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2 Structured and Simple Models of Enzymatic Lysis and Disruption of Yeast Cells

Separation, Recovery, and Purification in Biotechnology Downloaded from pubs.acs.org by UNIV LAVAL on 07/13/16. For personal use only.

J. B. Hunter and J. A. Asenjo Biochemical Engineering Laboratory, Department of Chemical Engineering and Applied Chemistry, Columbia University, New York, NY 10027

Microbial cell-wall-lytic enzymes are widely used in the laboratory for cell breakage, proto-plasting of yeasts and bacteria, and for studies of the structure and composition of microbial cell walls (1). Recently lytic systems have come under consideration as a specific and chemically mild way to rupture microbial cells on an industrial scale (2,3). There appear to be attractive commercial applications of lytic systems for the recovery of enzymes, antigens and other recombinant products accumulated within cells, for upgrading of microbial biomass for food and feed uses (4,5) and for the manufacture of functional biopolymers from cell wall carbohydrates (6). This paper presents two models of enzymatic lysis of yeast cells; a simplified two-step model, accounting for protein release at cell lysis followed by proteolysis, and a more complex mechanistic model which describes the removal of the two layers of the yeast wall and the extrusion and rupture of the protoplast and organelles. The use of these models in predicting the release and breakdown of microbial proteins, and the application of the structured model to enzyme recovery will also be discussed. One p r o b l e m i n p r o d u c t i o n o f r e c o m b i n a n t p r o t e i n s i s r e c o v e r y o f the f i n i s h e d product from the c e l l s which accumulate i t . T h i s problem i s p a r t i c u l a r l y acute i n the case o f y e a s t s and f u n g i , which have tough, t h i c k c e l l w a l l s which are d i f f i c u l t t o r u p t u r e mechanic a l l y o r by s o n i c a t i o n . Product s e c r e t i o n i s not always f e a s i b l e , even f o r l o w - m o l e c u l a r - w e i g h t p r o d u c t s , a l t h o u g h a newly developed s e c r e t i o n p r o c e s s f o r y e a s t (7) a p p e a r s p r o m i s i n g . 0097-6156/ 86/ 0314-0009$06.50/ 0 © 1986 American Chemical Society


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SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

There a r e numerous examples of overproduced r e c o m b i n a n t p r o t e i n s w h i c h p r e c i p i t a t e i n t r a c e l l u l a r l y i n E_. c o l i , f o r m i n g d e n s e i n c l u s i o n b o d i e s ( 8 ) ; t h e s e p r o d u c t s i n c l u d e i n s u l i n and s o m a t o s t a t i n , b o t h very small p r o t e i n s . In yeast, recombinant v i r a l surface antigen p r o t e i n s a r e n o t s e c r e t e d , b u t a s s e m b l e i n t o p a r t i c l e s ( 9 ) . Subc e l l u l a r s t r u c t u r e s such as m i t o c h o n d r i a , l y s o s o m e s o r t h e v a c u o l e must a l s o be r e c o v e r e d by c e l l b r e a k a g e , f o r use e i t h e r as b i o c a t a l y s t s (10) o r as an i n i t i a l s t e p i n t h e p u r i f i c a t i o n o f enzymes a s s o c i a t e d w i t h s u c h s t r u c t u r e s . U n t i l now, t h e s e p r o d u c t s h a v e g e n e r a l l y b e e n h a r v e s t e d by m e c h a n i c a l l y r u p t u r i n g t h e c e l l s i n a homogenizer, bead m i l l or French p r e s s . The h i g h s h e a r f i e l d s , e l e v a t e d t e m p e r a t u r e s and g a s - l i q u i d i n t e r f a c e s g e n e r a t e d i n t h e s e d e v i c e s can denature p r o t e i n s , e s p e c i a l l y multi-enzyme complexes and m e m b r a n e - l i n k e d p r o t e i n s (11) . M o r e o v e r , t h e s e p a r a t i o n of c e l l d e b r i s from the products i s e s p e c i a l l y complicated i f the product i s p a r t i c u l a t e , f r a g i l e or membrane-associated. L y t i c enzyme s y s t e m s p r o v i d e a c h e m i c a l l y m i l d , l o w - s h e a r and c a t a l y t i c a l l y s p e c i f i c a l t e r n a t i v e t o m e c h a n i c a l c e l l d i s ruption. D e p e n d i n g on t h e p a r t i c u l a r l y t i c s y s t e m e m p l o y e d and i t s p u r i t y , t h e e n z y m e s may be e n g i n e e r e d t o a t t a c k c e l l w a l l comp o n e n t s a l o n e , w i t h o u t p r o d u c t damage. The e n z y m e l y s o z y m e , active a g a i n s t some b a c t e r i a l c e l l w a l l s , h a s b e e n u s e d t o h a r v e s t b o v i n e g r o w t h h o r m o n e g r a n u l e s f r o m _E. c o l i ( 8 ) , a n d a m e m b r a n e - a s s o c i a t e d h y d r o x y l a s e c o m p l e x f r o m P. p u t i d a ( 1 1 ) ; u s e o f o t h e r b a c t e r i o l y t i c enzymes f r o m a v a r i e t y o f m i c r o b i a l s o u r c e s h a v e a l s o been reported (3). I n v e s t i g a t i o n s i n t o t h e s u b c e l l u l a r l o c a t i o n o f enzyme a c t i v i t i e s i n m i c r o b i a l c e l l s s u g g e s t t h a t one o r more enzyme p r o d u c t s c o u l d be s p e c i f i c a l l y f r a c t i o n a t e d f r o m a s i n g l e b a t c h o f c e l l s by p r o p e r l y c o n t r o l l i n g c e l l d i s r u p t i o n . Invertase i n yeast i s p o s s i b l y the best example of t h i s p r i n c i p l e . The s t u d i e s l e a d i n g to d i s c o v e r y of i t s l o c a t i o n ( i n the p e r i p l a s m i c space) have b e e n s u m m a r i z e d by P h a f f ( 1 2 ; p . 1 7 1 - 1 7 3 ) , and a s a m p l e p r o c e s s f o r i t s r e c o v e r y h a s b e e n p r o p o s e d ( 4 ) . The r e c o v e r y o f s e v e r a l d i f f e r e n t enzymes i n h i g h y i e l d and h i g h r e l a t i v e p u r i t y s h o u l d be p o s s i b l e u s i n g a c o m b i n a t i o n o f l y t i c enzymes, s u r f a c t a n t s and o s m o t i c s u p p o r t b u f f e r s t o s e l e c t i v e l y and s e q u e n t i a l l y r e l e a s e p r o t e i n s from p a r t i c u l a r s t r u c t u r e s . C e l l f r a c t i o n a t i o n b y m e c h a n i c a l r u p t u r e h a s a l r e a d y come u n d e r investigation. Two s e p a r a t e s t u d i e s o f m e c h a n i c a l r u p t u r e o f y e a s t showed d i f f e r e n t r a t e s o f r e l e a s e f o r enzymes i n d i f f e r e n t c e l l l o c a t i o n s (13,14). W a l l - l i n k e d and p e r i p l a s m i c enzymes w e r e r e leased r e l a t i v e l y f a s t e r than t o t a l p r o t e i n , s o l u b l e cytoplasmic e n z y m e s a t a b o u t t h e same r a t e , a n d t h e m i t o c h o n d r i a l e n z y m e f u m a r a s e l a t e r t h a n t o t a l p r o t e i n (13) . P r o t e o l y s i s by t h e y e a s t s own e n z y m e s was n o t f o u n d t o b e a p r o b l e m . A c t i v i t i e s of the r e l e a s e d e n z y m e s d e c l i n e d s l o w l y o r n o t a t a l l w h e n d i s r u p t i o n was cont i n u e d a f t e r t h e end o f p r o t e i n r e l e a s e , and t h e e f f e c t o f s h e a r was not separated from the e f f e c t of p r o t e o l y s i s . S h e t t y and K i n s e l l a (15) a l s o f o u n d a l o w r a t e o f p r o t e o l y s i s a f t e r m e c h a n i c a l d i s r u p t i o n , though t h i o l r e a g e n t s added t o weaken the c e l l w a l l s b e f o r e d i s r u p t i o n c a u s e d an i m p o r t a n t i n c r e a s e i n t h e e x t e n t of p r o t e i n b r e a k down . 1

2.

HUNTER AND ASENJO

Model

Enzymatic Lysis and Disruption of Yeast Cells

11

Background

Yeast c e l l structure. The e x t e n s i v e body o f l i t e r a t u r e on c e l l c o m p o s i t i o n and s t r u c t u r e has r e c e n t l y been reviewed b y B a l l o u and e a r l i e r by Phaff (12). A s a n e n g i n e e r i n g a p p r o x i m a t i o n , t h e c e l l w a l l o f y e a s t may b e c o n s i d e r e d a s a t w o - l a y e r s t r u c t u r e . ( F i g u r e 1) T h e i n n e r w a l l i s composed o f a m i x t u r e o f b r a n c h e d 3(1-3) a n d 3(1-6) l i n k e d g l u c a n s , glucose polymers s i m i l a r t o c e l l u l o s e (12). The o u t s i d e o f t h e g l u c a n l a y e r i s covered w i t h a mannan-protein complex c o n s i s t i n g of a c r o s s - l i n k e d n e t w o r k o f p r o t e i n m o l e c u l e s , t o w h i c h a r e a t t a c h e d two t y p e s o f mannan: a n a c i d i c o l i g o s a c c h a r i d e , a n d a h i g h e r m o l e c u l a r w e i g h t p h o s p h o m a n n a n h a v i n g a d.p. o f a b o u t 1 0 0 ( 1 7 ) . From t h e p e r s p e c t i v e o f c e l l l y s i s , t h i s m a n n o p r o t e i n l a y e r s e r v e s to p r o t e c t t h e g l u c a n s from h y d r o l y t i c enzymes (18,19,20). Within t h e t w o w a l l l a y e r s i s t h e p r o t o p l a s t , c o m p r i s e d o f a p l a s m a membrane e n c l o s i n g the c y t o s o l and the s u b c e l l u l a r s t r u c t u r e s .


wall (16)

Enzymes o f t h e l y t i c system. M i c r o b i a l y e a s t - l y t i c enzyme systems are w i d e l y d i s t r i b u t e d i n n a t u r e , and have been i s o l a t e d from R h i z o c t o n i a sp., ( 4 ) , B a c i l u s c i r c u l a n s (21), Coprinus macrorhizus (22), a n d C y t o p h a g a s p . ( 2 3 ) , among o t h e r s o u r c e s . C r u d e y e a s t l y t i c enzyme s y s t e m s c o m p r i s e s e v e r a l h y d r o l y t i c a c t i v i t i e s , o f t e n i n c l u d i n g c h i t i n a s e , mannanase, and a v a r i e t y o f p r o t e a s e s a n d g l u c a n a s e s ( 1 ) . Only two o f t h e s e a c t i v i t i e s , a l y t i c protease anda l y t i c glucanase, are e s s e n t i a l f o r l y s i s (19,24,20). L y t i c glucanases u s u a l l y b i n d p r e f e r e n t i a l l y t o l o n g c h a i n s o f 3(1-3) g l y c o s i d i c l i n k a g e s , such a s those found i n m i c r o f i b r i l l a r y e a s t w a l l glucan. I n g e n e r a l , the l y t i c g l u c a n a s e s have a n endo- a c t i o n p a t t e r n b u t some a t t a c k e x o - w i s e , r e l e a s i n g o l i g o s a c c h a r i d e s o f 5 glucose u n i t s from the s t r u c t u r a l yeast glucan. Other glucanases, with d i f f e r e n t substrate s p e c i f i c i t y anda c t i o n p a t t e r n s , are u s u a l l y p r e s e n t i n t h e l y t i c s y s t e m and a c t s y n e r g i s t i c a l l y t o d e g r a d e i n s o l u b l e y e a s t g l u c a n t o g l u c o s e a n d d i s a c c h a r i d e s (25) . Lytic p r o t e a s e s have a c h a r a c t e r i s t i c h i g h a f f i n i t y f o r the y e a s t w a l l s u r f a c e , and o f t e n have anomalously low a c t i v i t i e s a g a i n s t c o n v e n t i o n a l protein substrates. Their role i n l y s i s of viable yeast c e l l s cannot be s u b s t i t u t e d by o r d i n a r y p r o t e a s e s . (20,26). We u s e d a l y t i c s y s t e m f r o m O e r s k o v i a x a n t h i n e o l y t i c a L L - G 1 0 9 f r o m t h e c o l l e c t i o n o f M. L e c h e v a l i e r , a t R u t g e r s U n i v e r s i t y . F i l t e r e d c u l t u r e b r o t h was u s e d a s t h e enzyme s o u r c e . Details of t h e enzyme p r o d u c t i o n a r e g i v e n e l s e w h e r e ( 2 7 , 2 8 ) . The l y t i c a c t i v i t y o f the O e r s k o v i a system i s due t o a l y t i c p r o t e a s e and an endo 3 ( 1 , 3 ) g l u c a n a s e ( 2 0 ) , p o s s i b l y s u p p l e m e n t e d w i t h a n exo 3 ( 1 - 3 ) glucanase removing a 5-sugar u n i t from the c h a i n (29). Sequence o f c e l l l y s i s . Enzymatic c e l l l y s i s begins w i t h b i n d ing o f the l y t i c protease t o the outer mannoprotein l a y e r o f t h e wall. The p r o t e a s e opens up t h e p r o t e i n s t r u c t u r e , r e l e a s i n g w a l l p r o t e i n s and mannans, a n d e x p o s i n g t h e g l u c a n s u r f a c e b e l o w ( F i g u r e 2). Next, the glucanase a t t a c k s the i n n e r w a l l and s o l u b i l i z e s the g l u c a n ( 1 9 ) . When t h e c o m b i n e d a c t i o n o f p r o t e a s e a n d g l u c a n a s e has opened a s u f f i c i e n t l y l a r g e h o l e i n the c e l l w a l l , the plasma membrane a n d i t s c o n t e n t s a r e e x t r u d e d a s a p r o t o p l a s t ( 1 ) . I n o s m o t i c a l l y s u p p o r t e d b u f f e r s c o n t a i n i n g .55 t o 1.2M s u c r o s e o r

12



Mannoprotein Units

Cell Membrane Figure 1

Structural Glucan Units

Double-layered structure o f the yeast w a l l , t h e c e l l membrane

Figure

2

Schematic o f l y s i n g

yeast

cell

enclosing

2.

HUNTER AND

ASENJO


13

m a n n i t o l , the p r o t o p l a s t remains i n t a c t but i n d i l u t e b u f f e r s i t l y ses i m m e d i a t e l y , r e l e a s i n g c y t o p l a s m i c p r o t e i n s and the organelles w h i c h may t h e m s e l v e s l y s e . Meanwhile, p r o t e i n s r e l e a s e d from the w a l l and the cytoplasm are subject t o a t t a c k by product-degrading p r o t e a s e contaminants i n the l y t i c system (28,30).


Models Mathematical models w i t h d i f f e r e n t l e v e l s o f s t r u c t u r e are usef u l f o r the d e s i g n o f r e a c t o r s , t o c a r r y out s i m u l a t i o n s t u d i e s , f o r p r o c e s s o p t i m i z a t i o n and f o r i n c r e a s i n g our u n d e r s t a n d i n g o f t h e mechanistic, b i o l o g i c a l behavior o f biochemical systems. H i s t o r i c a l l y t h e r e has been l i t t l e p u b l i s h e d work on models o f microbial cell lysis. The models proposed f o r o v e r a l l c e l l l y s i s have been e l e m e n t a r y and t h e i r a p p l i c a t i o n has been l i m i t e d . Firsto r d e r and M i c h a e l i s - M e n t e n m o d e l s h a v e b e e n u s e d t o e s t i m a t e t h e performance o f a sample l y s i s p r o c e s s (2 3) , L y s i s o f f r e e z e - d r i e d M i c r o c o c c u s l y s o d e i k t i c u s c e l l s b y l y s o z y m e was modeled w i t h a second-order rate expression (31) . A t t h e o t h e r e n d o f t h e s p e c t r u m of mathematical c o m p l e x i t y i s a model o f l y s o z y m e - c a t a l y z e d degradat i o n o f s o l u b l e b a c t e r i a l c e l l - w a l l o l i g o s a c c h a r i d e s , f o c u s i n g on the d e g r e e o f p o l y m e r i z a t i o n o f t h e s u b s t r a t e a n d t h e b i n d i n g modes o f enzyme t o s u b s t r a t e s ( 3 2 ) . A c c o u n t i n g f o r one enzyme a n d c a r b o h y d r a t e o l i g o m e r s u p t o d.p. 9, i t h a s n i n e d i f f e r e n t i a l e q u a t i o n s a n d t e n p a r a m e t e r s , and was t e s t e d o n p u r i f i e d r a d i o l a b e l e d oligosaccharides. A l t h o u g h u s e f u l f o r e l u c i d a t i n g enzyme a c t i o n p a t t e r n s , s u c h models are too d e t a i l e d t o be r e a d i l y a p p l i e d t o a multi-enzyme, m u l t i - s u b s t r a t e system. The t w o m o d e l s o f y e a s t l y s i s p r e s e n t e d h e r e have been d e v e l o p ed t o s e r v e t w o d i f f e r e n t p u r p o s e s . The s i m p l e model i s a lumped, t w o - s t e p m o d e l w h i c h f o l l o w s t h e m a j o r f e a t u r e s o f t h e d a t a a n d may prove u s e f u l f o r design o f l y s i s reactors. The s t r u c t u r e d model, w h i c h can a c c o u n t f o r the s o u r c e o f p r o t e i n w i t h i n the c e l l , was developed t o g a i n a m e c h a n i s t i c b a s i s f o r p r e d i c t i n g the e f f e c t s o f u n t e s t e d p r o c e s s c o n d i t i o n s , and t o a i d i n s i g h t i n t o the p h y s i c a l p r o c e s s e s a t work d u r i n g l y s i s . S

Simple model. The s i m p l e model was b u i l t f o r compact d e s c r i p t i o n o f t h e d a t a i n a p r e - d e t e r m i n e d r a n g e o f y e a s t a n d enzyme c o n c e n trations. I t t r e a t s c e l l l y s i s and p r o t e o l y s i s as s i n g l e - s t e p r e a c t i o n s i n sequence. Both r e a c t i o n s are modeled w i t h M i c h a e l i s Menten k i n e t i c s , even though y e a s t , the s u b s t r a t e o f the f i r s t r e a c t i o n , i s p a r t i c u l a t e and the p r o t e i n s are s o l u b l e . The d i f f e r e n t enzymes o f t h e l y t i c s y s t e m a r e g r o u p e d t o g e t h e r i n t o a n a l l - i n c l u s i v e s i n g l e enzyme, E , b e a r i n g b o t h t h e p r o t e o l y t i c and y e a s t - l y t i c activities. A l l o f the c e l l s t r u c t u r e s are a l s o considered together a s a u n i f i e d y e a s t c e l l m a s s , Y. When a c e l l i s a t t a c k e d b y e n z y m e s i t i s p r e s u m e d t o d i s s o l v e i n s t a n t a n e o u s l y , r e l e a s i n g i t s e n t i r e mass a s s o l u b l e p r o t e i n s , p e p t i d e s andcarbohydrates. The assumption o f i n s t a n t a n e o u s s o l u t i o n o f t h e c e l l mass c o n s t r a i n s t h e m o d e l f o r use w h e r e t h e l y s i s medium i s hypo-osmotic andp r o t o p l a s t s cannot s u r v i v e i n t a c t .

14

SEPARATION, RECOVERY, A N D PURIFICATION IN BIOTECHNOLOGY

O n l y two i n d e p e n d e n t v a r i a b l e s a r e u s e d : y e a s t ( Y ) a n d e n z y m e (E); t h e measured v a r i a b l e s a r e y e a s t , T C A - i n s o l u b l e p r o t e i n (Ρ), T C A - s o l u b l e p r o t e i n ( p e p t i d e s , S ) , and c a r b o h y d r a t e s (C); a l l are e x p r e s s e d a s g/1 d r y b a s i s . E n z y m e c o n c e n t r a t i o n was e x p r e s s e d a s t h e v o l u m e p e r c e n t o f c r u d e l y t i c enzyme p r e p a r a t i o n added t o t h e reaction mixture. P r o t e o l y t i c and o t h e r c a u s e s f o r l y t i c enzyme d e a c t i v a t i o n ( e . g . , t h e r m a l ) h a v e been assumed t o be n e g l i g i b l e ( 2 8 ) , k E'(Y

dY dt

-k (Y-Y a


œ

(Y -

dP _

dY

>

f

" py

dt dS _

-f sy

dt dC

Yco)

r

=

_

+

Yco)

Κ

k E-P —Ρ

(2)

dt,

ρ + s + κ

dY/

k Ε·Ρ _J2 Ρ + S + K mp 1 + -

dt,

(1)

Ώ

m

P

1+

Y *

(3)

dY' "~fcy

dt

(4)

.dt,

V a r i a b l e names and p a r a m e t e r v a l u e s

are given

i n Table

I.

On t h e r i g h t - h a n d s i d e o f e q u a t i o n 1, t h e i n i t i a l t e r m r e p r e s e n t s a u t o l y s i s and t h e second t e r m , e n z y m a t i c l y s i s . Equation 2 d e s c r i b e s p r o t e i n breakdown by p r o d u c t - d e g r a d i n g proteases. The f i r s t t e r m on t h e r i g h t s i d e s t a n d s f o r t h e p r o t e i n r e l e a s e d f r o m l y s i n g c e l l s , and t h e second term, breakdown o f t h e p r o t e i n a l r e a d y in solution. E q u a t i o n 3 shows t h a t p e p t i d e s a r e r e l e a s e d f r o m l y s i n g y e a s t , b u t a l s o a r i s e f r o m b r e a k d o w n o f l o n g e r p r o t e i n s , P. Since the protease a c t i v i t y against s o l u b l e p r o t e i n s i s con s i d e r e d n o n - s p e c i f i c , b o t h l o n g - and s h o r t - c h a i n p r o t e i n s w i l l be a t t a c k e d b y t h e e n z y m e w i t h e s s e n t i a l l y t h e same a f f i n i t y p e r g r a m of s u b s t r a t e . Hence, S w i l l a c t as a c o m p e t i t i v e i n h i b i t o r o f t h e e n z y m e a c t i v i t y a g a i n s t P, w h e r e t h e i n h i b i t i o n c o n s t a n t i s e q u a l to t h e M i c h a e l i s c o n s t a n t K ^ . C a r b o h y d r a t e r e l e a s e i s shown i n equation 4. Parameters f o r t h e s i m p l e model were d e t e r m i n e d g r a p h i c a l l y by Eadie-Hofstee p l o t t i n g of i n i t i a l r e a c t i o n rates and s u b s t r a t e c o n centrations. D e t a i l s a r e g i v e n e l s e w h e r e (30). As has been ob served i n h y d r o l y s i s of other s o l i d s u b s t r a t e s , a r e s i d u e of nonl y s e d s u b s t r a t e was f o u n d a t e x t e n d e d r e a c t i o n t i m e s , w h e n d Y / d t tended toward zero. The e x t e n t o f r e a c t i o n was s t r o n g l y d e p e n d e n t on i n i t i a l s u b s t r a t e and enzyme c o n c e n t r a t i o n s ( 3 3 , 3 4 ) . An e m p i r i c a l f u n c i t o n f o r Y^ was f i t t e d t o t h e u l t i m a t e t u r b i d i t y d a t a f o r l y s i s r u n s a t a v a r i e t y o f i n i t i a l y e a s t and enzyme c o n c e n t r a t i o n s u s i n g a l e a s t squares method. The c a l c u l a t e d v a l u e s f o r Yoo w e r e u s e d i n t h e s i m u l a t i o n s (30) . F i g u r e 3 s h o w s r e s u l t s o f the simple model. S t r u c t u r e d model. T h i s model c o n s i d e r s l y s i s of t h e c e l l from the v i e w p o i n t of p r o g r e s s i v e breakdown of the c e l l s t r u c t u r e s , s t a r t i n g f r o m t h e o u t e r w a l l l a y e r and p r o g r e s s i n g t o t h e s u b c e l l u l a r s t r u c t u r e s i n s i d e t h e p r o t o p l a s t (35). Here the c e l l i s d i v i d e d i n t o

HUNTER AND ASENJO

2.

Table

L.


15

Lumped m o d e l v a r i a b l e s a n d p a r a m e t e r s

V a r i a b l e s - Simple Model Y Y e a s t , mg/1 Y Original yeast concentration Yoo U l t i m a t e y e a s t c o n c e n t r a t i o n , mg/1; p r o p o r t i o n a l 0


to

residual

turbidity.

^°°= a 4 b E + c Y

Ρ S C Ε

0

+

I

P r o t e i n ( T C A - i n s o l u b l e ) , mg/1 P e p t i d e s ( T C A - s o l u b l e ) , mg/1 C a r b o h y d r a t e s , mg/1 Enzyme, % ( v / v ) o f r e a c t i o n m i x t u r e Parameters-Simple

Yoo c o n s t a n t s :

a: b: c: d:

Model

3.6342 χ 1 0 "

1

- 2 . 6 5 8 4 χ 10 ~ 6

6.0442 χ ΙΟ"" -9.9603 χ 1 0

1

k

a

Rate constant

for autolysis

k

r

Rate constant

for lysis,

K

m

M i c h a e l i s constant

3.987 χ 1 0 - ^ m i n "

s i m p l e m o d e l 15.51 mg/L-min-%ez

for lysis,

for proteolysis,

1902 mg/L

kp

Rate constant

Κ

Michaelis constant, proteolysis,

4 5 9 8 mg/L

Inhibition

2 6 0 7 7 mg y e a s t / L

f

constant, proteolysis,

Fraction ofprotein

i n yeast

4.441

0.4048

f y

Fraction of peptides i n yeast

0.0777

f

Fraction of carbohydrates

0.3687

g

1

i n yeast

mg/L-min-%ez

16

SEPARATION, RECOVERY, AND

PURIFICATION IN BIOTECHNOLOGY


f o u r r e g i o n s ; t h e o u t e r w a l l o r w a l l p r o t e i n (WP); i n n e r w a l l o r w a l l g l u c a n (WG) ; t h e c y t o s o l (CS) a n d t h e o r g a n e l l e s , h e r e g r o u p e d t o g e t h e r as m i t o c h o n d r i a ( M I ) . The l y t i c s y s t e m i s a p p r o x i m a t e d a s t h r e e enzymes, a l y t i c g l u c a n a s e , Eg, w h i c h h y d r o l y z e s the i n n e r c e l l w a l l g l u c a n , a l y t i c p r o t e a s e , Ep, w h i c h a t t a c k s o n l y t h e o u t e r w a l l l a y e r and a d e s t r u c t i v e p r o t e a s e , E^, a c t i v e a g a i n s t s o l u b l e p r o t e i n s . P r o d u c t i n h i b i t i o n i s i n c l u d e d i n a l l enzyme r e a c t i o n s . Adsorption and d e s o r p t i o n o f t h e e n z y m e s t o t h e y e a s t w a l l i s n e g l e c t e d , since a d s o r p t i o n k i n e t i c s a p p e a r e d i n s t a n t a n e o u s on t h e t i m e s c a l e o f o u r measurements (35). A s c h e m a t i c o f t h e r e a c t i o n p a t h w a y s i s shown i n Figure 4. Special EGA

variables. =

(WG

-

r-WP)

The g l u c a n h y d r o l y s i s r a t e i s n o t r e l a t e d d i r e c t l y t o t o t a l g l u c a n c o n c e n t r a t i o n WG, b u t r a t h e r t o t h e a m o u n t o f g l u c a n made a c c e s s i b l e to a t t a c k through removal of w a l l p r o t e i n from the outside of the c e l l . EGA, " e x p o s e d g l u c a n , a c c e s s i b l e " r e p r e s e n t s t h e amount o f g l u c a n u n c o v e r e d by r e m o v a l o f t h e o u t e r w a l l . The p r o p o r t i o n a l i t y constant r i s the weight r a t i o of w a l l glucan to w a l l p r o t e i n .

PBR

The o v e r a l l r a t e o f s o l u b l e p r o t e i n h y d r o l y s i s , PBR, protein breakdown r a t e , a c c o u n t s f o r d e s t r u c t i o n o f s o l u b l e p r o t e i n by the destructive protease. The r e l e a s e o f c y t o s o l i n t o t h e m e d i u m d e p e n d s o n t h e o s m o t i c breakage of the p r o t o p l a s t s , which occurs at a r a t e approximately p r o p o r t i o n a l t o t h e o s m o t i c g r a d i e n t a c r o s s t h e p l a s m a membrane ( 3 6 ) . The i n t e r n a l o s m o l a l i t y o f t h e c e l l s was e s t i m a t e d t o b e 0.617 Os/L ( 3 5 ) , w h e r e 1 Os/L i s e q u i v a l e n t t o 1 M o l / L o f a n i d e a l s o l u t e . The e x t e r n a l o s m o l a l i t y i s t h e sum o f t h e c o n t r i b u t i o n f r o m t h e b u f f e r s y s t e m i n t h e m e d i u m ( a b o u t 0.02M i n o u r e x p e r i m e n t s ) a n d the s u b s t a n c e s r e l e a s e d by l y s i n g p r o t o p l a s t s . The s t a b i l i z a t i o n o f t h e r e m a i n i n g c e l l s by t h e s e s u b s t a n c e s i s f a r s t r o n g e r t h a n c o u l d be e x p e c t e d s o l e l y on t h e b a s i s o f o s m o t i c e f f e c t s , a n d c o u l d r e s u l t from the r e l e a s e of c a t i o n s which i n t e r a c t w i t h s p e c i f i c receptors o n t h e p l a s m a membrane ( 3 7 ) . The r e l e a s e o f s o l u b l e p r o d u c t s o f g l u c a n a n d p r o t e i n h y d r o l y s i s a r e a l s o e x p e c t e d t o add t o t h e s t a b i l i z i n g e f f e c t of the l y s a t e . The e f f e c t i v e o s m o l a l i t y o f c e l l l y s a t e was f i t t o a L a n g m u i r e x p r e s s i o n , w h e r e 0 S M i s t h e maximum s t a b i l i z i n g e f f e c t and Κ is the e q u i l i b r i u m constant f o r i n t e r a c t i o n of the s t a b i l i z e r s w i t n the protoplasts. The r e s u l t i n g e q u a t i o n , L

Q

S

K

C

S

C S

C S

V o s J * + o ~ > 1 + Κ ( C S * + CS - CS) osm ο e x p r e s s e s t o t a l e f f e c t i v e o s m o l a l i t y i n the l y s i s medium. B i s the o r i g i n a l o s m o l a l i t y o f t h e l y s i s b u f f e r a n d CS* i s t h e sum of p r o t e i n , p e p t i d e s and c a r b o h y d r a t e s p r e s e n t a t t h e s t a r t o f r e a c t i o n . 0SM

x

= B

0

+

Q

HUNTER AND ASENJO


en

s ο


5

TIME - MINUTES Figure

3

Simple model s i m u l a t i o n o f yeast l y s i s Y e a s t c e l l m a s s , mg/1 mg/1 P e p t i d e s , mg/1 h y d r a t e s , mg/1 0.78 g/1 y e a s t c o n c e n t r a t i o n ; 1 0 % enzyme

Oligopeptides Amino Acids

Soluble Proteins Cytoplasmic Enzymes

- Carbo-

1. Protease attack 2. Glucanase attack 3. Release of cell contents 4. Lysis of organelles 5. Glucan hydrolysis 6

Wall Protein Wall Enzymes Wall Mannan

Protein,

1

7. > Product proteolysis Ô ;

/-\ Proteins Organelles-- Organellar Enzymes

Carbohydrates

fl(l-3) Oligosaccharides Glucose Figure

4

Reaction

pathways f o r s t r u c t u r e d model

18


Based on t h e p r o d u c t OSM[/K the s t a b i l i z i n g e f f e c t o f c e l l l y s a t e a t l o w c o n c e n t r a t i o n s i s e q u i v a l e n t t o 4.4 χ 10 Os/mg c y t o s o l released (35). c

H

Wall

hydrolysis

equations.

d(WP) dt 1


WP (Km wjy_ WP - WP ο Ki wp

wp ρ WP Km wp

+

(1)

EGA Ε wg g (Km wg WG - WG ο l + # ^ + Km Km sg wg k

d(WG) dt

(2)

R e l e a s e o f c y t o s o l and m i t o c h o n d r i a . The o s m o t i c g r a d i e n t b e tween p r o t o p l a s t s and b u f f e r o r m i t o c h o n d r i a and b u f f e r d r i v e s t h e r e l e a s e o f p r o t e i n i n t o t h e medium. I f t h e o s m o l a l i t y o f t h e e x t e r n a l medium e x c e e d s t h e i n t e r n a l o s m o l a l i t y o f t h e p r o t o p l a s t o r o r g a n e l l e , no r u p t u r e o c c u r s . The o s m o l a l i t y d e c r e a s e s i n t e r n a l l y , and i n c r e a s e s e x t e r n a l l y , as m a t e r i a l i s r e l e a s e d from t h e p r o t o p l a s t . In addition, the r e l e a s e o f c y t o s o l i s p r o p o r t i o n a l t o t h e s i z e o f t h e opening i n t h e w a l l g l u c a n , up t o a maximum h o l e s i z e o f 1/3 o f t h e c e l l ^ sur face area. d(CS) dt

-

(CS).k

(CS)

d(MI) dt

f l

«k^max^OSM^

CS,

d(CS) ' dt

k

CS CS

r m

- 0SM )]· max(.33, 1 x

[ m a x ( 0 , 0.3 - OSK^.) ] ·ΜΙ

WG ) WG

(3)

(4)

Soluble products. V a l u e s f o r T C A - i n s o l u b l e p r o t e i n , p e p t i d e s and c a r b o h y d r a t e s r e l e a s e d w e r e e s t i m a t e d b y summing t h e c o n t r i b u t i o n t o each p o o l from t h e breakdown o f each c e l l u l a r s t r u c t u r e .

^ dt

- - f pwp

f pm

d(WP) [ dt

- f pes

'd(CS)'

I

dt

«k [ m a x ( 0 , 0.3 - OSM ) ] ·ΜΙ - PBR rm χ

(5)

2.

HUNTER AND ASENJO

'd(WP)' - - f [dt swp

^ dt

f sm


dÇ dt

m

19


- f

d(CS)l

ses

+

dt

-k [ m a x ( 0 , 0.3 - OSM ) ] - M I + P B R rm χ

_ d(WG) dt

_ f

(6)

-d(CS) ' dt

(

7

)

T o t a l y e a s t c e l l m a s s w a s e s t i m a t e a s t h e s u m o f WG, WP, C S , a n d M I ( s t r u c t u r e s r e m a i n i n g w i t h t h e c e l l ) , w i t h a n added f a c t o r a c c o u n t i n g for non-protein, non-carbohydrate substances i n the c e l l . T h e s e sums generate v a l u e s f o r y e a s t , p r o t e i n , p e p t i d e s and carbohydrates f o r comparison t o e x p e r i m e n t a l measurements. The v a r i a b l e s f o r t h e s t r u c t u r e d m o d e l a r e l i s t e d Parameter values are given i n Table I I I(35).

Table

II.

i n Table I I .

Structured Model V a r i a b l e s

EGA

Exposed glucan, a c c e s s i b l e f o r h y d r o l y s i s by glucanase

Y

Initial

Q

WG

Wall

WG

Original

0

CS CS

q u a n t i t y o f y e a s t , mg/1 d r y b a s i s

g l u c a n , mg/1 a m o u n t o f g l u c a n ; = Y « f W G , mg/1 0

Q

CS*

Original

quantity of cytosol

Long-chain

Oligopeptides

M

M i t o c h o n d r i a l m a s s , mg/1 Q

Q

Eg

Q

material i n

Q

W a l l p r o t e i n , mg/1

S

B

0

0

Ρ

M

= Y « f C S , mg/1

I n i t i a l amount o f o s m o t i c a l l y s t a b i l i z i n g r e a c t i o n medium: P + S + C Q

WP

0

C y t o s o l , mg/1

p r o t e i n (TCA - i n s o l u b l e ) , mg/1 (TCA - s o l u b l e ) , mg/1

O r i g i n a l mass o f m i t o c h o n d r i a Osmotic s t r e n g t h o f b u f f e r , G l u c a n a s e enzyme o f l y t i c protease

enzyme,

i n cell

= Y » f M , mg/1 0

0

Os/kg

system, %(V/V) o f m i x t u r e

Ep

Lytic

E(j

Destructive o r product-degrading

%(V/V) o f m i x t u r e

WE

Yeast

e n z y m e i n w a l l s , mg/1

CE

Yeast

e n z y m e i n c y t o s o l , mg/1

ME

Yeast

e n z y m e i n m i t o c h o n d r i a , mg/1

protease,

%(V/V)


20

Table I I I ,

S t r u c t u r e d model parameters and t h e i r

Ratio of wall

to wall

R a t e c o n s t a n t , g l u c a n a s e on

Kg

Michaelis Km, sg


glucan

wp Kin wp Ki wp

mg/L-min-%ez

4424

mg/L

800

Rate constant, p r o t e o l y s i s

4.441 m g / L - m i n ~ % e z

Michaelis

o f WP

constant

Inhibition o f WP

constant,

mg/L

459.8

mg/1

919.6

mg/1

proteolysis

of Ρ

constant

Rate constant

"rm

WG

M i c h a e l i s c o n s t a n t , g l u c a n a s e on soluble glucan

Michaelis

^ p

1.326 mg WG/mg WP 10.58

WG

c o n s t a n t , g l u c a n a s e on

Rate constant, p r o t e o l y s i s

Ρ

protein

1.441 m g / L - m i n - % e z 4598

f o r CS

leakage

mg/L

3.987 χ 1 0 - " m i n "

Rate constant

f o r CS

release

1.6667 m i n "

Rate constant

f o r MI

breakage

0.6 m i n "

protein

i n wall

Ε pes Ε

Fraction

protein

i n cytosol

0.3753

Fraction

protein

i n mitochondria

0.75

pm

Fraction

peptides i n wall

^swp

Fraction

peptides

Fraction

peptides i n mitochondria

0.05

Fraction

of carbohydrates

0.3145

CCS

protein

protein

i n cytosol

0.1170

i n cytosol

Initial

fraction

fWG

Q

Initial

fraction of wall

glucan

0.1922

fWP

Q

Initial

fraction

protein

0.1450

Initial

fraction of mitochondria

fM

0

of cytosol

of wall

OSMi

Internal osmolality

0SM

Maximum e f f e c t i v e o s m o l a l i t y released l y s i s products

T

ewp

of yeast

1

0.0566

0

fCS

1

1

0.9434

Fraction

pwp

values

i n yeast

cell

0.5612

0.7652 0.617

Os/L

0.539

Os

of

Equilibrium constant f o r osmotic s t a b i l i z a t i o n of protoplasts

8.135 χ l O - ^ L / m g CS

P r o p o r t i o n o f enzyme i n w a l l

0.01

protein

P r o p o r t i o n o f enzyme i n c y t o s o l

0.01

P r o p o r t i o n o f enzyme i n m i t o c h o n d r i a

0.01

2.

HUNTER AND ASENJO


21

The s t r u c t u r e d m o d e l s i m u l a t e s t h e p r o g r e s s o f l y s i s i n t e r m s of t h e c e l l ' s s t r u c t u r a l components d u r i n g l y s i s . The decrease i n WP s t a r t s i m m e d i a t e l y , a s i t i s t h e f i r s t c o m p o n e n t a t t a c k e d by t h e l y t i c enzymes. WG b r e a k d o w n l a g s WP r e m o v a l , a n d c y t o s o l r e l e a s e l a g s g l u c a n breakdown, as suggested by the s e q u e n t i a l i t y b u i l t i n t o the model. The m i t o c h o n d r i a a r e r e l e a s e d l a s t , and t e n d t o a c c u m u l a t e b e c a u s e t h e y a r e more r e s i s t a n t t o o s m o t i c r u p t u r e than the p r o t o p l a s t s . A t y p i c a l g r a p h i s shown i n F i g u r e 5 a . I n F i g u r e 5b, s t r u c t u r e d m o d e l e s t i m a t e s o f y e a s t c e l l m a s s , p r o t e i n , p e p t i d e s a n d c a r b o h y d r a t e s a r e p r e s e n t e d f o r t h e same e n z y m e a n d yeast concentrations.


Results The s i m p l e a n d s t r u c t u r e d m o d e l s i m u l a t i o n s f o r y e a s t m a s s a n d s o l u b l e p r o t e i n , p e p t i d e s and c a r b o h y d r a t e s a r e compared i n F i g u r e 6 f o r t h e y e a s t a n d e n z y m e c o n c e n t r a t i o n s h o w n i n F i g u r e s 3 a n d 4, a n d in Figure 7 for a concentrated yeast c e l l slurry. The simple model f i t s the data f a i r l y w e l l a t both yeast c o n c e n t r a t i o n s , i n every v a r i a b l e except the p e p t i d e s . The f i t f o r a l l v a r i a b l e s a t l o n g e r r e a c t i o n times i s d i r e c t l y r e l a t e d t o use o f the e x t e n t - o f - r e a c t i o n t e r m Yoo i n t h e y e a s t l y s i s e q u a t i o n . The s t r u c t u r e d m o d e l p r o v i d e s a d i s t i n c t i m p r o v e m e n t o v e r t h e simple model, i n the i n i t i a l stages o f the r e a c t i o n . The i n i t i a l l a g s i n t h e h y d r o l y s i s o f t o t a l y e a s t mass a n d c a r b o h y d r a t e i n f i g ures 6 and 7 are very w e l l represented. The p o s s i b i l i t y remains t h a t the i n i t i a l l a g s r e l a t e p a r t l y t o a d s o r p t i o n o f l y t i c enzymes t o t h e c e l l w a l l . On t h e t i m e s c a l e o f o u r e x p e r i m e n t s , h o w e v e r , a d s o r p t i o n appeared t obe instantaneous (35). A t h i g h y e a s t c o n c e n t r a t i o n ( f i g u r e 7) a t t h e l a t e r s t a g e s o f r e a c t i o n , the carbohydrates c o n t i n u e t o r i s e though t u r b i d i t y i s l e v e l l i n g o f f . A p p a r e n t l y some w a l l h y d r o l y s i s i s o c c u r r i n g e v e n though the t o t a l yeast s o l i d s c o n c e n t r a t i o n i s not v i s i b l y d e c l i n i n g . T h i s r e s u l t i s i n c o n t r a s t t o f i g u r e 6, w h e r e t h e s t r u c t u r e d m o d e l f o l l o w s carbohydrate data c l o s l e y , and the h y d r o l y s i s o f w a l l glucan i s e s t i m a t e d t o go e s s e n t i a l l y t o c o m p l e t i o n . Presumably the glucanase a t t a c k s t h e more s u s c e p t i b l e amorphous g l u c a n a t a h i g h e r r a t e than the f i b r i l l a r glucan f r a c t i o n o f the w a l l . Such dependence on p h y s i c a l s t r u c t u r e i s w e l l known t o o c c u r i n e n z y m a t i c h y d r o l y s i s o f c e l l u l o s e (38,34). I f t h e a n a l o g y i s c o r r e c t , t h e amorphous c a r b o hydrateds c o u l d be s o l u b i l i z e d without s u b s t a n t i a l l y changing the m i c r o f i b r i l network s t r u c t u r e i n the w a l l , o r r e l e a s i n g p r o t o plasts. C e l l u l o s e / c e l l u l a s e system r e s u l t s a l s o suggest t h a t t h i s e f f e c t o u g h t t o b e more p r o n o u n c e d a t t h e h i g h e r y e a s t - t o - e n z y m e r a t i o shown i n F i g u r e 7, t h a n a t t h e l o w e r r a t i o o f F i g u r e 6. A l i m i t a t i o n o f b o t h m o d e l s i s o v e r e s t i m a t i o n o f t h e amount o f p r o t e i n r e l e a s e d . P e p t i d e p r e d i c t i o n s b y the s i m p l e model are too high as w e l l . The e f f e c t resembles a gap i n the m a t e r i a l b a l a n c e , as i f the models p r e d i c t a l a r g e r q u a n t i t y o f p r o t e i n a c i o u s m a t e r i a l than i s a c t u a l l y present i n t h e c e l l s . P o s s i b l y some c y t o p l a s m i c p r o t e i n s are not r e l e a s e d d u r i n g p r o t o p l a s t breakage. I n f i g u r e 6, i n s o l u b l e p r o t e i n s could account f o r a s u b s t a n t i a l p a r t o f t h e r e s i d u a l y e a s t a t 90 m i n u t e s d i g e s t i o n .



45

TIME - MINUTES ure

5

Structured

5a

Cell

model s i m u l a t i o n

structures CY

of yeast

cytosol

WP w a l l p r o t e i n

—

lysis

WG w a l l — - — —-MI

glucan

mitochondria

mg/1

45

TIME - MINUTES 5b

C e l l mass and r e l e a s e d compounds "Y e a s t c e l l m a s s , mg/1 Peptides,

mg/1

mg/1

- P r o t e i n , mg/1 Carbohydrates,

HUNTER A N D ASENJO



I

Figure

Figure

6

7

TIME- MINUTES

TIME-MINUTES

TIME-MINUTES

TIME-MINUTES

Comparison o f simple and s t r u c t u r e d models - I n t i t i a l y e a s t c o n c e n t r a t i o n 0.78 g/1 ( d . b ) , 1 0 % e n z y m e S t r u c t u r e d model — Simple model

Comparison o f simple and s t r u c t u r e d models - I n i t i a l y e a s t c o n c e n t r a t i o n 3 6 . 3 g/1 ( d . b ) 4 0 % e n z y m e Symbols as i n f i g u r e 6

24


W o r k c o n t i n u e s i n two a r e a s : p u r i f i c a t i o n of the l y t i c system to a l l o w p r o t e a s e and g l u c a n a s e l e v e l s t o be c o n t r o l l e d i n d e p e n d e n t l y and i n v e s t i g a t i o n o f t h e r e l e a s e o f s i t e - s p e c i f i c y e a s t enzymes and s u b c e l l u l a r f r a c t i o n s by e n z y m a t i c lysis. Applications


The s t r u c t u r e d model's d e t a i l e d a c c o u n t i n g o f t h e f a t e o f c e l l s t r u c t u r e s c a n b e u s e d t o make p r e d i c t i o n s a b o u t t h e e f f e c t s o f a number o f i m p o r t a n t p r o c e s s v a r i a b l e s , f o r e x a m p l e : The r a t i o o f l y t i c p r o t e a s e t o g l u c a n a s e i n t h e l y t i c system T h e e f f e c t o f pH o r t e m p e r a t u r e o n s y n e r g i s m b e t w e e n t h e l y t i c enzymes Elimination of "destructive" protease (for bioactive protein recovery) or supplementation with a d d i t i o n a l proteases ( f o r f o o d and f e e d a p p l i c a i t o n s ) The a d d i t i o n o f p r o t e a s e i n h i b i t o r s a t a p o i n t o r p o i n t s d u r i n g l y s i s , e f f e c t i v e l y l o w e r i n g k p o r i n c r e a s i n g K^p. Osmotic b u f f e r i n g s t r a t e g i e s f o r recovery of b i o a c t i v e p r o t e i n from d i f f e r e n t s i t e s i n the c e l l . C e l l F r a c t i o n a t i o n S i m u l a t i o n . The w a l l p r o t e i n , c y t o s o l and o r g a n e l l e s o f y e a s t e a c h c o n t a i n enzymes w h i c h a r e found nowhere else i n the c e l l . Some e x a m p l e s o f t h e s e e n z y m e s i n c l u d e i n v e r t a s e i n t h e w a l l s , g l y c o l y t i c pathway enzymes i n t h e c y t o s o l and f u m a r a s e i n t h e m i t o c h o n d r i a (13) . A m o d e l o f r e c o v e r y o f t h e s e enzymes i s offered here. Enzyme a c c u m u l a t i o n . F o r the purpose of s i m u l a t i o n , w a l l - l i n k e d a n d p e r i p l a s m i c e n z y m e s (WE) a r e c o n s i d e r e d t o b e a p a r t o f t h e outer wall protein. C y t o p l a s m i c a n d m i t o c h o n d r i a l e n z y m e s (CE,ME) a r e a s s u m e d t o b e some f r a c t i o n o f t h e c y t o p l a s m i c a n d m i t o c h o n d r i a l mass, r e s p e c t i v e l y . The e q u a t i o n s d e s c r i b i n g t h e i r r e l e a s e and h y d r o l y s i s are e x a c t l y analogous t o equation 6 f o r t o t a l l o n g - c h a i n protein. d(WE) dt

L

ewp

d(CE) dt d (ME) dt Variables

W ^ r m

and parameters

d(WP) dt

(WE) Ρ

Lies)' dt

CE Ρ

PBR

(8)

'PBR

[max(0,0.3-0SM )]'MI - M l x

(9) .PBR

(10)

are included i n Table I I I .

F i g u r e 8 shows a s i m u l a t i o n o f enzyme r e c o v e r y f r o m t h e w a l l , c y t o s o l and m i t o c h o n d r i a . The c o n c e n t r a t i o n s o f r e c o v e r a b l e enzyme a r e n o r m a l i z e d t o t h e i n i t i a l amount o f enzyme p r e s e n t i n t h e c e l l site. The c u r v e s r i s e a s enzyme i s r e l e a s e d f r o m a s i t e , t h e n f a l l as i t i s h y d r o l y z e d . I t may b e s e e n t h a t t h e l y t i c s y s t e m i s u s a b l e even as a crude p r e p a r a t i o n t o r e c o v e r w a l l l i n k e d yeast enz ymes i n 60 t o 8 0 % y i e l d . The y i e l d o f y e a s t w a l l enzyme d e p e n d s on

HUNTER AND ASENJO



100

Figure

8

Release o f s i t e

8a 8b

Initial Initial — — ·=

- specific

enzymes - S i m u l a t i o n

y e a s t c o n c e n t r a t i o n , 0.78 g/1 y e a s t c o n c e n t r a t i o n , 36.33 g/1 W a l l enzyme Cytoplasmic M i t o c h o n d r i a l enzyme

enzyme


26


p r o t e a s e a c t i v i t y and i s t h e r e f o r e d i r e c t l y r e l a t e d t o t h e q u a n t i t y of " d e s t r u c t i v e " protease present i n t h e l y t i c system. The p r o d u c t p u r i t y depends on t h e r e l e a s e o f p r o t e i n s f r o m o t h e r c e l l s i t e s , and hence on o s m o t i c f a c t o r s . A t t h e h i g h e r y e a s t c o n c e n t r a t i o n ( F i g . 8 b ) , few o f t h e p r o t o p l a s t s and none o f t h e m i t o c h o n d r i a r e l e a s e t h e i r enzymes i n t o s o l u t i o n . W h i l e t h e y i e l d o f w a l l enzyme i s n o t as good a s i n F i g u r e 8 a , t h e p u r i t y i s f a r h i g h e r . W i t h p r o p e r o s m o t i c s u p p o r t d u r i n g l y s i s , and b r e a k a g e o f o s m o t i c a l l y s t a b l e p r o t o p l a s t s by m e c h a n i c a l means, t h e w a l l , c y t o p l a s m i c and m i t o c h o n d r i a l f r a c t i o n s c a n be o b t a i n e d s e p a r a t e l y . A s i m u l a t i o n of s i t e - l i n k e d product recovery i s presented i n F i g u r e 9 a n d T a b l e s I V a n d V. The c a l c u l a t i o n s assume t h a t s i t e l i n k e d e n z y m e s WE, C E , a n d ME c o n s t i t u t e 1% o f t h e w a l l p r o t e i n , c y t o s o l and m i t o c h o n d r i a r e s p e c t i v e l y . I n t h e f i r s t l y s i s s t e p , u s i n g 2 0 % l y t i c enzyme b r o t h and o s m o t i c s u p p o r t , 9 3 % o f t h e w a l l p r o t e i n ( a n d w a l l enzyme) i s r e l e a s e d f r o m t h e c e l l w a l l . Some i s h y d r o l y z e d by t h e " d e s t r u c t i v e " p r o t e a s e , b u t 73.8% o f i t s u r v i v e s t o be r e c o v e r e d a t t h e end o f t h e f i r s t h o u r . S i n c e o n l y 3% o f t h e protoplasts burst during t h i s step, l i t t l e cytoplasm i s released. T h e p r o t e i n c o n c e n t r a t i o n i n s o l u t i o n a t t h e e n d o f t h e h o u r i s 3.83 g/1 o f w h i c h a b o u t 1% i s WE. I f t h e c e l l s were broken mechanically, t h e w a l l enzyme w o u l d c o n s t i t u t e o n l y 0.35% o f t h e t o t a l p r o t e i n , even assuming t h a t i t c o u l d be c o m p l e t e l y s o l u b i l i z e d . The s i m u l a t i o n a l s o s h o w s a r a t i o o f WE t o CE o f 7.8 i n t h e m e d i u m a t t h e e n d o f t h e f i r s t s t e p , w h i c h c o m p a r e s t o a r a t i o o f 0.258 o n a t o t a l - c e l l basis. Table IV. First

Process

lysis

c o n d i t i o n s f o r enzyme r e l e a s e s i m u l a t i o n

step:

Yeast Enzyme Buffer T o t a l volume

36.33g 40% 0.3 O s / L 1L

R e a c t i o n m i x t u r e c e n t r i f u g e d ; '5% o f s u p e r n a t a n t a n d 1 0 0 % o f p e l l e t r e t a i n e d and resuspended i n t w i c e t h e i n i t i a l volume of enzyme/buffer s o l u t i o n . Second l y s i s

step:

Digested yeast: Enzyme Buffer T o t a l volume Breakage:

by s t i r r i n g

26.90g 20% 0.3 O s / L 2-L o r p a s s a g e t h r o u g h a pump

Protoplast rupture Mitochondrial rupture

95% 0%

HUNTER AND ASENJO


25

A)

AFTER PROTOPLAST RUPTURE f

CS

CS 60

120


TIME-MINUTES B) \WP

\ LYSIS STEP 2 \ \

AFTER 1 PROTOPLAST RUPTURE

\

Ml

\

\

ο

\WG \

LYSIS STEP 1

\

\ Ν

\

«s.

Ml

Ml

< i 60

120

T I M E - M INUTES C)

100 CE

Lu > Ο /WE .*

50

Lu /

WE

f c

! 0

60

120

T I M E - MINUTES Figure 9 9a

Enzyme r e c o v e r y Cell

from s u b c e l l u l a r

s t r u c t u r e breakdown Wall protein Cytosol —

j

9b 9c

structures

- - Wall glucan • —Mitochondria

C e l l s t r u c t u r e : Close-up, showing m i t o c h o n d r i a Enzyme r e l e a s e , p e r c e n t o f o r i g i n a l enzyme i n c e l l W a l l enzyme C y t o p l a s m i c enzyme

28


Table Initial

End o f first step

Start of second step

End o f second step

After protoplast rupture

36332 8.1 760.7

26902 3831.5 2135.0

26902 191.6 106.7

21660 966.9 684.9

4766 7307.3 2661.5

655.0

4449.8

222.5

3321.4

8634.7

5268.2 6983.0 20389

371.0 3380.2 19779

371.0 908.9 19779

0.028 908.9 17783

0.028 908.9 889.2

II

2780.1

2696.9

2696.9

2424.7

121.0

II

0

83.2

83.2

355.4

2659.1

mg/1

0

38.6

.97

4.18

4.18

II

0

4.95

.12

15.24

184.2

II

0

0

0

0

0


Yeast s o l i d s mg/1 S o l u b l e p r o t e i n It Soluble peptides " Soluble II carbohydrates

Wall protein Wall glucan Cytosol Mitochondria (internal) Mitochondria (released)

W a l l enzyme Cytoplasmic enzyme Mitochondrial enzyme

V. Y i e l d s

II II It

T h e s e c o n d d i g e s t i o n was i n c l u d e d t o d e c r e a s e t h e amount o f s t r u c t u r a l g l u c a n from about 50% t o about 13% o f i t s o r i g i n a l mass, i n o r d e r t o make t h e c e l l s m o r e f r a g i l e , t h u s e a s i e r t o r u p t u r e mechanically. O n l y a s m a l l amount o f c y t o p l a s m i c p r o t e i n i s r e leased from the p r o t o p l a s t s during t h i s time - a d e s i r a b l e r e s u l t , s i n c e p r o t e i n s e q u e s t e r e d i n s i d e t h e p r o t o p l a s t s i s n o t a t t a c k e d by protease. A t t h e end o f t h e second h o u r t h e r e m a i n i n g p r o t o p l a s t s c a n be b r o k e n m e c h a n i c a l l y by s t i r r i n g o r c e n t r i f u g a t i o n . P r o t e a s e a c t i v i t y c a n be m i n i m i z e d by k e e p i n g t h e t e m p e r a t u r e l o w . Assuming t h a t 95% o f t h e p r o t o p l a s t s (and none o f t h e s t u r d i e r mitochondria) a r e b r o k e n b y s t i r r i n g , t h e f i n a l p r o t e i n c o n c e n t r a t i o n i s 7.3 g / 1 , o f w h i c h 2.5% i s c y t o p l a s m i c enzyme. Almost a l l of the mitochondria (95.6%) a r e r e l e a s e d d u r i n g t h e second l y s i s and p r o t o p l a s t r u p t u r e , but they remain whole because t h e b u f f e r o s m o l a l i t y i s kept above 0.3 O s / L . C e n t r i f u g a t i o n o f t h e f i n a l m i x t u r e p r o d u c e s 4.77 g/1 o f a p e l l e t , o f w h i c h 2.66 g o r 5 6 % i s m i t o c h o n d r i a a n d 0.89 g o r 19%, p r o t o p l a s t s . These s i m u l a t i o n s suggest an a d d i t i o n a l t e s t f o r t h e s t r u c t u r e d model: t h a t i s , t o compare i t s p r e d i c t i o n s t o d a t a on r e l e a s e o f s i t e - l i n k e d enzymes i n y e a s t . T e s t s f o r c y t o p l a s m i c and m i t o c h o n d r i a l enzyme r e l e a s e w i l l b e a i d e d by p r e p a r a t i o n o f a l o w - p r o t e a s e l y t i c system.

2.

HUNTER AND

ASENJO



Conclusions The s i m p l e m o d e l i s c o n c e p t u a l l y s t r a i g h t f o r w a r d a n d g i v e s a n approximate f i t t o the data over the e n t i r e range o f v a r i a b l e s s t u d i e d : y e a s t c o n c e n t r a t i o n , enzyme c o n c e n t r a t i o n a n d t i m e . The p r o d u c t d i s t r i b u t i o n depends on the r e l a t i v e r a t e s o f l y s i s and proteolysis. U s i n g a s i n g l e enzyme p r e p a r a t i o n , a s was done h e r e , the r e l a t i v e r a t e s change o n l y w i t h y e a s t c o n c e n t r a t i o n . I n prac t i c e , however, i n h i b i t i o n o f the p r o t e a s e a c t i v i t y , supplementation of the l y t i c a c t i v i t y w i t h p u r i f i e d glucanases o r m i x i n g o f l y t i c systems from d i f f e r e n t s o u r c e s can b r i n g about l a r g e changes i n t h e a c t i v i t y r a t i o , w h i c h may b e i n c o r p o r a t e d i n t o t h e s i m p l e m o d e l by a d j u s t i n g and k . The s t r u c t u r e d m o d e l i s c o n s i s t e n t w i t h f e a t u r e s o f l y t i c e n zyme a c t i o n a n d y e a s t s t r u c t u r e r e p o r t e d i n t h e l i t e r a t u r e . T h e s e q u e n t i a l r e m o v a l o f the two w a l l l a y e r s , f o l l o w e d b y p r o t o p l a s t r u p t u r e , a c c u r a t e l y d e s c r i b e s the e a r l y l a g i n p r o t e i n and carbo hydrate release. The p r e s e n c e o f r e s i d u a l s o l i d s a t l o n g r e a c t i o n t i m e s was a c c o u n t e d f o r s t a b i l i z a t i o n o f p r o t o p l a s t s b y s u b s t a n c e s released from l y s e d c e l l s . The s t r u c t u r e d model can be used t o e s t i m a t e t h e e f f e c t s o f s e v e r a l p r o c e s s a l t e r n a t i v e s , a s shown i n a s i m u l a t i o n o f a p r o c e s s f o r r e c o v e r y o f s i t e - l i n k e d enzymes f r o m yeast. r

Acknowledgments T h i s work was s u p p o r t e d b y a g r a n t f r o m the N a t i o n a l S c i e n c e F o u n d a t i o n , (NSF) t o whom t h a n k s a r e d u e . One o f t h e a u t h o r s (JBH) was s u p p o r t e d b y g r a d u a t e f e l l o w s h i p s f r o m NSF a n d t h e J o s e p h i n e de Karman F o u n d a t i o n d u r i n g p a r t o f t h i s work. This support i s a l s o g r a t e f u l l y acknowledged.

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Received March 26, 1986

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