Usage of Enzyme Substrate to Protect the Activities of Cellulase, Protease and α-Amylase in Simulations of Monogastric Animal and Avian Sequential Total Tract Digestion

Cellulase from Aspergillus niger, (α-amylase from Bacillus sp. and protease from Bacillus globigii were used as enzyme sources in this study to examine how their respective substrates protect them in two kinds of simulated gastrointestinal tract digesting processes. Avian total digest tract simulation test showed that filter paper, Avicel and cellulose resulted in 7.7, 6.4 and 7.4 times more activity than of unprotected cellulose, respectively. Protease with addition of casein, gelatin or soybean protein showed no significant protection response. Starch protected amylase to be 2.5 times activity of the unprotected one. Monogastric animal total tract digestion simulation test showed that filter paper, Avicel and cellulose resulted in 5.9, 9.0 and 8.8 times activity of unprotected cellulase, respectively. Casein, gelatin and soybean protein resulted in 1.2, 1.3 and 2.0 times activity of unprotected protease, respectively. Starch did not protect amylase activity in monogastric animal total tract simulation. Protection of mixed enzymes by substrates in two animal total tract simulation tests showed that filter paper in combination with soybean protein resulted in 1.5 times activity of unprotected cellulose, but all substrates tested showed no significant protection effect to protease. Soybean protein and starch added at the same time protected the amylase activity to be two times of the unprotected one. Test of non-purified substrate protection in two animal total digest tract simulation showed that cellulase activity increased as BSA (bovine serum albumin) concentration increased, with the highest activity to be 1.3 times of unprotected enzyme. However, BSA showed no significant protection effect to protease. Amylase activity increased to 1.5 times as BSA added more than 1.5% (w/v). Cellulase activity increased to 1.5 times as soybean hull was added higher than 1.5%. Amylase had a significant protection response only when soybean hull added up to 2%. Protease activity was not protected by soybean hull to any significant extent. (


INTRODUCTION
Many polysaccharides can form viscous gel-like structure in the small intestine which will trap nutrients and hinder the action of the animal's digestive enzymes.Cellulases and xylanases added to the diet can break down these gels and release the trapped nutrients (Pettersson and Aman, 1989).However, positive responses were not achieved all the time for fibrolytic enzymes' application in pigs or broilers (Kim et al., 2004;Qiao et al., 2005).Some cellulases, expressed by bacteria and fungi, are glycosylated by post-translational modification which often protects enzymes from inactivation by heat or protease (Olsen and Thomsen, 1991).The stability of cellulase can also be improved by modification with synthetic copolymers over a wide range of pH (Jin, 1996).Fontes et al. (1995) indicated that labile cellulases were resistant to proteolytic attack in the presence of their appropriate substrate.Many commercial feed enzyme additives are mixtures of protease, cellulase and amylase.In this case, protease may attack other enzymes and decrease their efficiency.To be fully functional in the digestive tract, exogenous enzymes should be resistant to attack of protease in the small intestine, and able to exhibit catalytic activity in the pH range 6 to 8. To maintain the activity of exogenous enzymes, substrate addition seems to be a convenient and cheap method to protect enzymes.In this study, purified microbial protease, cellulase and amylase were used alone or mixed for various tests of substrates' protection ability.Common protease substrates (casein, gelatin or soybean protein), cellulase substrates (Avicel, cellulose or filter paper) and amylase substrate (corn starch) were tested for their protection ability on their respective enzymes.Finally, the nonpurified substrate BSA and soybean hull were tested for their protection ability toward all three kinds of enzymes.The objective of the study was to find a better way to supply mixed enzyme feed additives and exploit low cost enzyme protection materials for various enzymes.

Enzyme activity in different pH
To study the individual and mixed enzymes activity under different pH conditions, glycine-HCl buffer (pH 3), sodium-phosphate buffer (pH 7) and HEPES buffer (pH 8) were used.Cellulase (3 mg/ml, 3.5 units/ml), protease (0.0438 mg/ml, 0.45 units/ml), α-amylase (5 mg/ml, 250 units/ml) or mixtures of all three enzymes were dissolved in different pH buffers and incubated at room temperature for 10 minutes, followed by immersion in ice to stop the reaction and enable assay of residual activities..

Enzyme activity tests in simulation of stomach and small intestine conditions
To simulate the stomach conditions, pepsin solution was prepared by dissolving porcine pepsin (EC 3.4.23.1, Sigma P-7000) in 50 mM glycine-HCl buffer (pH 2 or 3) to a final concentration of 75 units/ml.Trypsin and chymotrypsin solution for simulating the small intestinal conditions were prepared by dissolving trypsin (Sigma, T-0303) and chymotrypsin (Sigma, C-4129) in 50 mM Tris-HCl buffer to a final concentration of 10 mg/ml.The concentrations of cellulase, protease and α-amylase used were the same as in the previous pH test.

Substrate protection ability tests
Casein, soybean protein and gelatin were tested for their protective efficacy on protease.Cellulose, Avicel PH-101 and filter paper (Whatman No.1) were tested for their protective efficacy on cellulase.Corn starch, the common  starch source was used to test its protective efficacy on αamylase.Cellulase, protease and α-amylase were diluted five times to final concentrations of 0.6 mg/ml, 0.00876 mg/ml and 1 mg/ml, respectively.All tested substrates were dissolved in water before mixing with enzyme solution.
Enzymes were dissolved in 100 mM sodium phosphate buffer (pH 6.8) and mixed with the substrate solution at a 1:1 ratio.Lists and abbreviations of substrates used in the protection ability test for cellulase-protease or amylaseprotease mixtures are shown in Table 1.To determine the protection ability for individual or mixed enzymes, two kinds of in vitro digestion simulation procedures were employed (Figure 1).Remaining activities of exogenous enzymes in both stomach and sequential total tract simulation condition were analyzed to determine the protection effect from different substrates.

Non-purified substrate protection ability tests
Soybean hull and BSA were individually mixed with three kinds of enzymes to test their protection ability for cellulase, protease and α-amylase.Soybean hull was ground through the 30 mesh screen before use.Enzyme concentration and in vitro test procedures were performed as previously described for substrate protection ability tests.
to ensure complete precipitation of the remaining azocasein.The samples were centrifuged at 15,000×g for 10 min, 100 µl supernatant fluid transferred to a microplate and an equal volume of 1 N NaOH added.Absorbance was determined at 450 nm.

Statistical analysis
Data were analyzed by analysis of variance (ANOVA) procedures using the SAS system for Windows (SAS 8.0, SAS Institute).Differences between treatment means were determined using Duncan's test with significance of difference set at p<0.05.

The effects of pH and digestion enzyme
Cellulase : Cellulase activity in different pH buffers with or without pepsin or pancreatic enzyme is shown in Figure 2. Residual cellulase activity in mixed enzymes was higher than cellulase applied alone in all tested conditions.Cellulase had high residual activity at neutral pH, but activity decreased toward basic conditions.The cellulase activity decreased in low pH or pepsin treatment (Figure 2A and 2B), but trypsin and chymotrypsin treatment had no significant effect on cellulase (Figure 2C).Cellulase in mixed enzyme tests was more stable in trypsin and chymotrypsin treatment or pancreatin treatment than cellulase applied alone (Figure 2C and 2D).
The cellulase used in our study was from Aspergillus niger with an optimal pH at 5.0.In the present study, cellulase of Aspergillus niger was more stable at pH 7.0 than pH 3.0.Cellulase activity in individual and mixed enzyme tests increased immediately after pancreatin treatment, but longer treatment time did not enhance the activity further.The phenomenon of increasing cellulase activity may be due to pancreatin's interference with the color presentation of DNS reagent.
Protease : Activities of protease alone or in mixed enzymes were all significantly depressed by low pH (pH 2 or 3) and pepsin treatment (Figure 3A and 3B), with only 10% activity left in comparison to neutral pH conditions.Exogenous protease was inactivated by trypsin and chymotrypsin (Figure 3C), whereas pancreatin treatment had no significant effect on protease, and retained >85% residual protease activity (Figure 3D).
Amylase : The amylase in mixed enzymes was more stable than amylase alone at low pH or pepsin treatment (Figure 4A and 4B), but amylase activity in mixed enzymes dropped to 20% of its original activity at neutral pH (Figure 4A).This may be related to the higher protease activity in mixed enzymes at pH 7 (Figure 3A).The amylase activity increased after trypsin, chymotrypsin and pancreatin treatment (Figure 4C and 4D).After pancreatin treatment, amylase activity was higher for amylase alone than for mixed enzymes.This may be due to the combined action of protease from mixed enzymes and pancreatin.
Comparing the activity of mixed enzymes with enzyme alone implied that α-amylase in mixed enzymes was more stable in low pH or pepsin treatment than amylase alone.It is possible that enzymes in mixtures have less opportunity to be attacked by pepsin than when applied alone.

Substrate protection ability tests for individual enzyme
Cellulase : All kinds of cellulase substrates can protect cellulase activity in simulations of stomach and total tract digestion (Figure 5A and 5B).In the simulation of total tract digestion, cellulose and Avicel had better protective ability on cellulase (Figure 5B).Both avian and monogastric animal digestive simulation procedures showed that cellulase activity was well protected by its substrates.Residual activities of cellulase with substrate addition were five times higher than without substrate protection.
It is possible that the substrate protected the enzyme activity through occupying the active site of the enzyme and preventing the gastrointestinal tract's enzyme attack.Alternatively, the binding of the substrate to enzyme may cause a conformational change to a much tighter tertiary structure that is more resistant to proteolysis.Addition of 0.2% β-glucan was shown to significantly decrease the sensitivity of protease-labile cellulase or xylanase and increase the residual activity about 20% (Fontes et al., 1995).Many kinds of cellulase have serine-rich sequences which are very sensitive to protease if they are not protected in any way (Gilkes et al., 1991).
Amylase : Starch addition protected amylase activity insimulation of stomach digestion, but had no protective effect in simulation of total tract digestion conditions (Figure 7A and 7B).The pancreatin used in this study contained amylases which may affect the outcome of total amylase activity in the digestion simulation test.

Substrate protection ability test for mixed enzymes
The arrangement of enzyme mixtures and their substrate combinations is shown in Table 1.Two kinds of enzyme mixtures, (1) cellulase and protease and (2) amylase and protease, were tested.
Cellulase and protease : All cellulose substrates could protect the cellulase activity in stomach digestion simulation as shown in Table 2.The combination of Avicel and soybean protein (AS) had higher protection ability than others, the residual cellulase activity was four times higher than that without addition of any substrate (EN).Compared with enzyme applied alone without substrate, soybean protein addition increased the residual activity of protease about two times in simulation of stomach digestion for both animal modules.Total tract digestion simulation with both animal modules (Table 2) showed that filter paper with soybean protein protected cellulase activity appreciably and maintained 1.5 times more residual cellulase activity.In  contrast, no substrate mixture had a good protection effect on protease activity in simulations of total tract digestion.Amylase and protease : Starch and soybean protein mixed substrate (SS) provided the best protection for amylase in simulations of monogastric animal and avian stomach and avian total tract digestion (Table 3); the residual amylase activity was about 10 times higher than that of the enzyme mixture without substrate addition (EN).No any substrate mixture could provide effective protection to protease in simulations of total tract digestion for both animal modules (Table 3).

Non-purified substrate protection ability tests
BSA : In simulations of stomach digestion, individual cellulase, protease or amylase were stabilized by addition of >1.5% BSA (Table 4).However, BSA addition had no protection on protease activity in simulation of total tract digestion for both animal modules.Protection was greater for cellulase than other enzymes when BSA was added with mixed enzymes, especially in simulations of total tract digestion.Addition of 1.5% BSA seemed capable of protecting mixed enzymes in simulation of stomach digestion Compared with other protease substrates in this study (casein, gelatin or soybean protein), BSA had a greater protective effect on cellulase than other individual protein substrates in mixed enzymes containing protease (Table 2; EC, EG and ES treatment).BSA is a soluble protein known to be resistant to  proteolysis, and has a complex tertiary structure with 6% cysteine and disulphide bonds (Broderick and Craig, 1989).Casein has essentially a linear secondary and tertiary structure without disulphide bonds, and was therefore sensitive to degradation (Mangan, 1972).This may explain why BSA had better protection of enzyme residual activity than casein.As BSA was added to the mixture of cellulase, protease and amylase, it is possible that the BSA could bind to protease and prevent protease from attacking cellulase or amylase.
Soybean hull : The simulation of stomach digestion indicated that soybean hull addition was useful to protect the residual activity of all three kinds of enzyme in the avian module (Table 5), but it had no significant protection effect on amylase in the monogastric animal module.Addition of 1.5% soybean hull appeared to significantly protect cellulase activity applied alone or in mixed enzymes in simulations of total tract digestion for both animal modules (Table 5).Compared to BSA, soybean hull seems to have greater potential for protecting exogenous cellulase in digestion simulation tests (Tables 4 and 5).There was no noticeable protection of protease and amylase residual activity when soybean hull was added to individual or mixed enzymes.
There are a number of components present in soybeans that can exert a negative impact on animal nutrition.These include protease inhibitors, lectins, saponins, tannins, phytate and other factors (Liener, 1994).Saponin content of soybean hull was reported to be about 2% on a dry matter basis (Price et al., 1987).Soybean saponins can inhibit pancreatic enzymes by a nonspecific interaction (Ishaaya and Birk, 1965).Ikedo et al. (1996) indicated that BSA susceptibility to chymotrypsin digestion was decreased by soybean saponins and that the N-terminal peptide fragment obtained from the hydrolysate of BSA-soya saponin complex could interact with soya saponin to form a protease-resistant moiety that had low sensitivity to chymotrypsin.It is possible that the protective effect of soybean hull observed in the present study may be related to the inhibition of digestive enzyme by saponins.High levels of pectin in soybean hull might be another protection factor because pectin can increase the viscosity of solution and hinder the process of digestion (Atallah and Melnik, 1982).

Figure 1 .
Figure 1.In vitro digestion simulation procedures for substrate protection test.(a) Simulation of monogastric animal digestion sequence.(b) Simulation of avian digestion sequence.

Figure 6 .
Figure 6.The protection effects of substrates on protection of protease activity.(A) Simulation of avian digestion sequence.(B) Simulation of monogastric animal digestion sequence.Superscripts a, b and c denote different in stomach digestion simulation (p<0.05).Superscripts A and B denote different in total tract digestion simulation (p<0.05).

Figure 5 .Figure 7 .
Figure 5.The protection effects of substrates on protection of cellulase activity.(A) Simulation of avian digestion sequence.(B) Simulation of monogastric animal digestion sequence.Superscripts a, b and c denote different in stomach digestion simulation (p<0.05).Superscripts A, B and C denote different in total tract digestion simulation (p<0.05).

Table 1 .
List and abbreviations of substrates used for protection ability tests for enzyme mixture Cellulase and protease mixture protection test

Table 2 .
The protective effects of cellulose substrates and protease substrates in simulations of monogastric animal and avian digestive Vaules in the same column without the same superscripts are significantly different (p<0.05,n = 8).

Table 3 .
The protective effects of starch substrates and protease substrates in simulations of monogastric animal and avian digestive a, b, c, d, e Vaules in the same column without the same superscripts are significantly different (p<0.05,n = 8).

Table 4 .
The protective effects of BSA Cellulase (glucose µg/mg/min) , b, c, d, e, f Means in the same column without the same superscripts are significantly different (p<0.05,n = 8). a