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The Effect of a Purified Guar Degrading Enzyme on Chick Growth1
The Effect of a Purified Guar Degrading Enzyme on Chick Growth1 SWAPNA RAY, MERTON H. PUBOLS, and JAMES MCGINNIS Department of Animal Sciences, Washington State University, Pullman, Washington 99164 (Received for publication July 6, 1981) ABSTRACT Guar gum, a galactomannan, is growth depressing when fed to chicks. As part of the continuing studies on the mode of action of this and other gums in the intestine, a mannanase has been isolated which degrades guar gum. A commercial hemicellulase was separated into several protein fractions by chromatography on DEAE-cellulose. Only one of these proteins effectively reduced the viscosity of a guar gum solution. Activity towards pectin, rye water extract, and barley water extract was not present in the fraction which degraded guar gum but was found in other fractions at lower concentrations. Graded levels of this purified guar-degrading enzyme were incorporated into chick diets having 2% guar gum. This purified enzyme was effective at levels as low as .6 mg protein/kg diet in preventing growth depression caused by the guar gum and thus permitting growth equivalent to controls. Purity of this mannanase was confirmed by chromatography on QAE-Sephadex at pH 6.0 and by slab-gel electrophoresis at pH 8.4. (Key words: guar gum, guar-degrading enzyme, chick growth, sticky feces) 1982 Poultry Science 61:488-494 INTRODUCTION Guar meal from t h e guar bean (Cyamopsis tetragonoloba) has been used in p o u l t r y rations, b u t it has a deleterious effect on growth rate and feed efficiency for broiler chicks (Borcher and Ackerson, 1 9 5 0 ; V o h r a and Kratzer, 1964a; Saxena and Pradhan, 1 9 7 4 ; T h a k u r a n d Pradhan, 1 9 7 5 ; T h a k u r and Saxena, 1976). Guar gum, a g a l a c t o m a n n a n from t h e endosperm (Whistler and H y m o w i t z , 1 9 7 9 ) , is p r o b a b l y t h e detrimental c o m p o n e n t (McCleary, 1979; White et al, 1978). It is one of m a n y p l a n t gums which causes growth depression when fed t o chicks. O t h e r s include locust bean gum, g u m tragacanth, gum karaya, and carrageenin ( V o h r a and Kratzer, 1 9 6 4 b ) . Feeding guar gum reduced nitrogen r e t e n t i o n , fat absorption, and metabolizable energy (Kratzer et al, 1967). A d d i t i o n of 2% guar gum t o a corn diet reduced liver fat, serum cholesterol, and b o d y weight in chicks (Patel et al, 1 9 8 1 ) . The deleterious effects of guar gum can be reduced by several m e t h o d s . O n e is t o treat t h e guar gum with enzymes from either s p r o u t e d guar beans o r from a commercial source (Anderson and Warnick, 1 9 6 4 ; V o h r a and Kratzer, 1965). A commercial pectinase also was effec-
1 Scientific Paper No. 5959. College of Agriculture Research Center, Washington State University, Pullman, WA. Project No. 0311.
tive (Patel et al, 1980). A n o t h e r m e t h o d is t o subject guar gum to gamma-irradiation. If this latter t r e a t m e n t was c o m b i n e d with the pectinase, then the g r o w t h depression was nearly eliminated (Patel et al, 1980). These observations indicate t h a t the d e t r i m e n t a l effects of guar gum are i n h e r e n t t o the polysaccharide structure and can be elimimated by the action of enzymes t h a t degrade it. T h e means by which guar gum causes detrimental effects w h e n fed t o t h e chicks is n o t k n o w n . T h e effects are observed only with guar g u m t h a t has n o t been degraded by enz y m e s or gamma-irradiation. O n e approach t o t h e d e t e r m i n a t i o n of the m o d e of action of guar gum is t o establish the m i n i m u m chain length of guar gum which can cause depression. A m a n n a n a s e was purified which degrades t h e guar g u m , as measured by r e d u c t i o n of viscosity. This purified e n z y m e was fed t o chicks and found t o inactivate t h e g r o w t h depressing p r o p e r t i e s of this g a l a c t o m a n n a n .
MATERIALS AND METHODS A guar-degrading e n z y m e was purified from a commercial hemicellulase p r e p a r a t i o n by ion-exchange c h r o m a t o g r a p h y , as assayed by t h e reduction in viscosity of a guar gum solution. This purified m a n n a n a s e was added to chick diets having 2% guar gum. Enzyme. A hemicellulase p r o d u c t , CE1 0 0 , 0 0 0 , was o b t a i n e d from Miles Laboratories, Elkhart, IN. It is 4 3 . 6 % p r o t e i n (N X 6.25).
488
PURIFIED GUAR DEGRADING ENZYME AND CHICK GROWTH Substrates. Guar g u m was o b t a i n e d from either Sigma Chemical C o m p a n y , St. Louis, MO (catalogue # G - 4 1 2 9 ) or Celanese Specialties C o m p a n y , Louisville, KY. Pectin was obtained from Sunkist C o m p a n y , Ontario, CA. R y e extract was p r e p a r e d b y stirring g r o u n d rye with 10 volumes (w/v) of 80% e t h a n o l and heating t o boiling. After cooling t o r o o m t e m p e r a t u r e , t h e suspension was filtered through Whatman # 1 filter paper using a Buchner funnel. T h e residue collected by filtration was e x t r a c t e d with 10 volumes of water b y heating t o 70 to 8 0 C, t h e n cooling t o r o o m t e m p e r a t u r e before centrifuging at 1 0 , 0 0 0 X g. T h e s u p e r n a t a n t was used for viscosimetric assays. Barley e x t r a c t was prepared by t h e same p r o c e d u r e . Ion-Exchange Chromatography. The hemicellulase p r e p a r a t i o n ( C E - 1 0 0 , 0 0 0 ) (64.3 g, 5 6 9 0 total A 2 8 o ) was dissolved in 2 0 0 ml .05 M Tris pH 9.0, t h e n centrifuged at 4 0 0 0 X g for 30 min. T h e clear s u p e r n a t a n t was applied t o a column (7.6 X 7.0 cm high) of DEAE-cellulose (Whatman DE-52, Clifton, N J ) , which had been equilibrated with .05 M Tris buffer p H 9.0. After t h e b r e a k t h r o u g h peak of protein h a d eluted, a . 5 M KC1 gradient was applied t o t h e column. Protein was m e a s u r e d by the absorbance at 2 8 0 n m . T h e fractions were assayed for viscosity reducing activity against the various substrates. Pools of fractions were m a d e
489
according t o e n z y m e activity with Pool C, t u b e s 175 t o 196, having the guar-degrading activity. A p o r t i o n of t h e fraction containing t h e guar-degrading e n z y m e , Pool C (5.3 A 2 8 0 units), was passed through a c o l u m n of G-25 Sephadex which had been equilibrated with .05 M a m m o n i u m acetate, p H 6. T h e p r o t e i n peak was then applied t o a column of QAE-Sephadex (1.0 b y 10 c m ) equilibrated with t h e same buffer. A single, sharp peak was eluted with a .5 M KC1 gradient in .05 M a m m o n i u m acetate, pH 6.0. It had t h e guar-degrading activity, according t o viscosity reduction, and was labeled Pool X. Viscosity. An Ostwald viscometer was used t o assay t h e e n z y m e activity in t h e various fractions. T h e substrates were p r e i n c u b a t e d with t h e fractions for a specified t i m e a t r o o m t e m p e r a t u r e . T h e flow rate was c o m p a r e d to t h e flow rates for water and substrate blanks. T h e results are expressed in t e r m s of the specific viscosity which is defined as:
%p =-2m._i where n m is viscosity of the reaction m i x t u r e and n w is viscosity of water. A l i q u o t s of t h e e n z y m e were selected which w o u l d reduce the flow time t o a b o u t one-half t h a t of t h e s u b t r a t e blank. T h e reciprocal of the specific viscosity, -~— was used t o determine peaks of various r %p
TABLE 1. Composition of the diet Ingredient
(%)
Fish meal (65% protein) Isolated soy protein (88% crude protein) Dehydrated alfalfa meal (17% crude protein) Meat and bone meal (50% crude protein) Soybean meal (47% crude protein) Dicalcium phosphate Iodized salt Vitamin premix a Trace mineral mix"3 DL-methionine Animal fat Total Glucose and/or 2% guar gum
8.00 1.25 2.25 4.00 30.60 .90 .30 .25 .05 .15 2.25 50.00 50.00
Vitamin premix at .25% of the diet supplies the following per kilogram of the diet: vitamin A, 5500 IU; vitamin D 3 , 1650 ICU; vitamin E, 6.4 IU; riboflavin, 3.3 mg; calcium pantothenate, 9.8 mg; niacin, 22 mg; choline chloride, 577 mg; vitamin B 1 2 , .011 mg; biotin, .05 mg; pyridoxine, 2.0 mg; thiamine, 1.0 mg; and ethoxyquin, 62.2 mg. Mineral premix at .05% of the diet supplies the following (mg/kg) of the diet: Mn, 50: Fe, 50; Cu, 5; Zn, 50; I, 1.5; Ca, 60 mg; Co, .5.
RAY ET AL.
490
TABLE 2. Specific activities of the enzymes in the pooled fractions from the DEAE-cellulose
column"
Sample
Tube
Volume (ml)
A 28 „/pool
Guar
Pectin
Barley
Rye
CE-100,000 Pool A Pool B PoolC PoolD PoolE
120-150 152-166 170-196 216-248 293-306
1100 565 1000 96 560
532.4 591.5 1488. 41.8 162.4
121.5 .7 .2 343 .5 1.5
1.7 4.3 .3 4.5 .2 .2
92.4 2.0 3.4 1.7 103.0 275.8
1.1 .4 .3 .3 .1 .2
Specific activity = units/A 2
enzyme activities in the column effluents (Sandegren and Enebo, 1952). The substrate solutions, . 1 % guar gum, .2% pectin, 2% rye water extract, or 2% barley extract were clarified by centrifugation before use in die viscometer. The substrates were dissolved in distilled water. Slab-Gel Electrophoresis. The procedures of Smith (1968) and Maurer (1971) were followed. The samples were dialyzed against water to remove salt, lyophilized to dryness, then resuspended in buffer. A 10% polyacrylamide gel was used in a Beckmann microzone electrophoresis system. The dimensions of the cell were 6.5 X 10 X .4 cm. The gel had eight wells for simultaneous analysis. The amount of protein applied into the wells was as follows: hemicellulase, 97.8 Mg; "Pool C", 99.0 £(g; "Pool X", 59.4 /Ug. A Tris-glycine buffer (pH 8.4) was used in preparing the slab and also served as the electrolyte. Electrophoresis was at 450 V with a current of 44 to 35 raA for 54 min. The gel was stained with Coomassie Brilliant Blue G (Smith, 1968) for 1 hr. The stained gel was rinsed with 2% acetic acid and then electrolytically destained for 1 hr in 2% acetic acid solution. The destained gel was scanned in a Beckman R-110 microzone densitometer at 570 nm. Feeding Trial. A feeding trial was used to test the effectiveness of the purified guardegrading enzyme on growth of chicks fed 2% guar gum. The diet listed in Table 1 is based on the bioassay diet of McGinnis et al. (1980). In this trial, 10 female, day-old broiler chicks were assigned randomly to a pen, and three pens were assigned randomly to each treatment. The birds were maintained in electrically heated battery brooders equipped with raised, wirescreen floors. Feed and water were supplied ad libitum with 14 hr of light per day. The birds
were weighed in groups after 2 weeks. Feed consumption was measured. At the end of the trial, the wire floors were scored visually between 0 and 5 for adhering feces, the higher score indicating a greater amount of feces sticking to the floor. The guar-degrading enzyme in lyophilized Pool C was added to the diet at 5, 10, 15, or 20 mg/kg diet. Since the lyophilized Pool C had only 6.4% protein, this represented .32, .64, .96, or 1.28 mg protein/kg diet, respectively. Data from the experiments were treated by analysis of variance according to Steel and Torrie (1960) and the differences analyzed by multiple range test of Duncan (1955). RESULTS AND DISCUSSION
The guar-degrading enzyme activity was concentrated in Pool C (Table 2) from the DEAE-cellulose ion-exchange fractionation (Fig. 1). This fraction had relatively no activity on pectin, rye water extract, or barley water extract. Activity toward these substrates was found in other fractions. Pool C had 52% of the total eluted protein and represented about a threefold purification of the enzyme (Table 2). Activity toward barley extract was found in Pools D and E, whereas only small amounts of activity towards pectin and rye were detected. The lyophilized powder from Pool C was incorporated into chick diets containing 2% guar gum and was found to be effective in stimulating growth at levels as low as about .6 mg enzyme protein/kg diet (Table 3). The reduction of the sticky feces, as measured by screen score, may require a greater amount of enzyme, approaching 1 mg/kg. The single sharp peak, Pool X, which eluted from QAE-Sephadex (Fig. 2), having 3.4 total A28o units, had the guar-degrading enzyme.
PURIFIED GUAR DEGRADING ENZYME AND CHICK GROWTH
TUBE N O . - * 20 POOL
40
60
80
•
100 120 140
491
160 180 200 220 240 260 280 300 320
I—A—li-BH I—C—I
I—D—I
I-EH
FIG. 1. Fractionation of a hemicellulase by DEAE-cellulose chromatography, pH 9.0. Units for each of the enzymes are reciprocals of the specific viscosities (see text). Conductivity units are mmhos.
Slab-gel electophoresis of this p r o t e i n at p H 8.4 indicated this p r o t e i n to be essentially pure. T h e densitometric scans of t h e e l e c t r o p h o r e t i c p a t t e r n s of t h e proteins during t h e stages of purification are shown in Figure 3. T h e guardegrading e n z y m e in Pool C was a b o u t 6 8 % of t h e dye b o u n d t o the gel, whereas in Pool X
a b o u t 8 3 % was b o u n d . The guar-degrading e n z y m e of t h e commercial hemicellulase C E - 1 0 0 , 0 0 0 is a b o u t 50% of t h e total protein. Guar gum is a p o l y m e r of D-mannose linked (3-1, 4 with D-galactose attached t o alternate m a n n o s e u n i t s ce-1, 6. Presumably, this e n z y m e would degrade o t h e r
TABLE 3. Average weight gain, feed efficiency and screen score (SCSC) of chicks fed diets containing 2% guar gum supplemented with graded levels of pool C from the DEAE-cellulose column Supplement Guar gum
(%)
Enzyme (mg protein/kg)
Gain (g)
Gain/feed
SCSC
None (glucose) 2 2 2 2 2 2
None None + 10.91 HC1 + 0.32 Pool C + 0.64 Pool C + 0.96 Pool C + 1.28 Pool C
168.5 a b 123.2 C 182.6 a 163.lb 167.6 a b 169.5 a b 172.3 a b 5.78
.675 a .509 d .650 b .620° .638 b c .640 b c .643bc 2.12
.67 d 3.50 a 1.33 c d 2.50 b 2.00 b c 1.50 b «l 1.67 b c d 28.42
CV %
a ' ' c ' Means having the same letter in a column do not differ significantly (P>.05) according to Duncan's (1955) multiple range test. 1
Hemicellulase (CE-100,000).
492
RAY ET AL.
>•
=>
i
o o T40
135 30 25 H20
15 10 5 20
40
60
80
100 120 140 160 180 200 220 240
TUBE NUMBER FIG. 2. Further purification of "Pool C" by QAE-Sephadex chromatography at pH 6.0. "Guarase" units are reciprocals of the specific viscosities (see text). Conductivity units are mmhos.
polymers with a similar structure, such as locust bean gum. We could not detect any galactose liberated by the enzyme (M.Pubols, 1981, unpublished data). This strongly suggests that the enzyme is a mannanase, cleaving the polymer randomly. The effect of other fractions purified from this hemicellulase have not been tested in feeding trials with diets having pectin, barley, or rye. However, based on the relative rate of reduction of viscosity by these other fractions on pectin, rye extract, or barley extract, this commercial hemicellulase probably would be much less effective on a weight basis in chick diets incorporating these complex carbohydrates. Viscosity of gum solutions has been considered an important property with respect to growth depression. Indeed, our own data (Table 3) would support this view since screen score, which is associated with viscosity, is reduced
and gain is increased with increased dietary levels of the purified enzyme. The data of Vohra and Kratzer (1964a) indicate that both locust bean gum and guar gum reduce the growth rate of chicks to about the same extent, 73 and 67% of controls, respectively. These two gums are both galactomannans, differing only in the frequency of a-l,6-galactose linkages, yet they have great differences in viscosity. Whistler and Hymowitz (1979) report the viscosities of 1% solutions, in centipoises, to be only 100 for locust bean gum but 4200 for guar gum. Changes in several parameters are observed when guar gum is incorporated into the diets. For example, it lowers total cholesterol of plasma in fasting rats (Chen and Anderson, 1979), chickens (Patel and McGinnis, unpublished data), and in humans witli Type II hyperlipidemia (Jenkins et al, 1979). Another very interesting effect of dietary guar gum is
PURIFIED GUAR DEGRADING ENZYME AND CHICK GROWTH 100-i
DISTANCE (cm) FIG. 3. Densitometric scan of the electrophoretic patterns of 1) hemicellulase, 2) Pool C, and 3) Pool X. Peak height is percent of full-scale deflection at 570 nm.
the reduction of blood glucose, plasma insulin, and plasma gastric inhibitor p o l y p e p t i d e in h u m a n s (Morgan et al, 1979). T h e m o d e of action of guar gum in causing changes in these specific c o m p o n e n t s of t h e blood as well as t h e general g r o w t h depression is n o t u n d e r s t o o d . Several suggestions can be m a d e , such as increased viscosity of the digesta, lectin-like interaction with t h e intestinal m u c o s a , changes in intestinal microflora, o r interactions with g u t h o r m o n e s . It is o u r i n t e n t to s t u d y these possibilities by examining t h e effects of degraded guar gum p r o d u c e d b y t h e action of this highly purified mannanase, which apparently cleaves t h e g a l a c t o m a n n a n b a c k b o n e r a n d o m l y t o reduce viscosity. REFERENCES Anderson, J. O., and R. E. Warnick, 1964. Value of enzyme supplement in rations containing certain legume seed meals or gums. Poultry Sci. 43: 1091-1097. Borcher, R., and C. W. Ackerson, 1950. The nutritive
493
value of legume seeds. X. Effect of autoclaving and trypsin inhibitor test for 17 species. J. Nutr. 41:339-345. Chen, W. -J. L., and J. W. Anderson, 1979. Effects of guar gum and wheat bran on lipid metabolism of rats. J. Nutr. 109:1028-1034. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics. 11:1—42. Jenkins, D.A.J., A. R. Leeds, B. Slavin, J. Mann, and E. M. Jepson, 1979. Dietary fiber and blood lipids: reduction of serum cholesterol in Type II hyperlipidemia by guar gum. Amer. J. Clin. Nutr. 32:16. Kratzer, F. H., R.W.A.S.B. Rajagurer, and P. Vohra, 1967. The effect of polysaccharides on energy utilization, nitrogen retention and fat absorption in chickens. Poultry Sci. 46:1489-1493. Maurer, H. R., 1971. Disc electrophoresis and related techniques of polyacrylamide gel electrophoresis. 2nd ed. Walter de Gruyter & Co., Berlin, Germany. McCleary, B. V., 1979. Enzymic hydrolysis, fine structure, and gelling interaction of legume-seed D-galacto-D-mannans. Carbohydr. Res. 71:205— 230. McGinnis, J., S. J. Reddy, M. B. Patel, and D. Zaviezo, 1980. A bioassay procedure of studying unidentified chick growth depressing factors in feedstuffs. Poultry Sci. 59:1637. Morgan, L. M., T. J. Goulder, D. Tsiolakis, V. Marks, and K.G.M.M. Alberti, 1979. The effect of unabsorbable carbohydrate on gut hormones. Modification of post-prandial GIP secretion by guar. Diabetologia 17:85-89. Patel, M. B., M. S. Jami, and J. McGinnis, 1980. Effect of gamma irradiation, penicillin, and/or pectic enzyme on chick growth depression and fecal stickiness caused by rye, citrus pectin and guar gum. Poultry Sci. 59:2105-2110. Patel, M. B., J. McGinnis, and M. H. Pubols, 1981. Effect of dietary cereal grain, citrus pectin and guar gum on liver fat in laying hens and young chicks. Poultry Sci. 60:631-636. Sandegren, E., and L. Enebo, 1952. Cell wall decomposing enzymes of barley and malt. I. Determination and stability investigations. J. Inst. Brewing 58:198-203. Saxena, U. C , and K. Pradhan, 1974. Effect of high protein levels on the replacement value of guar-meal (Cyamopsis tetragonoloba) in layer's ration. Indian J. Anim. Sci. 44:190—193. Smith, I., 1968. Acrylamide gel disc electophoresis. Pages 365—418 in Chromatographic and electophoretic techniques. I. Smith, ed. Pittman Press, Bath, Great Britain. Steel, R.G.D., and J. H. Torrie, 1960. Principles and procedures of statistics. McGraw-Hill Book Corp., New York, NY. Thakur, R. S., and K. Pradhan, 1975. Guar-meal (Cyamopsis tetragon oloba) inclusion in broiler ration. Effect on carcass yield and meat composition. Indian J. Anim. Sci. 45:880— 884. Thakur, R. S., and U. P. Saxena, 1976. Guar-meal in poultry ration, a review. Indian Poultry Rev. 7:9-12.
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Vohra, P., and F. H. Kratzer, 1964a. The use of guar-meal in chicken rations. Poultry Sci. 4 3 : 502-503. Vohra, P., and F. H. Kratzer, 1964b. Growth inhibitory effect of certain polysaccharides for chickens. Poultry Sci. 43:1164-1170. Vohra, P., and F. H. Kratzer, 1965. Improvement of guar meal by enzymes. Poultry Sci. 44:1201—
1205. Whistler, R. L., and T. Hymowitz, 1979. Guar: agronomy, production, industrial use and nutrition. Purdue Univ. Press, West Lafayette, IN. White, W. B., H. R. Bird, M. L. Sunde, N. Prentice, and W. C. Burger, 1978. Effects of gums and grit on barley and corn based diets. Poultry Sci. 57: 1170-1171.
1 Scientific Paper No. 5959. College of Agriculture Research Center, Washington State University, Pullman, WA. Project No. 0311.
tive (Patel et al, 1980). A n o t h e r m e t h o d is t o subject guar gum to gamma-irradiation. If this latter t r e a t m e n t was c o m b i n e d with the pectinase, then the g r o w t h depression was nearly eliminated (Patel et al, 1980). These observations indicate t h a t the d e t r i m e n t a l effects of guar gum are i n h e r e n t t o the polysaccharide structure and can be elimimated by the action of enzymes t h a t degrade it. T h e means by which guar gum causes detrimental effects w h e n fed t o t h e chicks is n o t k n o w n . T h e effects are observed only with guar g u m t h a t has n o t been degraded by enz y m e s or gamma-irradiation. O n e approach t o t h e d e t e r m i n a t i o n of the m o d e of action of guar gum is t o establish the m i n i m u m chain length of guar gum which can cause depression. A m a n n a n a s e was purified which degrades t h e guar g u m , as measured by r e d u c t i o n of viscosity. This purified e n z y m e was fed t o chicks and found t o inactivate t h e g r o w t h depressing p r o p e r t i e s of this g a l a c t o m a n n a n .
MATERIALS AND METHODS A guar-degrading e n z y m e was purified from a commercial hemicellulase p r e p a r a t i o n by ion-exchange c h r o m a t o g r a p h y , as assayed by t h e reduction in viscosity of a guar gum solution. This purified m a n n a n a s e was added to chick diets having 2% guar gum. Enzyme. A hemicellulase p r o d u c t , CE1 0 0 , 0 0 0 , was o b t a i n e d from Miles Laboratories, Elkhart, IN. It is 4 3 . 6 % p r o t e i n (N X 6.25).
488
PURIFIED GUAR DEGRADING ENZYME AND CHICK GROWTH Substrates. Guar g u m was o b t a i n e d from either Sigma Chemical C o m p a n y , St. Louis, MO (catalogue # G - 4 1 2 9 ) or Celanese Specialties C o m p a n y , Louisville, KY. Pectin was obtained from Sunkist C o m p a n y , Ontario, CA. R y e extract was p r e p a r e d b y stirring g r o u n d rye with 10 volumes (w/v) of 80% e t h a n o l and heating t o boiling. After cooling t o r o o m t e m p e r a t u r e , t h e suspension was filtered through Whatman # 1 filter paper using a Buchner funnel. T h e residue collected by filtration was e x t r a c t e d with 10 volumes of water b y heating t o 70 to 8 0 C, t h e n cooling t o r o o m t e m p e r a t u r e before centrifuging at 1 0 , 0 0 0 X g. T h e s u p e r n a t a n t was used for viscosimetric assays. Barley e x t r a c t was prepared by t h e same p r o c e d u r e . Ion-Exchange Chromatography. The hemicellulase p r e p a r a t i o n ( C E - 1 0 0 , 0 0 0 ) (64.3 g, 5 6 9 0 total A 2 8 o ) was dissolved in 2 0 0 ml .05 M Tris pH 9.0, t h e n centrifuged at 4 0 0 0 X g for 30 min. T h e clear s u p e r n a t a n t was applied t o a column (7.6 X 7.0 cm high) of DEAE-cellulose (Whatman DE-52, Clifton, N J ) , which had been equilibrated with .05 M Tris buffer p H 9.0. After t h e b r e a k t h r o u g h peak of protein h a d eluted, a . 5 M KC1 gradient was applied t o t h e column. Protein was m e a s u r e d by the absorbance at 2 8 0 n m . T h e fractions were assayed for viscosity reducing activity against the various substrates. Pools of fractions were m a d e
489
according t o e n z y m e activity with Pool C, t u b e s 175 t o 196, having the guar-degrading activity. A p o r t i o n of t h e fraction containing t h e guar-degrading e n z y m e , Pool C (5.3 A 2 8 0 units), was passed through a c o l u m n of G-25 Sephadex which had been equilibrated with .05 M a m m o n i u m acetate, p H 6. T h e p r o t e i n peak was then applied t o a column of QAE-Sephadex (1.0 b y 10 c m ) equilibrated with t h e same buffer. A single, sharp peak was eluted with a .5 M KC1 gradient in .05 M a m m o n i u m acetate, pH 6.0. It had t h e guar-degrading activity, according t o viscosity reduction, and was labeled Pool X. Viscosity. An Ostwald viscometer was used t o assay t h e e n z y m e activity in t h e various fractions. T h e substrates were p r e i n c u b a t e d with t h e fractions for a specified t i m e a t r o o m t e m p e r a t u r e . T h e flow rate was c o m p a r e d to t h e flow rates for water and substrate blanks. T h e results are expressed in t e r m s of the specific viscosity which is defined as:
%p =-2m._i where n m is viscosity of the reaction m i x t u r e and n w is viscosity of water. A l i q u o t s of t h e e n z y m e were selected which w o u l d reduce the flow time t o a b o u t one-half t h a t of t h e s u b t r a t e blank. T h e reciprocal of the specific viscosity, -~— was used t o determine peaks of various r %p
TABLE 1. Composition of the diet Ingredient
(%)
Fish meal (65% protein) Isolated soy protein (88% crude protein) Dehydrated alfalfa meal (17% crude protein) Meat and bone meal (50% crude protein) Soybean meal (47% crude protein) Dicalcium phosphate Iodized salt Vitamin premix a Trace mineral mix"3 DL-methionine Animal fat Total Glucose and/or 2% guar gum
8.00 1.25 2.25 4.00 30.60 .90 .30 .25 .05 .15 2.25 50.00 50.00
Vitamin premix at .25% of the diet supplies the following per kilogram of the diet: vitamin A, 5500 IU; vitamin D 3 , 1650 ICU; vitamin E, 6.4 IU; riboflavin, 3.3 mg; calcium pantothenate, 9.8 mg; niacin, 22 mg; choline chloride, 577 mg; vitamin B 1 2 , .011 mg; biotin, .05 mg; pyridoxine, 2.0 mg; thiamine, 1.0 mg; and ethoxyquin, 62.2 mg. Mineral premix at .05% of the diet supplies the following (mg/kg) of the diet: Mn, 50: Fe, 50; Cu, 5; Zn, 50; I, 1.5; Ca, 60 mg; Co, .5.
RAY ET AL.
490
TABLE 2. Specific activities of the enzymes in the pooled fractions from the DEAE-cellulose
column"
Sample
Tube
Volume (ml)
A 28 „/pool
Guar
Pectin
Barley
Rye
CE-100,000 Pool A Pool B PoolC PoolD PoolE
120-150 152-166 170-196 216-248 293-306
1100 565 1000 96 560
532.4 591.5 1488. 41.8 162.4
121.5 .7 .2 343 .5 1.5
1.7 4.3 .3 4.5 .2 .2
92.4 2.0 3.4 1.7 103.0 275.8
1.1 .4 .3 .3 .1 .2
Specific activity = units/A 2
enzyme activities in the column effluents (Sandegren and Enebo, 1952). The substrate solutions, . 1 % guar gum, .2% pectin, 2% rye water extract, or 2% barley extract were clarified by centrifugation before use in die viscometer. The substrates were dissolved in distilled water. Slab-Gel Electrophoresis. The procedures of Smith (1968) and Maurer (1971) were followed. The samples were dialyzed against water to remove salt, lyophilized to dryness, then resuspended in buffer. A 10% polyacrylamide gel was used in a Beckmann microzone electrophoresis system. The dimensions of the cell were 6.5 X 10 X .4 cm. The gel had eight wells for simultaneous analysis. The amount of protein applied into the wells was as follows: hemicellulase, 97.8 Mg; "Pool C", 99.0 £(g; "Pool X", 59.4 /Ug. A Tris-glycine buffer (pH 8.4) was used in preparing the slab and also served as the electrolyte. Electrophoresis was at 450 V with a current of 44 to 35 raA for 54 min. The gel was stained with Coomassie Brilliant Blue G (Smith, 1968) for 1 hr. The stained gel was rinsed with 2% acetic acid and then electrolytically destained for 1 hr in 2% acetic acid solution. The destained gel was scanned in a Beckman R-110 microzone densitometer at 570 nm. Feeding Trial. A feeding trial was used to test the effectiveness of the purified guardegrading enzyme on growth of chicks fed 2% guar gum. The diet listed in Table 1 is based on the bioassay diet of McGinnis et al. (1980). In this trial, 10 female, day-old broiler chicks were assigned randomly to a pen, and three pens were assigned randomly to each treatment. The birds were maintained in electrically heated battery brooders equipped with raised, wirescreen floors. Feed and water were supplied ad libitum with 14 hr of light per day. The birds
were weighed in groups after 2 weeks. Feed consumption was measured. At the end of the trial, the wire floors were scored visually between 0 and 5 for adhering feces, the higher score indicating a greater amount of feces sticking to the floor. The guar-degrading enzyme in lyophilized Pool C was added to the diet at 5, 10, 15, or 20 mg/kg diet. Since the lyophilized Pool C had only 6.4% protein, this represented .32, .64, .96, or 1.28 mg protein/kg diet, respectively. Data from the experiments were treated by analysis of variance according to Steel and Torrie (1960) and the differences analyzed by multiple range test of Duncan (1955). RESULTS AND DISCUSSION
The guar-degrading enzyme activity was concentrated in Pool C (Table 2) from the DEAE-cellulose ion-exchange fractionation (Fig. 1). This fraction had relatively no activity on pectin, rye water extract, or barley water extract. Activity toward these substrates was found in other fractions. Pool C had 52% of the total eluted protein and represented about a threefold purification of the enzyme (Table 2). Activity toward barley extract was found in Pools D and E, whereas only small amounts of activity towards pectin and rye were detected. The lyophilized powder from Pool C was incorporated into chick diets containing 2% guar gum and was found to be effective in stimulating growth at levels as low as about .6 mg enzyme protein/kg diet (Table 3). The reduction of the sticky feces, as measured by screen score, may require a greater amount of enzyme, approaching 1 mg/kg. The single sharp peak, Pool X, which eluted from QAE-Sephadex (Fig. 2), having 3.4 total A28o units, had the guar-degrading enzyme.
PURIFIED GUAR DEGRADING ENZYME AND CHICK GROWTH
TUBE N O . - * 20 POOL
40
60
80
•
100 120 140
491
160 180 200 220 240 260 280 300 320
I—A—li-BH I—C—I
I—D—I
I-EH
FIG. 1. Fractionation of a hemicellulase by DEAE-cellulose chromatography, pH 9.0. Units for each of the enzymes are reciprocals of the specific viscosities (see text). Conductivity units are mmhos.
Slab-gel electophoresis of this p r o t e i n at p H 8.4 indicated this p r o t e i n to be essentially pure. T h e densitometric scans of t h e e l e c t r o p h o r e t i c p a t t e r n s of t h e proteins during t h e stages of purification are shown in Figure 3. T h e guardegrading e n z y m e in Pool C was a b o u t 6 8 % of t h e dye b o u n d t o the gel, whereas in Pool X
a b o u t 8 3 % was b o u n d . The guar-degrading e n z y m e of t h e commercial hemicellulase C E - 1 0 0 , 0 0 0 is a b o u t 50% of t h e total protein. Guar gum is a p o l y m e r of D-mannose linked (3-1, 4 with D-galactose attached t o alternate m a n n o s e u n i t s ce-1, 6. Presumably, this e n z y m e would degrade o t h e r
TABLE 3. Average weight gain, feed efficiency and screen score (SCSC) of chicks fed diets containing 2% guar gum supplemented with graded levels of pool C from the DEAE-cellulose column Supplement Guar gum
(%)
Enzyme (mg protein/kg)
Gain (g)
Gain/feed
SCSC
None (glucose) 2 2 2 2 2 2
None None + 10.91 HC1 + 0.32 Pool C + 0.64 Pool C + 0.96 Pool C + 1.28 Pool C
168.5 a b 123.2 C 182.6 a 163.lb 167.6 a b 169.5 a b 172.3 a b 5.78
.675 a .509 d .650 b .620° .638 b c .640 b c .643bc 2.12
.67 d 3.50 a 1.33 c d 2.50 b 2.00 b c 1.50 b «l 1.67 b c d 28.42
CV %
a ' ' c ' Means having the same letter in a column do not differ significantly (P>.05) according to Duncan's (1955) multiple range test. 1
Hemicellulase (CE-100,000).
492
RAY ET AL.
>•
=>
i
o o T40
135 30 25 H20
15 10 5 20
40
60
80
100 120 140 160 180 200 220 240
TUBE NUMBER FIG. 2. Further purification of "Pool C" by QAE-Sephadex chromatography at pH 6.0. "Guarase" units are reciprocals of the specific viscosities (see text). Conductivity units are mmhos.
polymers with a similar structure, such as locust bean gum. We could not detect any galactose liberated by the enzyme (M.Pubols, 1981, unpublished data). This strongly suggests that the enzyme is a mannanase, cleaving the polymer randomly. The effect of other fractions purified from this hemicellulase have not been tested in feeding trials with diets having pectin, barley, or rye. However, based on the relative rate of reduction of viscosity by these other fractions on pectin, rye extract, or barley extract, this commercial hemicellulase probably would be much less effective on a weight basis in chick diets incorporating these complex carbohydrates. Viscosity of gum solutions has been considered an important property with respect to growth depression. Indeed, our own data (Table 3) would support this view since screen score, which is associated with viscosity, is reduced
and gain is increased with increased dietary levels of the purified enzyme. The data of Vohra and Kratzer (1964a) indicate that both locust bean gum and guar gum reduce the growth rate of chicks to about the same extent, 73 and 67% of controls, respectively. These two gums are both galactomannans, differing only in the frequency of a-l,6-galactose linkages, yet they have great differences in viscosity. Whistler and Hymowitz (1979) report the viscosities of 1% solutions, in centipoises, to be only 100 for locust bean gum but 4200 for guar gum. Changes in several parameters are observed when guar gum is incorporated into the diets. For example, it lowers total cholesterol of plasma in fasting rats (Chen and Anderson, 1979), chickens (Patel and McGinnis, unpublished data), and in humans witli Type II hyperlipidemia (Jenkins et al, 1979). Another very interesting effect of dietary guar gum is
PURIFIED GUAR DEGRADING ENZYME AND CHICK GROWTH 100-i
DISTANCE (cm) FIG. 3. Densitometric scan of the electrophoretic patterns of 1) hemicellulase, 2) Pool C, and 3) Pool X. Peak height is percent of full-scale deflection at 570 nm.
the reduction of blood glucose, plasma insulin, and plasma gastric inhibitor p o l y p e p t i d e in h u m a n s (Morgan et al, 1979). T h e m o d e of action of guar gum in causing changes in these specific c o m p o n e n t s of t h e blood as well as t h e general g r o w t h depression is n o t u n d e r s t o o d . Several suggestions can be m a d e , such as increased viscosity of the digesta, lectin-like interaction with t h e intestinal m u c o s a , changes in intestinal microflora, o r interactions with g u t h o r m o n e s . It is o u r i n t e n t to s t u d y these possibilities by examining t h e effects of degraded guar gum p r o d u c e d b y t h e action of this highly purified mannanase, which apparently cleaves t h e g a l a c t o m a n n a n b a c k b o n e r a n d o m l y t o reduce viscosity. REFERENCES Anderson, J. O., and R. E. Warnick, 1964. Value of enzyme supplement in rations containing certain legume seed meals or gums. Poultry Sci. 43: 1091-1097. Borcher, R., and C. W. Ackerson, 1950. The nutritive
493
value of legume seeds. X. Effect of autoclaving and trypsin inhibitor test for 17 species. J. Nutr. 41:339-345. Chen, W. -J. L., and J. W. Anderson, 1979. Effects of guar gum and wheat bran on lipid metabolism of rats. J. Nutr. 109:1028-1034. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics. 11:1—42. Jenkins, D.A.J., A. R. Leeds, B. Slavin, J. Mann, and E. M. Jepson, 1979. Dietary fiber and blood lipids: reduction of serum cholesterol in Type II hyperlipidemia by guar gum. Amer. J. Clin. Nutr. 32:16. Kratzer, F. H., R.W.A.S.B. Rajagurer, and P. Vohra, 1967. The effect of polysaccharides on energy utilization, nitrogen retention and fat absorption in chickens. Poultry Sci. 46:1489-1493. Maurer, H. R., 1971. Disc electrophoresis and related techniques of polyacrylamide gel electrophoresis. 2nd ed. Walter de Gruyter & Co., Berlin, Germany. McCleary, B. V., 1979. Enzymic hydrolysis, fine structure, and gelling interaction of legume-seed D-galacto-D-mannans. Carbohydr. Res. 71:205— 230. McGinnis, J., S. J. Reddy, M. B. Patel, and D. Zaviezo, 1980. A bioassay procedure of studying unidentified chick growth depressing factors in feedstuffs. Poultry Sci. 59:1637. Morgan, L. M., T. J. Goulder, D. Tsiolakis, V. Marks, and K.G.M.M. Alberti, 1979. The effect of unabsorbable carbohydrate on gut hormones. Modification of post-prandial GIP secretion by guar. Diabetologia 17:85-89. Patel, M. B., M. S. Jami, and J. McGinnis, 1980. Effect of gamma irradiation, penicillin, and/or pectic enzyme on chick growth depression and fecal stickiness caused by rye, citrus pectin and guar gum. Poultry Sci. 59:2105-2110. Patel, M. B., J. McGinnis, and M. H. Pubols, 1981. Effect of dietary cereal grain, citrus pectin and guar gum on liver fat in laying hens and young chicks. Poultry Sci. 60:631-636. Sandegren, E., and L. Enebo, 1952. Cell wall decomposing enzymes of barley and malt. I. Determination and stability investigations. J. Inst. Brewing 58:198-203. Saxena, U. C , and K. Pradhan, 1974. Effect of high protein levels on the replacement value of guar-meal (Cyamopsis tetragonoloba) in layer's ration. Indian J. Anim. Sci. 44:190—193. Smith, I., 1968. Acrylamide gel disc electophoresis. Pages 365—418 in Chromatographic and electophoretic techniques. I. Smith, ed. Pittman Press, Bath, Great Britain. Steel, R.G.D., and J. H. Torrie, 1960. Principles and procedures of statistics. McGraw-Hill Book Corp., New York, NY. Thakur, R. S., and K. Pradhan, 1975. Guar-meal (Cyamopsis tetragon oloba) inclusion in broiler ration. Effect on carcass yield and meat composition. Indian J. Anim. Sci. 45:880— 884. Thakur, R. S., and U. P. Saxena, 1976. Guar-meal in poultry ration, a review. Indian Poultry Rev. 7:9-12.
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Vohra, P., and F. H. Kratzer, 1964a. The use of guar-meal in chicken rations. Poultry Sci. 4 3 : 502-503. Vohra, P., and F. H. Kratzer, 1964b. Growth inhibitory effect of certain polysaccharides for chickens. Poultry Sci. 43:1164-1170. Vohra, P., and F. H. Kratzer, 1965. Improvement of guar meal by enzymes. Poultry Sci. 44:1201—
1205. Whistler, R. L., and T. Hymowitz, 1979. Guar: agronomy, production, industrial use and nutrition. Purdue Univ. Press, West Lafayette, IN. White, W. B., H. R. Bird, M. L. Sunde, N. Prentice, and W. C. Burger, 1978. Effects of gums and grit on barley and corn based diets. Poultry Sci. 57: 1170-1171.