7 Enzymes: Specific Tools Coming of Age DONALD F. DURSO and ANITA VILLARREAL Department of Forest Science, College of Agriculture, Texas A&M University, College Station, Tex. 77840
Abstract C e l l u l o s e chemistry has long represented a difficult battleground f o r researchers who have s u c c e s s f u l l y a p p l i e d science to other fileds. The seeming s i m p l i c i t y and the true complexity of the c e l l u l o s e "package"---the f i b e r s t r u c t u r e---have served to entrap those who would unravel its mysteries and use if efficiently. Combining the mysteries of c e l l u l o s e and enzymes has produced an area c o n t a i n i n g l i t t l e f a c t and much fiction. The s t u d i e s of t h i s paper provide methods to p r e d i c t enzymatic h y d r o l y s i s of c e l l u l o s e and i n d i c a t e a p r a c t i c a l method f o r r a p i d conversion of cellulosic substrates i n t o glucose. * * * As p a r t of a p r o j e c t aiming a t some u s e f u l d i s p o s i t i o n for mesquite, means have been devised f o r the production and the c h a r a c t e r i z a t i o n of a potent c e l l u l a s e system obtained as an exo-enzyme from Trichoderma viride (1, 2). A sample of the enzyme and the p r e f e r r e d s t r a i n of T. v i r i d e were k i n d l y s u p p l i e d by Mary Mandels s e v e r a l years ago. The methods to produce the cell-free enzyme and to measure its activity are e s s e n t i a l l y those revealed by Mandels (1,3). The r e s u l t s presented i n t h i s paper stem from an e f f o r t to standardize the enzyme system so that i t can be used as a r e l i a b l e , r e p r o d u c i b l e y a r d s t i c k to measure progress i n the p r o j e c t to make mesquite wood t o t a l l y a c c e s s i b l e to c e l l u l a s e s . Although t h i s work i s based on pure c e l l u l o s e ( f i l t e r paper) and c e l l - f r e e enzyme, the conclusions appear to be a p p l i c a b l e to the d i g e s t i o n of l i g n o c e l l u l o s e m a t e r i a l by i n t a c t organisms i n the rumen of c a t t l e and sheep ( 4 ) . Methods devised f o r the c o n c e n t r a t i o n of the enzyme, the measurement of i t s a c t i v i t y v i a DNS c o l o r r e a c t i o n and the assay of i t s a c t i o n on f i l t e r paper are o u t l i n e d i n Appendix B. With the standard enzyme c o n c e n t r a t i o n , the r a t e of weight l o s s i s shown as the "Normal" curve i n F i g . 1. The n o n - l i n e a r e f f e c t of changing only the enzyme concentration i s a l s o i n d i c a t e d . Here the response seems to be l o g a r i t h m i c , agreeing 106
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HYDROLYSIS F I L T E R
PAPER
DAY Figure 1.
"NormaF' curve for weight loss rate
ι
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12
14
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D A Y
Figure 2.
Decrease in enzyme activity
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with some (5) while disagreeing with others (6); i n any case, s e t t i n g the enzyme a c t i v i t y at a d e s i r e d l e v e l (DNS = 2.0 mg of glucose) r e q u i r e s t r i a l and e r r o r r a t h e r than c a l c u l a t i o n . In attempting to understand the shape of the weight l o s s curve and to develop methods f o r p r e d i c t i n g substrate r e a c t i v i t y , c e r t a i n r e s u l t s were obtained and are presented i n t h i s paper i n the hope of s t i m u l a t i n g d i s c u s s i o n and s t u d i e s by others. A knowledge of the i n t e r a c t i v e e f f e c t s of f a c t o r s such as surface area, p u r i t y , a c c e s s i b i l i t y , and concentration of the substrate upon enzyme a c t i v i t y i s b a s i c i n attempts to use enzymes i n a p r a c t i c a l manner. Without i t , there can be only incomplete understandings of complex mixtures (_7) or highl y d e t a i l e d a t t e n t i o n to s e l e c t e d s i n g l e f a c t o r s (8). The l a t t e r are subject to i n s t a n t c o n t r a d i c t i o n (9) while the former can only be taken as a whole which d e f i e s a n a l y s i s . Following the l e a d of Z e f f r e n and H a l l (10), i t was discovered that the a c t i v i t y of the enzyme decreases as shown i n F i g . 2 i f i t i s merely heated i n the absence of substrate. Some p o r t i o n of the c e l l u l o l y t i c a b i l i t y of the enzyme d i s appears i n a p r e d i c t a b l e manner such that the known a c t i v i t y at the s t a r t of the r e a c t i o n can be used to c a l c u l a t e the r e s u l t s of h y d r o l y s i s t e s t s as shown i n Table 1. However, i t i s not p o s s i b l e to p r e d i c t the d a i l y increment of weight l o s s using only the enzyme a c t i v i t y and the substrate concentration a t any given day. In a d d i t i o n , i f one c a l c u l a t e s the incremental increase i n weight l o s s by an apparently l o g i c a l method (See Case I , Appendix A), i t i s found to be much l a r g e r than the a c t u a l increment. On the other hand, i f the b a s i c increment i s chosen a f t e r the major r a t e change between Days 0 and 2 (See Case I I , Appendix A) a l l of the curve i n F i g . 1 i s p r e d i c t a b l e . But, t h i s represents an e m p i r i c a l approach rather than r i g o r o u s c a l c u l a t i o n . Another f a c t o r to be considered i s the p o s s i b l e i n h i b i t i o n of enzymatic a c t i o n by the accumulation of the end-product, glucose, i n the h y d r o l y s i s mixture. I t i s shown i n Figure 3 t h a t , while enzymatic h y d r o l y s i s i s not r e v e r s i b l e , i t i s i n h i b i t e d i n some way by the a d d i t i o n , to the i n i t i a l mixture, of glucose at l e v e l s approximating that a f t e r 2 days or 15 days of h y d r o l y s i s . Further, b a l l - m i l l dust (from s e l f - a t t r i t i o n of g r i n d i n g media) i s shown i n Figure 4 to cause a s l i g h t reduct i o n i n h y d r o l y s i s r a t e . However, s i n c e here the r a t i o of enzyme to substrate i s a c t u a l l y 2X that i n the standard, the weight l o s s should have been 1.3X r a t h e r than 0.9X STD; thus, t h i s i n e r t component i s " d i s t r a c t i n g " the enzyme. From these i n h i b i t i o n data, another e m p i r i c a l approach to p r e d i c t a b i l i t y i s i n d i c a t e d . The d a i l y increments, a f t e r Day 1, were read f o r a l l the runs i n F i g u r e 3. Then they were p l o t t e d against the glucose concentration during that day and the i n t e r e s t i n g r e s u l t i s shown i n Figure 5. I t must be remembered that d i f f e r e n t substrate and glucose concentrations,
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Table I.
REACTION TIME,
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Enzymes
Enzyme Activity on Filter Paper
ENZYME CONDITION** AT START OF HYDROLYSIS VS. WT. LOSS
DAYS
FRESH, S TP.
4
41.1
4-DAY** F 29.5
10-DAY** C*" f
29.4
15-DAY** F 21.8
22.5 28.0
7
53.1
28.1
10
59.9
31.7
30.9
15
70.2
37.2
35.4
DNS ACTIVITY
50.4
47.4
1.22
1.7
38.0
41.3
0.9
1.0
* CALCULATED » STD. VALUE X DNS ACTIVITY * 1.7 e
** PRE-HEAT DAYS AT 50 C, pH 5 IN SEALED CONTAINER
Figure 3.
Inhibition of enzymatic
hydrolysis
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•o
DAYS Figure 4.
Cellulase hydrolysis of filter paper.Effect of ball-mill "rocW
9
20
40 GLUCOSE
Figure 5.
60 IN
REACTION
80 M I X , *
Glucose concentration vs. daily hydrolysis increment
100
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and enzyme a c t i v i t i e s are included. Yet, using only the glucose concentration at any given degree of h y d r o l y s i s to read the next d a i l y weight l o s s increment, i t becomes p o s s i b l e to pre d i c t the e n t i r e "Normal" curve of Figure 1 from Day 1 through Day 15. Further, i t i s i n t e r e s t i n g to note that t h i s r e l a t i o n (Figure 5) p r e d i c t s the c e s s a t i o n of h y d r o l y s i s at the 80% level. T h i s Figure 5 a l s o l e d to the conclusion that something v i t a l l y d e c i s i v e happens during the f i r s t day of h y d r o l y s i s . Therefore, the r e a c t i o n s were c a r r i e d out at s h o r t e r i n t e r v a l s and the data are given i n Figure 6. I t i s c l e a r that a remarkable decrease i n substrate degradation occurs a f t e r the f i r s t hour. In t h i s f i r s t hour, there has occurred only a minor change i n each of the primary f a c t o r s (DNS enzyme a c t i v i t y and substrate concentration) and the glucose e f f e c t i s minor (note that even 70% added glucose only s l i g h t l y a f f e c t s the i n i t i a l h y d r o l y s i s r a t e ) ; yet the hourly increment drops r a p i d l y from almost 9% to much l e s s than 1%. In the hopes of b e t t e r d e f i n i n g the p o s s i b l e e f f e c t s of substrate s t r u c t u r e or enzyme d e s t r u c t i o n (absorption?), the experiments summarized i n Figure 7 were c a r r i e d out. The standard was continued f o r 5 hours while each of 12 other r e a c t i o n s were given a l t e r n a t e treatments at the end of the 3rd hour. In A, the residue was recovered by f i l t r a t i o n and washing; then, assuming about 10% weight l o s s , i t was r e dispersed i n 90% of the i n i t i a l volume of enzyme to r e s t o r e the o r i g i n a l substrate to enzyme r a t i o . This was done i n d u p l i c a t e and the r e a c t i o n progress measured at the end of 1 and 2 more hours. In B, 100% f r e s h enzyme was added to s e l e c t e d tubes and again these were analyzed at 1 and 2 hours. In C, 100% f r e s h substrate was added and then the analyses were c a r r i e d out as before. In Figure 7, A i n d i c a t e s that enzymatic h y d r o l y s i s had been stymied by some attack on a f a c t o r i n the enzyme r a t h e r than i n the s u b s t r a t e . Β and C are somewhat confused by the presetnce of glucose and " o l d " substrate and enzyme; however, from the appearance of the r e a c t i o n mix i t can be deduced that r e a c t i o n had ceased i n C a f t e r about 1 hour while Β s t i l l con tinued at a renewed pace. I t i s thus i n f e r r e d but not proven that some secondary e f f e c t of substrate on the enzyme system i s mainly r e s p o n s i b l e f o r the l o s s of the i n i t i a l r a t e of r e a c t i o n . Substrate s t r u c t u r e per se, i f important i s minor under the c o n d i t i o n s of these experiments. Conclusions There are some p o s i t i v e f e e l i n g s from these s t u d i e s d e s p i t e the doubts and questions which r e q u i r e f u r t h e r work. I t i s p o s s i b l e by r i g o r o u s and e m p i r i c a l methods, combined, to
Figure 6.
First-hour glucose effect
Figure 7.
Results of enzymatic hydrolysis experiments
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p r e d i c t the r a t e of h y d r o l y s i s of c e l l u l o s e from about 10% r e a c t i o n to i t s end. I t i s shown that glucose, rock and other a t t r a c t i o n s f o r the enzyme w i l l d i s t r a c t i t from i t s purpose. I t appears these f i n d i n g s can be put to use f o r the production of glucose a t very p r a c t i c a l r a t e s , i f one can maint a i n the i n i t i a l c o n d i t i o n s . These can be obtained by 1. D i a l y s i s to maintain zero glucose i n the r e a c t o r . 2. A d d i t i o n of substrate at the r a t e i t i s h y d r o l y z i n g . 3. A d d i t i o n of f r e s h enzyme to c o r r e c t f o r a c t i v i t y l o s s . On t h i s b a s i s the data i n t h i s paper can be used by p r a c t i c a l chemists or chemical engineers to design a r e a c t o r i n t o which one would add continuously the raw m a t e r i a l s and continuously remove the glucose through a s u i t a b l e membrane. I f there i s need f o r such glucose, economic and p i l o t p l a n t s t u d i e s can now begin to develop t h i s system. I t w i l l give continuous r e d u c t i o n of carbohydrate wastes to glucose i n hours, r a t h e r than the u s u a l l y p r e s c r i b e d days of r e a c t i o n time (11). Literature Cited 1.
2. 3.
4. 5.
6.
7. 8. 9. 10.
11.
V i l l a r r e a l , A n i t a , The A c t i o n of T. v i r i d e C e l l u l a s e on P u r i f i e d and P a r t i a l l y P u r i f i e d C e l l u l o s i c Substrates, M.S. T h e s i s , Texas A&M U n i v e r s i t y , December 1972. G o l d s t e i n , I r v i n g S. and V i l l a r r e a l , A n i t a , Wood S c i . (1972), 5, 15-20. Mandels, Mary and Weber, James, The Production of C e l l u l a s e s , i n " C e l l u l a s e s and t h e i r A p p l i c a t i o n s , Advances in Chemistry S e r i e s 95", 391-414, American Chemical Society, Washington, D.C., 1969. Mellenberger, R.W., e t a l , J . Animal S c i . (1970), 30, 1005-1011. Reese, E.T. and Mandels, Mary, Enzymatic Degradation, i n " C e l l u l o s e and C e l l u l o s e D e r i v a t i v e s , Part V", 1088, WileyInterscience , New York, 1971. H a l l i w e l l , G., Measurement of C e l l u l a s e and Factors A f f e c t i n g i t s A c t i v i t y , i n " E n z y m i c H y d r o l y s i s of C e l l u l o s e and Related M a t e r i a l s , " 83, Pergamon, London, 1963. Davis, C . L . , et al, Conference on C e l l u l o se Utilization in the Rumen, Federation Proc. (1973), 32, 1803-1825. C a u l f i e l d , D.F. and Moore, W.E., Wood S c i . (1974), 6, 375-379. McKeown, J.U. and Lyness, W.I., J . Polymer S c i . (1960), 47, 9-17. Z e f f r e n , E. and H a l l , P.L., K i n e t i c s I & I I I , in "The Study of Enzyme Mechanisms," 62; 63-67; 87-95, W i l e y - I n t e r s c i e n c e , New York, 1973. Anon., C&EN, May 27, 1974, page 20; Moore, W.E., et a l , J . Agr. Food Chem. (1972), 20 ( 6 ) , 1173-5.
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Appendix A C a l c u l a t i o n o f D a i l y Weight Loss Increment (Example: Day 7 to Day 8 ) . I. Using Day 0 to Day 1 as the b a s i c increment Enzyme A c t i v i t y (DNS): I n i t i a l = 1.7; Day 7 = 1.08 Substrate Concentration: At Day 7 = 48% o f O r i g i n a l I n i t i a l Increment = 25% Weight Loss C a l c u l a t e d Day 7 to Day 8 Increment = I n i t i a l Increment X R e l a t i v e Substrate Cone. X Enzyme Activity = 25 X 0.48 X 1.08/1.7 = 7.62% II.
Using Day 2 t o Day 3 as the b a s i c increment Enzyme A c t i v i t y (DNS): Day 2 - 1.38; Day 7 = 1.08 Substrate C o n e : At Day 2 = 69%; A t Day 7 = 48% Basic Increment = 37-31 = 6% D a i l y Weight Loss C a l c u l a t e d Day 7 to Day 8 Increment - 6 X 48/69 X 1.08/1.38 - 3.27%
Note:
Using the case I I procedure, the e n t i r e Normal weight l o s s curve o f F i g u r e 1 can be constructed from Day 4 through Day 15.
Appendix Β Enzyme P r e p a r a t i o n . Per the procedure i n Reference 3 except that 14.7 g/1 o f sodium c i t r a t e were added to the n u t r i e n t medium. Enzyme Concentration. I f the enzyme s o l u t i o n needs to be concentrated, the Amicon u l t r a f i l t r a t i o n apparatus i s used. Nitrogen i s used as the source o f pressure. The f i l t e r used i s an Amicon membrane (PM 30, 62mm). The enzyme s o l u t i o n i s t r a n s f e r r e d to the r e s e r v o i r tank. The n i t r o g e n i s connected to the i n l e t o f the r e s e r v o i r tank. The o u t l e t connection i s attached to the f i l t e r i n g c e l l which has the membrane i n p l a c e (glossy s i d e up). The o u t l e t hose of the f i l t e r i n g c e l l , i s allowed to s i t i n a c o l l e c t i n g f l a s k . The r e s e r v o i r tank i s pressured to 60 p s i with n i t r o g e n and then the n i t r o g e n source i s removed. The s t i r r e r i s turned on a t a speed which produces a v o r t e x o f 1/3 the volume i n the c e l l . When the d e s i r e d volume has been c o l l e c t e d , the s t i r r e r i s stopped and the vent o f the r e s e r v o i r tank i s r e leased slowly. A f t e r the pressure i s completely down, the s t i r r e r i s turned on a t a slow speed f o r about 5 minutes. The concentrated enzyme i n the c e l l and the tank i s saved and
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refrigerated.
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Adjust the pH to 4.8-5.0 before using.
C e l l u l a s e Assay by DNS Color Reaction Per the procedure i n Reference 3. C e l l u l a s e Assay by Weight Loss Method Apparatus Screw-cap c u l t u r e tubes, 30 ml. 20x150 mm Tared s i n t e r e d g l a s s c r u c i b l e s , 30ml, coarse p o r o s i t y 50°C H ° Vortex mixer b
a
t
h
2
Reagents Trichoderma v i r i d e c e l l u l a s e adjusted to a pH of 5.0 and approximate a c t i v i t y of 2.0 mg glucose (DNS method). The DNS a c t i v i t y l e v e l i s obtained by concentration or d i l u t i o n . Procedure 1.
2. 3. 4.
Weight out 0.5 g of 1x6 cm Whatman #1 f i l t e r paper s t r i p s . Place i n screw-cap c u l t u r e tubes ( s t e r i l i z a t i o n not necessary) as i n t a c t s t r i p s . Add 10 ml of T. v i r i d e c e l l u l a s e (pH 5.0 and 2.0 mg activity). Incubate a t 50°C f o r 15 days and mix d a i l y on a v o r t e x mixer. At the end of the i n c u b a t i o n p e r i o d , f i l t e r the r e s i d u e on a tared s i n t e r e d glass c r u c i b l e , r i n s e w e l l with c o l d water, and oven dry a t 105°C. Determine the weight l o s s as a percentage of the o r i g i n a l sample weight.
