. In conclusion, in order to obtain the maximum amount of the valuable digoxin compound, the cultivation of Digitalis lanata cell should be ceased at 2 days after dig/toxin injection. The results were obtained in this study can be a useful framework in developing opti- real operation strategies to increase digoxin produc-

10o

80 -~

6 0 -

~ o o

~ 4 0 -

2 0 -

[ I I I 2 4 6 8 10

Time (days)

Fig. 4. The experimental and kinetic model results of sub- strate consumption time course change in batch culture. The circle represents experimental results, and the solid line represents model prediction results.

~00

E

2O0

J

i t. t "

~. , i

! 2 4 6 8 10

Time (days)

Fig. 5. The experimental and kinetic model results of car- diac glycosides time course change in batch culture. The symbols (digitoxin (O), digoxin ([~), purpureaglycoside A (~), deacetyllanatoside C (A)) represent experimental results, and the lines represenr model prediction results.

tion from digitoxin.

C O N C L U S I O N

A kinetic model was proposed to predict cell growth, substrate uptake, and biotransformation of dig/toxin in the suspension culture of Digitalis lanata cells. It was assumed that the biotransformation of dig/toxin occurred in the cells. In order to consider interaction between extracellular and intracellular compounds, the active transport of extracellular compounds by carrier molecules needed to be described by the pro- posed model. Because the carrier molecules might be considered as transport proteins, the active trans- port could have been considered as enzyme reaction. It was assumed that biotransformation of digitoxin is achieved by two different pathways; one was the digoxin intermediated reaction and the other was purpureaglycoside A intermediated reaction. The kinetic model was established based on the above

286

assumption. The proposed model fairly predicted the cell growth, substrate consumption and biotrans- formation of digitoxin. Thus, the model can be used in developing an operating strategy and control scheme to increase the production of digoxin.

NOMENCLATURE

A concentration of purpureaglycoside A (mg/L or rag/g-cell)

C concentration of deacetyllanatoside C (mg/L or mg/g-cell)

Dg concentration of digoxin (mg/L or rag/g-cell) Dt concentration of digitoxin (mg/L or mg/g-cel]) G concentration of glucose (g/L or g/g-cell) k rate constant (g/g-cell or rag/g-cell/day) Km Monod constant (g/g-cell) K i inhibition constant (mg/g-cell) M concentration of carrier molecule (g/L or g/g-cell) R overall transport rate (mg/g-cell/day) t time (day) X cell mass (g/L) Y yield coefficient ic rate constant (mg/g-cell/day, 1/day or g/L) ~z specific growth rate (1/day)

Subscr ipts

A purpureaglycoside A C deacetyllanatoside C Dg digoxin Dt digitoxin E extracellular G glucose I intracellular max maximum o total

R E F E R E N C E S

[1] Kutney, J. P. (1998) Biotechnology and synthetic chemist ry-routes to clinically impor tan t com- pounds. Pure Appl. Chem. 70: 2093-2100.

[2] Krings, U. and R. G. Berger (1998) Biotechno- logical product ion of f lavours and fragrances.

Biotechnol. Bioprocess Eng. 1999, Vol. 4, No. 4

Appl. Microbiol. Biotechnol. 49: 1-8. [3] Prenosil, J. E. and H. Pedersen (1983) Immobi-

lized plant cell reactors. Enzyme Microb. Tech- nol. 5: 323-331.

[4] Panda, A. K., S. Mishira, V. S. Bisaria, and S. S. Bhojwani, (1989) Plant cell reactors - a perspec- tive. Enzyme Microb. Technol. 11: 386-397.

[5] Bu i t e l aa r , R. M. and J. M. T r a m p e r (1992) Strategies to improve the production of second- dary m e t a b o l i t e s wi th p l an t cell cu l tu re ; a literature review. J. Biotechnol. 23: 111-141.

[6] Kutney, J. P. (1997) Plant cell cul ture combined with chemis t ry- routes to clinically impor tant compounds. Pure Appl. Chem. 69: 431-436.

[7] Kreis, W. and E. Reinhard (1992) 12~-Hydro- xylat ion of digi toxin by suspens ion-cul tured Digitalis lanata cells: Production of digoxin in 20-1itre and 300-1itre air-lift bioreactors . J.

Biotechnol. 26: 257-273. [8] Kreis, W. and E. Reinhard (1988) 12[3-Hydro-

xylation of digitoxin by suspension-cultured Digi- talis lanata cells: Production of deacetyl lanto- side C using a two-stage culture method. Planta Med. 54: 143-148.

[9] Kreis, W., W. Zhu, and E. Reinhard (1989) 12~- H y d r o x y l a t i o n of d ig i toxin by s u s p e n s i o n - cul tured Digitalis lanata cells: Product ion of deacetyllantoside C in a 20-1itre air-lift bioreac- tors. Biotechnol. Lett. 11: 325-330.

[10] Lee, J.-E., S.-Y. Lee, and D.-I. Kim (1999) In- creased production of digoxin by digitoxin bio- t rans format ion using cyclodextrin polymer in Digitalis lanata cell cultures. Biotechnol. Bio- process Eng. 4: 32-35.

[11] Bailey, J. E. and D. F. Ollis (1986) Biochemical Engineering Fundamentals. p. 265-269, 2nd ed. McGraw-Hill Inc. New York, NY, U.S.A.

[12] Mulchandani, A. and J. H. T. Luong (1989) Micro- bial kinetics revisited. Enzyme Microb. Technol. 11: 66-73.

[13] Metzler, C. M., G. L. Elfring, and A. J. McEwen (1974) A package of computer programs for phar- maco-kinetic modeling. Biometrics 30: 562-563.

[14] Seinfeld, J. and L. Lapidus (1970) Process Mod- eling Estimation and Identification. Prentice- Hall Inc. Englewood Cliffs, NJ, U.S.A.