Biocatalysis in Agricultural Biotechnology - ACS Publications

Chapter 5

Downloaded by UNIV OF TEXAS AT AUSTIN on October 4, 2017 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch005

Plant Cells for Secondary Metabolite Production Dietrich Knorr Department of Food Technology, Berlin University of Technology, D—1000 Berlin 33, Federal Republic of Germany, and Biotechnology Group, Department of Food Science, University of Delaware, Newark, DE 19716 Plant cell cultures represent a potentially rich source of secondary metabolites of commercial importance and have been shown to produce them in higher concentrations than the related intact plants. However, plant cell cultures often produce metabolites in lower concentrations than desired and commonly store them intracellularly. These limitations can be overcome by product yield enhancement procedures, including immobilization of cultured cells, and permeabilization, or ideally using a combined immobilization/ permeabilization process with retained plant cell viability. Complex coacervate capsules consisting of chitosan and alginate or carrageenan proved to be effective biomaterials for entrapment, controlled permeabilization of cells and to allow control of capsule membrane diffusivity. Using immediate precursors of desired food aroma compounds also increased metabolite yields. For example, by applying ferulic acid to cultured Vanilla planifolia cells, vanillin concentration could be enhanced as compared to untreated cells. Vanilla concentration was also increased in green vanilla bean extracts when the cells were treated with β-glucosidase. M i c r o b i a l p o l y s a c c h a r i d e s have been shown t o s t r e s s p l a n t c e l l s , r e s u l t i n g i n ' e l i c i t a t i o n ' ( i n d u c t i o n ) and increased metabolite synthesis. Induction of various enzymes has been r e p o r t e d . Chitosan s u c c e s s f u l l y e l i c i t e d c h i t i n a s e p r o d u c t i o n i n c a r r o t (Daucus c a r o t a ) c e l l c u l t u r e s and e l i c i t a t i o n o f d e s i r e d food i n g r e d i e n t s and p r o c e s s i n g a i d s v i a c h i t o s a n has been a t t e m p t e d .

β

0097-6156/89/0389-0065$06.00/0 1989 American Chemical Society

Whitaker and Sonnet; Biocatalysis in Agricultural Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


66

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

The b a s i c concept o f t o t i p o t e n c y l e a d i n g t o t h e r e c e n t advances o f p l a n t c e l l c u l t u r e was f o r m u l a t e d i n 1838 (1) and 1839 ( 2 ) . I n 1902 H a b e r l a n d t ( 3 ) r e p o r t e d h i s p i o n e e r i n g e x p e r i m e n t s on m a i n t a i n i n g p l a n t c e l l s i n a v i a b l e s t a t e away from t h e p a r e n t body by b a t h i n g them i n s i m p l e n u t r i e n t s o l u t i o n s . S i n c e t h e n , t h e s y n t h e t i c p o t e n t i a l o f p l a n t c e l l c u l t u r e s was e s t a b l i s h e d ( 4 ) . Large s c a l e p r o d u c t i o n o f p l a n t biomass from c u l t u r e d c e l l s was suggested as e a r l y a s 1962 ( 5 ) . P l a n t c e l l c u l t u r e s can now be e s t a b l i s h e d f o r a wide range o f h i g h e r p l a n t s s p e c i e s ( 6 ) and r e p r e s e n t a p o t e n t i a l l y r i c h s o u r c e of c o m m e r c i a l l y i m p o r t a n t secondary m e t a b o l i t e s (7_,8). P l a n t c e l l and organ c u l t u r e s c a n produce h i g h e r m e t a b o l i t e c o n c e n t r a t i o n s t h a n found i n t h e c o r r e s p o n d i n g i n t a c t p l a n t organs ( 6 , 9 ) . However, p l a n t c e l l s grown i n c u l t u r e may a l s o produce lower q u a n t i t i e s o f t h e d e s i r e d secondary m e t a b o l i t e s w h i c h a r e commonly stored i n t r a c e l l u l a r l y . The c h a l l e n g e s t o i n c r e a s e p r o d u c t y i e l d and t o enhance t h e r e l e a s e o f secondary m e t a b o l i t e s c a n be met i n v a r i o u s ways (7_). These i n c l u d e i m m o b i l i z a t i o n ( 9 ) , p e r m e a b i l i z a t i o n (10,11), t h e use o f p r e c u r s o r s ( 1 2 , 1 3 ) , and t h e i n d u c t i o n o f secondary m e t a b o l i t e p r o d u c t i o n v i a e l i c i t o r s ( 1 4 ) . Immobilization

of Cultured P l a n t

Cells

Nunez and Lema (15) viewed t h e main o b j e c t i v e i n i m m o b i l i z i n g a b i o l o g i c a l l y a c t i v e agent as t h e c o n c e n t r a t i o n o f i t s a c t i v i t y i n as s m a l l a volume as p o s s i b l e . T h i s r e q u i r e s a p r o c e d u r e ( 1 6 ) w h i c h c o n f i n e s a c a t a l y t i c a l l y a c t i v e enzyme o r c e l l i n s i d e a r e a c t o r system t h a t p r e v e n t s i t s e n t e r i n g t h e m o b i l e phase c a r r y i n g t h e s u b s t r a t e and t h e p r o d u c t . Some o f t h e advantages o f p l a n t c e l l i m m o b i l i z a t i o n a r e l i s t e d i n T a b l e I . The advantages o f c e l l i m m o b i l i z a t i o n have l o n g been r e c o g n i z e d i n c l u d i n g i t s importance i n n i t r o g e n f i x a t i o n i n legumes, t h e adherence o f f l u v i a l m i c r o o r g a n i s m s t o sand and s t o n e o r t h e p r o d u c t i o n o f v i n e g a r u s i n g woodshavings as s u p p o r t medium ( 1 5 ) . T a b l e I . Some advantages o f p l a n t c e l l Advantage

immobilization

a

Reason/Consequence

Reuse o f b i o c a t a l y s t

entrapment

Maintenance o f s t a b l e and active biocatalyst

e l i m i n a t i o n of non-productive growth phase; reduced r i s k o f d e c l i n i n g p r o d u c t i v i t y from a selected c e l l l i n e

P r o t e c t i o n o f c e l l s by m a t r i x

s i m p l i f i c a t i o n o f s c a l e up; p r o t e c t i o n from p h y s i c a l damage

Employment o f c o n t i n u o u s p r o c e s s

c o n t i n u o u s removal o f m e t a b o l i c i n h i b i t o r s ; improved p r o c e s s c o n t r o l and p r o d u c t r e c o v e r y

Increased

increased c e l l d e n s i t i e s ; e l i m i n a t i o n o f growth phase

a

productivity

Adapted from ( 9 , 17)



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Plant Cells for Secondary Metabolite Production

67

Polymers commonly used f o r p l a n t c e l l i m m o b i l i z a t i o n i n c l u d e agar, agarose, a l g i n a t e , carrageenan, c h i t o s a n , g e l a t i n , polya c r y l a m i d e , p r e p o l y m e r i z e d p o l y a c r y l a m i d e (Γ7,18) and p o l y u r e t h a n e foam ( 1 9 ) . Complex c o a c e r v a t e g e l c a p s u l e s have a l s o been developed (8,10,20-22) c o n s i s t i n g of c o u p l e d c h i t o s a n - a l g i n a t e or c h i t o s a n carrageenan networks ( 2 3 ) . Entrapment methods have been used almost e x c l u s i v e l y t o i m m o b i l i z e p l a n t c e l l s ( 1 7 ) . They can be c l a s s i f i e d i n t o t h r e e g e n e r a l groups (24,25): a. g e l f o r m a t i o n by i o n i c c r o s s l i n k i n g of a charged polymer ( i o n o t r o p i c ) ; b. g e l f o r m a t i o n by c o o l i n g of a heated polymer ( t h e r m a l ) , and c. g e l f o r m a t i o n by c h e m i c a l r e a c t i o n (cross-linking, r a d i c a l polymerization). I o n i c c r o s s l i n k i n g i n v o l v i n g marine p o l y s a c c h a r i d e s , such as a l g i n a t e , c a r r a g e e n a n , or c h i t o s a n , as charged p o l y e l e c t r o l y t e s w i t h c a l c i u m , p o t a s s i u m or polyphosphate i o n s as low m o l e c u l a r weight c o u n t e r i o n s , have r e c e i v e d much a t t e n t i o n ( 9 ) . The p o l y c a t i o n i c n a t u r e of c h i t o s a n , as opposed t o the p o l y a n i o n i c p r o p e r t i e s of most o t h e r marine polymers, p r o v i d e s v a r i o u s unique p r o p e r t i e s and o f f e r s a wide range of a p p l i c a t i o n s i n food b i o t e c h n o l o g y (26,27). The p o l y c a t i o n i c p r o p e r t i e s o f c h i t o s a n p e r m i t i t s use as a h i g h m o l e c u l a r weight c o u n t e r i o n i n p o l y e l e c t r o l y t e complex f o r m a t i o n r e s u l t i n g i n a c o u p l e d network c o n s i s t i n g of c h i t o s a n and one or more p o l y a n i o n i c polymers ( F i g u r e 1). M e c h a n i c a l s t a b i l i t y of the complex c o a c e r v a t e c a p s u l e s ( w i t h a l i q u i d c o r e ) was improved by a c o m b i n a t i o n o f h i g h and low m o l e c u l a r weight c o u n t e r i o n s , as w e l l as by the a d d i t i o n of g l u c o s e (Table I I ) . The use o f g l u c o s e has a d d i t i o n a l p r a c t i c a l relevance a l l o w i n g shorter r e a c t i o n times f o r bead f o r m a t i o n w h i c h i s e s s e n t i a l f o r the v i a b i l i t y of the entrapped c e l l s , and lower a l g i n a t e c o n c e n t r a t i o n s , which - due t o the d e c r e a s e d v i s c o s i t y of the a l g i n a t e s o l u t i o n - p r o v i d e s b e t t e r c o n d i t i o n s f o r capsule formation. The m e c h a n i c a l s t a b i l i t y of the c h i t o s a n / a l g i n a t e g e l capsules decreases w i t h i n c r e a s i n g e s t e r i f i c a t i o n of a l g i n a t e but g e l s t a b i l i t y was improved by a u t o c l a v i n g the c a p s u l e s i n T r i z m a ( t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ) b u f f e r s o l u t i o n (Table I I I ) . T h i s i s of i m p o r t a n c e f o r food b i o t e c h n o l o g y a p p l i c a t i o n s of the complex c o a c e r v a t e c a p s u l e s (e.g. m i c r o e n c a p s u l a t i o n ) where p r o d u c t s r e q u i r e a u t o c l a v i n g . S i n c e the g e l m a t r i x p r o v i d e s a mass t r a n s p o r t b a r r i e r , i t can a f f e c t v i a b i l i t y of entrapped p l a n t c e l l s as w e l l as the r e l e a s e of desired plant metabolites. C o n s e q u e n t l y , adequate d i f f u s i v i t y of the polymer network i s e s s e n t i a l f o r development of i m m o b i l i z e d p l a n t c e l l r e a c t o r s . C a p s u l e w a l l p r o p e r t i e s of c h i t o s a n - a l g i n a t e c o a c e r v a t e g e l c a p s u l e s a r e a f f e c t e d by pH, b u f f e r t r e a t m e n t i n c l u d i n g p h t h a l a t e , borax and t r i s ( h y d r o x y m e t h y l ) aminomethane b u f f e r , as w e l l as by v a r i o u s t y p e s of polymers (20, 21_, Pandya, Y. and K n o r r , D., U n i v . of Delaware, u n p u b l i s h e d d a t a ) . F o r example, degree o f e s t e r i f i c a t i o n of a l g i n a t e s used ( 2 0 ) , c h i t o s a n c o n c e n t r a t i o n and degree o f d e a c e t y l a t i o n of the c h i t o s a n ( r e s u l t i n g i n c h i t o s a n p r o d u c t s of d i f f e r e n t m o l e c u l a r weight and v i s c o s i t y ) were shown t o a l t e r g e l d i f f u s i v i t y . The e f f e c t s of c h i t o s a n c o n c e n t r a t i o n and v i s c o s i t y of a l g i n a t e - c h i t o s a n c o a c e r v a t e c a p s u l e s on the r e l e a s e o f low m o l e c u l a r w e i g h t food dyes from such c a p s u l e s , i s shown i n F i g u r e 2. A l s o , s i g n i f i c a n t d i f f e r e n c e s i n g e l p e r m e a b i l i t y o c c u r r e d when the a l g i n a t e o f the complex c o a c e r v a t e g e l




Plant cells (20 - 30 percent loading density)

(2.5 - 3 percent chitosan in 260 mM citric acid plus 450 mM CaCI ) or 2

Polymer solution

(2 - 3 percent Na alginate plus 3 percent sodium hexametaphosphate) droplet formation via syringe or peristaltic pump

counterion solution

0.75 percent alginate plus 2.25 percent glucose or 1 percent chitosan in 3 percent ascorbic acid

stir for 30 minutes

wash with sterile, distilled water •

transfer into nutrient medium ,

immobilized cell reactor Figure 1. S i m p l i f i e d protocol of chitosan/alginate or a l g i n a t e / chitosan complex coacervate capsule formation (adapted from 10.20.22)


5.

KNORR

Plant Cells for Secondary Metabolite Production

69

T a b l e I I . E f f e c t of C a l c i u m C h l o r i d e and G l u c o s e on M e c h a n i c a l S t a b i l i t y o f C h i t o s a n - a l g i n a t e Coacervate G e l C a p s u l e s 3


Polyelectrolyte (capsule core)

Setting Time (hrs)

Force t o Burst (Newtons)^

Counterion ( o u t e r membrane)

Reaction Time (min)

3.0% c h i t o s a n

2.0% N a - a l g i n a t e

20

3.0

0.73+0.30

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e

5

3.0

0.10+ 0.05

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e

15

3.0

0.97+0.61

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e

20

3.0

10.8 + 5.70

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e + 6.0% g l u c o s e

10

0.5

9.8 + 6 . 1 0

3.0% c h i t o s a n + 460 mM C a C l


15

0.5

16.5 + 3.00

3.0% c h i t o s a n + 460 mM C a C l


20

0.5

11.6 + 5.70

3.0% c h i t o s a n + 460 mM C a C l


15

0.5

24.5 + 2.40

3.0% c h i t o s a n + 460 mM C a C l


15

3.0

24.8 +10.50

3.0% c h i t o s a n + 460 mM C a C l


15

0.5

19.4 + 7.10

3.0% c h i t o s a n + 460 mM C a C l


15

24.5

17.2 +10.20

2

2

2

2

2

2

2

2

2

2

Average c a p s u l e diameter 5.7 mm; adapted from ( 2 0 ) Mean ±_ s t a n d a r d d e v i a t i o n (N > 5)


70


c a p s u l e s was r e p l a c e d by kappa-carrageenan (Pandya, Y. and K n o r r , U n i v . of Delaware, u n p u b l i s h e d d a t a ) .

D.,

T a b l e I I I . E f f e c t of b u f f e r t r e a t m e n t and e s t e r i f i c a t i o n of a l g i n a t e on the m e c h a n i c a l s t a b i l i t y of c h i t o s a n / a l g i n a t e c o a c e r v a t e c a p s u l e s 3

Setting s o l u t i o n P e r c e n t

of a l g i n a t e c a r b o x y l groups e s t e r i f i e d ^


0

5 Pressure

D i s t i l l e d water 100 mM

CaCl

2

100 mM T r i z m a + 100 mM C a C l

d

to

12.5 burst

0

_

23.6

6.0

172.5

119.6

37.2

149.9

108.9

37.8

189.3

176.6

100.1

2

d

100 mM T r i z m a + 100 mM C a C l

e 2

Adapted from (21) b E s t e r i f i c a t i o n a c h i e v e d by s u b s t i t u t i n g 50% e s t e r i f i e d p r o p y l e n e g l y c o l a l g i n a t e f o r 0, 10 and 25% of sodium a l g i n a t e c χ 102 P a s c a l ; Per i n i t i a l c r o s s - s e c t i o n a l a r e a of c a p s u l e T r i s (hydroxymethyl)aminomethane C a p s u l e s were a u t o c l a v e d a

d

e

P e r m e a b i l i z a t i o n of P l a n t C e l l s Secondary m e t a b o l i t e s produced by p l a n t c e l l c u l t u r e a r e commonly accumulated i n the c e l l s . W i t h few e x c e p t i o n s such as Capsicum f r u t e s c e n s , T h a l i c t r u m minus (9) and V a n i l l a p l a n i f o l i a ( K n o r r , D. and Romagnoli, L., U n i v . of Delaware, u n p u b l i s h e d d a t a ) c u l t u r e s , which r e l e a s e v a l u a b l e compounds such as c a p s a i c i n , b e r b e r i n e and v a n i l l i n , r e s p e c t i v e l y , i n t o the medium, p r o c e d u r e s t o i n d u c e p r o d u c t r e l e a s e are r e q u i r e d to develop continuous production processes. R e p o r t e d p e r m e a b i l i z a t i o n methods i n c l u d e t r e a t m e n t w i t h d i m e t h y l s u l f o x i d e (DMS0), i s o p r o p a n o l , t o l u e n e , p h e n e t h y l a l c o h o l or c h l o r o f o r m (9_, 28). But as F o n t a n e l and Tabata (9) p o i n t e d o u t , such t r e a t m e n t s w i t h o r g a n i c s o l v e n t s are s e v e r e and o t h e r methods of p e r m e a b i l i z a t i o n need t o be developed. Based on the p i o n e e r i n g work on c h i t o s a n and i n d u c e d p l a n t membrane p e r m e a b i l i t y i n c u l t u r e d G l y c i n e max c e l l s conducted by Young et a l . ( 2 9 ) , the r e l e a s e of secondary m e t a b o l i t e s from v a r i o u s c e l l c u l t u r e s i n t o the r e l a t e d n u t r i e n t medium was made p o s s i b l e i n the presence of c h i t o s a n (18, 22, 30, 31^, Beaumont, M. and K n o r r , D., U n i v . of Delaware, u n p u b l i s h e d d a t a ) . Leuba and S t o s s e l (32) suggested t h a t the r e l e a s e of C a and subsequent e f f l u x of + +



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