Comparative studies on the in vitro properties of …...shaking (220 rpm) for 26 h at 37 C. After pelleting cells by centifuga-tion at 7000 g for 30 min the supernatant was 50, fold

Arch. Anim. Nutr., 2000, Vol. 53, pp. 353-373 © 2000 OPA (Overseas Publishers Association) N.V.Reprints available directly from the publisher Published by license underPhotocopying permitted by license only the Harwood Academic Publishers imprint,

part of The Gordon and Breach Publishing Group.Printed in Malaysia.

COMPARATIVE STUDIESON THE IN VITRO PROPERTIESOF PHYTASES FROM VARIOUS

MICROBIAL ORIGINS

F. A. IGBASANa, K. MÄNNERa, G. MIKSCHb, R. BORRISSc,A. FAROUKc and O. SIMONa,*

aInstitute of Animal Nutrition, Faculty of Veterinary Medicine,Free University, Berlin, Germany;

bChair of Fermentation Engineering, Faculty of Technical Sciences,University of Bielefeld, Bielefeld, Germany;

cInstitute of Biology, Humboldt University, Berlin, Germany

(Received 3 April 2000)

The physical and chemical properties of six crude phytase preparations were compared.Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T ) wereproduced at commercial scale for the use as feed additives while the other two (E. coliand Bacillus) were produced at laboratory scale. The encoding genes of the enzymes werefrom different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coliand Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed intransgenic rape. The enzymes were studied for their pH behaviour, temperatureoptimum and stability and resistance to protease inactivation. The phytases were foundto exhibit different properties depending on source of the phytase gene and theproduction organism. The pH profiles of the enzymes showed that the fungal phytaseshad their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also itspH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzymewas identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal andbacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueoussolutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°Cin the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coliphytases were more heat stable compared to other enzymes (Bacillus enzyme was notincluded). At a temperature of 70°C in the conditioner, these enzymes maintained a

Address for correspondence: Dr. F. A. Igbasan, Freie Universität Berlin, Institut fürTierernährung. Fachbereich Veterinärmedizin, Brummer Strasse 34, D-14195 Berlin,Germany. Tel.: 030/8385 2256.

*e-mail: [email protected]

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354 F. A. IGBASAN et al

residual activity of approximately 70% after pelleting compared to approximately30% determined for the other enzymes. Incubation of enzyme preparations with porcineproteases revealed that only E. coli phytase was insensitive against pepsin andpancreatin. Incubation of the enzymes in digesta supernatants from various segmentsof the digestive tract of hens revealed that digesta from stomach inactivated the enzymesmost efficiently except E. coli phytase which had a residual activity of 93% after 60 minincubation at 40°C. It can be concluded that phytases of various microbial originsbehave differently with respect to their in vitro properties which could be of importancefor future developments of phytase preparations. Especially bacterial phytases containproperties like high temperature stability (Bacillus phytase) and high proteolytic stability(E. coli phytase) which make them favourable for future applications as feed additives.

Keywords: Phytases; Properties; Temperature stability; Proteolytic stability; Feed addi-tives

1. INTRODUCTION

Poultry and pig diets are based primarily on cereals, legumes, andoilseed products. About two-thirds of phosphorus (P) present in thesefeedstuffs occur as phytates (myo-inositol hexakisphosphate, InsP6),the salts of phytic acid (Jongbloed et al., 1993). Phytate P in plants isa mixed calcium-magnesium-potassium salt of phytic acid that ispresent as chelate and its solubility is very low (Pallauf and Rimbach,1997). Phosphorus in this form is poorly digestible/available forsimple-stomached animals (Van Der Klis and Versteegh, 1996).

For the utilisation of phytate P and minerals and trace elementsbound in phytic acid complexes, hydrolysis of the ester-type bondedphosphate groups of phytic acid by phytase is necessary (Rimbachet al., 1994). Phytases (myo-inositol hexakisphosphate-phosphohy-drolase) belong to a special group of phosphatases which are capableof hydrolyzing phytate to a series of lower phosphate esters of myo-inositol and phosphate. Two types of phytases are known: 3-phytase(EC 3.1.3.8) and 6-phytase (EC 3.1.3.26), indicating the initial attackof the susceptible phosphate ester bond. Monogastric animals lackintrinsic phytase which is necessary for hydrolysis of phytate present inthe plant feedstuffs (Cooper and Gowing, 1983; Williams and Taylor,1985). However, many fungi, bacteria and yeasts can produce thisenzyme. With the industrial production of phytase, application of thisenzyme to poultry and pig diets to increase P availability and improveanimal performance, as well as reducing environmental pollution hasgained widespread attention.

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IN VITRO PROPERTIES OF VARIOUS PHYTASES 355

The beneficial effects of supplementary phytases on P digestibilityand animal performance have been well documented (Mroz et al.,1994; Kornegay et al, 1996; Rao et al, 1999; Ravindran et al, 1999).However, most of these studies have been performed on an ad hocbasis with often only superficial information of the enzymes providedas marketing strategies by the manufacturers. Most often these infor-mation are on the type, inclusion rate and the overall activity of theenzymes.

The efficacy of any enzyme preparation depends not only on thetype, inclusion rate and level of activity present, but also on the abilityof the enzyme to maintain its activity in the different conditionsencountered through the gastrointestinal tract and the conditions usedfor the pre-treatment of a feedstuff or diet. To evaluate an enzymepreparation, it is important to characterise the enzymes in terms ofpH stability, behaviour during technological processing of feeds,resistance to proteolytic attack, and stability in the digestive tract ofthe host animal. The data on the above mentioned characteristicswould not only provide information on the stability of the enzymewithin the digestive tract but would also support the development ofimproved enzymes.

By comparison of different microbial enzyme preparations for theirphysical and chemical characteristics including stability against diges-tive enzymes, the aim of this study was to draw conclusions for futuredevelopments of desirable enzyme cocktails designed for maximalsupporting the degradation of phytic acid within the digestive tract ofmonogastric farm animals.

2. MATERIALS AND METHODS

2.1. Enzymes

Six crude phytase preparations (Tab. I) from different microbialorigins were used in the present study. Two of these enzymes,Aspergillus A and Aspergillus T, are produced by genetically modifiedfungal expression systems and have been approved for regular use inpoultry and pig diets by the European Union. The Peniophora phytaseis also expressed by a transformed fungal strain, but is not approved

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356 F. A. IGBASAN et al.

TABLE I Description of different phytase preparations

Name of phytase2

Aspergillus A3

Aspergillus R3

Peniophora^Aspergillus T3

E. coli4

Bacillus5

Production strain/organism

Aspergillus nigerBrassica napusAspergillus oryzaeTrichoderma reesiEscherichia coliBacillus subtilis

Origin of phytasegene

Aspergillus ficuumAspergillus ficuumPeniophora lyciiAspergillus awamoriEscherichia coliBacillus subtilis

ActivitylFTU/gf

5171106

2662501720354.66

1 Activity was as determined in our laboratory.2 Name of phytase used in this study.3 Products of industrial scale.4 Produced at laboratory scale by Dr. Miksch; University of Bielefeld, Bielefeld, Germany.5 Produced at laboratory scale by Dr. Farouk and Prof. Borriss; Humboldt University Berlin,Germany.6 Activity was expressed as FTU/ml.

yet by the European commission. In case of Aspergillus R, the phytasegene of Aspergillus ficuum was transferred to rape. The enzymeproduct consists of seeds of this transgenic plant, which are treated ina way to prevent their ability to germinate. To our knowledge, theproperties of these transgenic seeds have not been published.According to the manufacturer, Peniophora phytase is a 6-phytaseunlike Aspergillus phytases which are 3-phytases. The four fungalenzymes are produced at commercial scale and were obtained fromtheir manufacturers.

Among different Bacillus strains isolated from plant-pathogen-infected soils (Krebs et al., 1998), Bacillus subtilis strain 45 was selectedfor its high extracellular phytase activity in low phosphate medium.The enzyme has been characterized as a highly thermostable 3-phytaseacting between pH 6 to 8 (Greiner, Farouk and Borris, unpublisheddata). Wheat bran medium containing 2% wheat bran, 0.2% CaC^,0.04% (NH4)2SO4, 0.02% MgSO4 • 7H2O was prepared and adjustedto pH 6.5. 10 ml Bacillus subtilis 45 grown in Luria broth containing5 g yeast extract, 10 g tryptone and 10 g of NaCl was used to inoculate500 ml wheat bran medium. The cells were cultivated under vigorousshaking (220 rpm) for 26 h at 37°C. After pelleting cells by centifuga-tion at 7000 g for 30 min the supernatant was 50, fold concentrated bydiaultrafiltration (Millipore) against 50 mM Tris, 5mM CaC^ and1 mM MgSO4. The concentrate contained a specific phytase activity of4.6 Uml"1.

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The E. coli phytase was produced by E. coli as recombinant protein,i.e., the phytase gene (from E. coli K12 ATCC33965) was cloned ona multi copy plasmid and overexpressed. Through the use of a specialsecretion system (Miksch et al., 1997), the E. coli phytase can berecovered from the fermentation medium and produced with highefficiency. The enzyme has been characterized as 6-phytase (Greineret al., 1993).

2.2. Estimation of Phytase Activity

Determination of phytase activities based on the estimation ofinorganic orthophosphate released on hydrolysis of phytic acid wasroutinely performed at 37°C following the method described byEngelen et al. (1994). One unit of enzyme activity was defined as theamount of enzyme that liberates 1 umol of inorganic orthophosphateper min under assay conditions. All measurements were performedin duplicate and repeated in some cases if there was discrepancy induplicate values.

2.3. pH Behaviour

For the study of pH behaviour, each phytase was diluted in 200 mMNa-acetate buffer, pH 5.5. Substrate solution was prepared in one ofthe following buffers: 200mM glycine, pH 2.0, 2.5 or 3.0; 200 mMNa-acetate buffer, pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5 and 200mMTris-HCl, pH 7.0, 7.5, 8.0, 8.5 or 9.0. All buffers were supplementedwith 1 mM CaCl2. The substrate solution contained lOmM phytic acidfrom rice (C6H6O24Nai2; Sigma-Aldrich Chemie GmbH, Steinheim,Germany).

Two millilitres of enzyme preparation were preincubated in awaterbath at the assay temperature for 5 min, and the enzyme reac-tions were initiated by adding 4 ml of the substrate solution. Since themixing ratio slightly altered the pH of the mixture, the pH of themixture was adjusted to the desired pH before incubation. The mixturewas incubated for 60 min at a temperature of 37°C. The incubationwas terminated by adding 4 ml of molybdovanadate reagent. Thereagent was prepared as described by Engelen et al. (1994). Then theactivities of the enzymes were determined.

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2.4. Thermal Behaviour

For the determination of the optimum temperature curves, pre-paration of enzyme and substrate solutions, as well as their mixingratio were as described above. However, the pH of the mixturescorresponded to the determined optimum pH of the respectiveenzyme. The mixtures were incubated for 60 min at each of thefollowing temperatures: 30, 40, 50, 55, 60, 65, 70 and 80°C. Theactivity was measured on the basis of inorganic orthophosphatereleased. For thermostability study in aqueous solutions, phytaseswere preincubated at 50, 60 and 70°C for 10, 20, 40, 60 and 120 min.After the preincubation periods, the samples were cooled on ice for30min. They were reincubated at 37°C and the residual activities ofthe enzymes were determined as described above.

For thermostability study in feed mixtures, a practical dietcontaining wheat as a major ingredient and fortified with vitaminsand minerals was chosen for pelleting experiments at different pelletingtemperatures. Since wheat contains an appreciable quantity of nativephytase activity, the diet was first pelleted at different temperatures inorder to measure the inactivation of the native phytase activity. Heattreatments were varied by modifying the steam introduction intothe conditioner and temperatures of 60, 70 and 80°C were adjusted inthe conditioner. Temperature control in the conditioner was madecontinuously by a sensor incorporated in the machine. For pelleting, adie with holes of 5 mm diameter and 15 mm length was used. Forcalculating the residual activity of added phytases, the native phytaseactivity at each temperature treatment was substracted from the totalactivity. Fungal phytases were added to the mash feed at 2,000FTU/kg and the E. coli phytase was added at 500 FTU/kg because oflimited amounts available of this enzyme. Bacillus phytase could notbe included in the pelleting experiments because of insufficientquantities. The pellets were cooled subsequently in a batch cooler.Samples of the resulting pellets were analysed for the level of phytaseactivity remaining relative to that added to the meal and taking intoconsideration native phytase activity at each temperature treatment. Incase of Peniophora phytase, temperature stability during pelletingwas measured at its pH optimum (pH 4.5) and at pH 5.5 as recom-mended by the manufacturer while other enzymes were measured attheir respective pH optima.

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2.5. Resistance to Protease Inactivation

The resistance of the phytases to protease inactivation was investi-gated using pepsin from porcine stomach mucosa and pancreatin fromporcine pancreas. The pepsin, Sigma P7012 (Sigma-Aldrich ChemieGmbH, Steinheim, Germany) contained 2,500 to 3,500 units ofactivity per mg protein and the pancreatin, Sigma PI500, from thesame source, contained activity equivalent to the United StatesPhamacopeia (U.S.P). Pepsin was suspended with 0.1 M HC1 (pH 2.0)and pancreatin was dispersed in 0.1 M NaHCO3 (pH 7.0).

For assays with pepsin, 1 ml of a freshly prepared pepsin solutioncontaining 3000 U/tnl was carefully mixed with 1 ml of a freshlyprepared phytase solution (0.02 and 0.08 FTU/2 ml after dilution withbuffer at the final stage of measuring phytase activity) in a test tube.The mixture was incubated for 0 and 45 min in a waterbath at 37°Cand pH 2.0 (optimum conditions for pepsin activity). After incubation,1 ml of the solution was diluted (1:9) with buffer solution (pH 5.5) andthoroughly mixed. 2 ml of the solution was incubated with 4 ml phyticacid substrate solution for 60 min at 40°C and pH 5.5 and phytaseactivity was determined. For assays with pancreatin, 1 ml of a freshlyprepared pancreatin solution containing 4.81 mg/ml was carefullymixed with 1 ml of phytase solution. The mixture was incubated for 0and 45 min at 40°C and pH 7.O. Dilutions and pH adjustments forphytase activity measurements were the same as described above.

2.6. Stability in Digesta Supernatants

Digesta samples were collected from laying hens. The birds were killedby cervical dislocation and their digestive tracts were removed. Digestasamples were collected from crop, stomach (proventriculus), duode-num (pylorus to entrance of bile ducts), jejunum (bile ducts entrance toMeckeFs diverticulum), and ileum (Meckel's diverticulum to the ileo-cecal junction). The pH of the digesta samples were determined using adigital pH meter (Ingold Messtechnik AG, Urdorf, Switzerland). ThepH readings of the various segments were 5.02, 2.75, 6.28, 6.63 and6.98 for crop, stomach, duodenum, jejunum and ileum, respectively.

The samples were either frozen at — 20°C until use or usedimmediately. Digesta samples were diluted 1:1 in distilled water,mixed thoroughly and centrifuged at 10,000 g for 10min. Supernatants

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were recovered and their pH values were adjusted to correspond to theinitial pH values of the different segments of the digestive tract. Therecovered digesta supernatants were held in an ice/waterbath untiluse. For assays, 1 ml of digesta supernatants was mixed with 1 ml ofenzyme solution and the mixture was incubated for 0 and 20 min at40° C. For measuring residual phytase activity, 1 ml of the solution wasdiluted (1:9) with buffer solution (pH 5.5). 2 ml of the solution wasthen mixed with 4 ml of substrate solution and incubated for 60 minat 40°C.

3. RESULTS AND DISCUSSION

3.1. pH Behaviour

The optimum pH of each phytase preparation studied is presented inTable II and the activities when exposed to range of pH conditions areshown in Figures 1A and B.

All Aspergillus phytases displayed considerable activities betweenpH 2.5 and 6.0 (Fig. 1 A). At least from 50 to 100% of their activities atthe pH optimum were found in this pH range, having their highestactivities measured at pH 5.5. The Aspergillus R phytase from thetransgenic seeds had similar pH behaviour to other phytases ofthe same genetic origin. Compared to the phytases from Aspergillus,the Peniophora phytase had a narrow pH optimum, between 3.5 and5.0 (Fig. 1A). At least 80% of the maximal activity was observed inthis pH range, with the highest activity determined at pH 4.5. Theenzymatic activity of the E. coli phytase occurred at low pH values(Fig. IB). The highest level of activity was measured at pH 4.5 (similarto the phytase from Peniophora), and at least 60% of activity occurred

TABLE II Comparison of pH and temperature optima of phytase preparations

Name of phytase pH optimum Temperature optimum [°C\

50.050.050.050.060.060.0

Aspergillus AAspergillus RPeniophoraAspergillus TE. coliBacillus

5.55.54.55.04.57.0

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B

I -A- Aspergillus A -M- Aspergillus R -it- Peniophora -B- Aspergillus T | -E-coli -e-Bacillus

FIGURE 1 A: pH behaviour of fungal phytase preparations; B: pH behaviour of bacterial phytase preparations. The data are expressed as percentageof the maximal activity.D

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between pH 3.5 and 5.5. In contrast to this bacterial phytase and to thefungal phytases, the Bacillus phytase displayed a narrow pH optimumaround pH 7.O.

The production of free phosphate by phytate hydrolysing enzymesin the gastrointestinal tract (GIT) of monogastric animals is heavilydependent on the optimal pH of the enzyme preparation. TheAspergillus and Peniophora phytases showed pH optima between 4.5and 5.5. This pH range is typical for fungal enzymes (McNab, 1993;Wyss et al., 1999; Vahjen and Simon, 1999). Such pH values occurgenerally in the upper part of the GIT of poultry and pigs, indicatingthat these enzymes may preferentially be active in these segments ofthe GIT of host animals. Although it should be noted that enzymaticactivities were determined using pure substrate and under standardisedconditions, conditions to be encountered by these enzymes in the hostanimals would probably be far different. With regard to pH behaviour,E. coli phytase is similar to fungal phytases. For the Bacillus phytase,the pH optimum was around neutral pH. At least 70% of its maximalactivities could be found between pH 5.5 and 7.5. Usually mostenzymes of bacterial origin exhibit pH optima close to neutrality.Apart from the gastric region of poultry and the pig stomach, the pHconditions present in the GIT of these animals vary between 5 and 7(McNab, 1993) and this indicates that phytases of Bacillus origin couldact especially in post gastric segments.

3.2. Thermal Behaviour

Table II shows the optimum temperature for each of the enzymeswhile Figures 2A and B are plots of relative enzyme activities atdifferent temperatures. Both bacterial phytases exhibited theiroptimum activities at higher temperatures than their fungal counter-parts. It worths mentioning here that the optimum temperatures (50 to60°C) of the phytases are higher than the temperature (about 38°C)obtainable in the body. Therefore, only between 40 to 60% of maximalphytase activities could occur in the body.

The results of further assays to investigate the stabilities of theenzymes in aqueous solutions after short and prolonged incubationat 50, 60 and 70°C are shown in Figures 3 to 5. With the exception ofthe Aspergillus T and Peniophera phytases, all other enzymes retained

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100 1

90

Ü 80 •

I" 70-I 60I 50 "& 40-u

•1 3 ° -I 20-

10 -

0 20 30 40 50 60

Temperature [°C]

70 80 90

|-A-Aspergillus A - » - Aspergillus R -A-Peniophora -B- Aspergillus T |

100-

90 •

^ 80

1 ™1< 60-

| 50-

£ 40-tu

•S 3 0 -

§ 20-|10

0

B

20 30 40 50 60 70

Temperature [°C]

I - • - E-coli -G- Bacillus I

80 90

FIGURE 2 A: Temperature profile of fungal phytase preparations; B: Temperature profile of bacterial phytase preparations. The data are expressed aspercentage of the maximal activity.D

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110 -i

40 60 80Incubation time [min]

20

I-Ar- Aspergillus A - « - Aspergillus R -A- Peniophora -B- Aspergillus T


|-*-E-coli - e - Bacillus]

100 120

FIGURE 3 Residual enzymatic activity of phytase preparations after exposure for different periods at 50°C. A: fungal phytase preparations; B:bacterial phytase preparations. The data are expressed as percentage of activity determined at 37°C for 60 min.

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I-A-Aspergillus A - » - Aspergillus R -A-Peniophora - S - Aspergillus T |


|-»-E-coli -©-Bacillus!

100 120

FIGURE 4 Residual enzymatic activity of phytase preparations after exposure for different periods at 60°C. A: fungal phytase preparations; B:bacterial phytase preparations. The data are expressed as percentage of activity determined at 37°C for 60 min.D

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B

20 40 60 80

Incubation time [min]

100 120 20

| -&- Aspergillus A -M- Aspergillus R -A- Peniophora -B- Aspergillus T |


I - • - E-coli - e - Bacillus I

100 120

FIGURE 5 Residual enzymatic activity of phytase preparations after exposure for different periods at 70°C. A: fungal phytase preparations; B:bacterial phytase preparations. The data are expressed as percentage of activity determined at 37°C for 60 min.D

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close to 100% of their original activities even after prolongedincubation for 120 min at 50°C (Figs. 3A and B). The Aspergillus Aand R phytases could still retain about 50% of their initial activitiesafter short incubation for 20 min at 60°C while at the same incubationconditions, the E. coli phytase could retain above 70% of its initialactivity and the Bacillus phytase was completely stable at thistemperature (Figs. 4A and B). The higher thermal stability of theBacillus phytase compared to the other enzymes can also be seen fromthe inactivation plot at 70°C (Figs. 5A and B). It should be noted thatthe ranking of the fungal phytases regarding their activities after heattreatment changed depending on incubation temperature. This ishowever, unexpected and no reason can be given for such behaviour.

Thermal stability in aqueous media does not properly reflectstability in the feed pelleting process. Therefore, those enzymes whichhave potential application in animal feed were pelleted at temperaturesof 60, 70 and 80°C adjusted in the conditioner. One should take intoaccount that the temperature of the die will increase for 7 to 10°Cabove the temperature in the conditioner. However, this temperaturewill not be reached in the entire pellet and temperature measurementin the die is very complicated, therefore temperature was controlled inthe conditioner. The results of the pelleting experiment are presentedin Table III. Significant differences exist amongst the enzymes tested intheir abilities to withstand each processing temperature applied. Whenthe feeds were conditioned at 60° C, the recovery of activities (relativeto the content in feed before pelleting) were 84.0, 92.6 and 98.8% forAspergillus R, Aspergillus A and E. coli phytase, respectively. Above70% of the enzymatic activities of Aspergillus A and E. coli phytasecould still be recovered at 70° C processing temperature whileAspergillus T phytases was less stable under those conditions. It hadlost more than 30% of their activities even at 60°C. None of theseenzymes could survive conditioning temperatures higher than 70° C ata considerable degree. Temperature stability of Peniophora phytasediffer considerably when activity was measured at pH 4.5 and 5.5,respectively (Tab. III). While the enzyme showed high temperaturesensitivity at pH 4.5 but at pH 5.5 it was relatively stable. It is possiblethat this preparation contains two phytase fractions, one with low pHoptimum which is highly susceptible to temperature denaturationand the other with pH optimum 5.5 which is considerably stable to

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TABLE III Effect of pelleting on percent recovery1 of phytase preparations in feed

Phytase

NativeAspergillus AAspergillus RPeniophora3

Peniophora4

Aspergillus TE. coli

Phytase addition[FTU/kg]

-20002000200020002000

500

Before pelleting

100 (550)2

100 (1850)100 (1830)100 (2670)100 (2370)100 (1750)100 (410)

After pelletingConditioning temperature

60° C

90.992.684.026.874.551.998.8

70° C

36.470.562.315.766.225.078.0

80° C

11.830.919.80

57.06.5

24.4

1 Values measured in feed. When microbial phytase was added, the native phytase activity estimatedfor each treatment was substracted.2 Values in brackets represent analyzed phytase activities [FTU/kg] before pelleting.3 Measured at pH 4.5 (pH optimum for Peniophora phytase).4 Measured at pH 5.5 (pH recommended by the manufacturer).

temperature denaturation. At 80°C Peniophora phytase was morestable compared to the other enzymes. The thermostability resultsobtained from feed pelleting and in aqueous phase followed the sametrend. It appeared that the phytase from transgenic seeds AspergillusR is more susceptible to heat denaturation than its counterpartAspergillus A from the same genetic origin but expressed in fungi.

For an enzyme to be attractive for widespread application as feedadditive, it should be able to withstand temperature conditionsnecessary for pre-treatment of feeds. One common pre-treatment ofanimal feeds is pelleting. The results on thermostability presented inthis study demonstrate that in general thermostability of phytases isnot satisfactory and that differences between the enzymes in theirability to withstand temperature conditions do exist. From tempera-ture stability measurements in aqueous solutions, it can be concludedthat phytases originating from the bacterial genus Bacillus will be morestable than the tested fungal phytases and E.coli phytase. Forscreening of enzyme stability, measurements in aqueous media areadequate, however, an accurate prediction of heat inactivation duringpelleting is not possible on that basis. Therefore, direct pelleting trialsare necessary. Unfortunately, at present the Bacillus phytase could notbe included in these measurements since Bacillus subtilis 45 does notproduce sufficient amounts of phytase under the conditions used here.Recently, a more efficient expression system has been developed basedon the cloned gene under control of an inducible strong Bacillus phase

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promotor (Farouk, unpublished data) also permitting to study the invivo efficacy of Bacillus phytases in near future. The pelleting resultsreveal that Aspergillus A, Peniophora and E. coli phytases are capableof maintaining high stability (at least 65% of their activities) atprocessing temperatures of 70°C. Assuming that most broiler andpiglet diets are pelleted at temperatures around 70° C, the stability ofthese enzymes could be regarded as reasonably good. Previous studies(Simons et al, 1990; Simoes-Nunes, 1993; Wyss et al, 1998) haveshown that pelleting feeds containing phytase at temperatures above60°C resulted in a significant loss of activity. The present studysupports, in part, this finding.

3.3. Resistance to Protease lnactivation

The resistance of phytases to inactivation when exposed for 60 minto pepsin or pancreatin are presented in Table IV. Both proteolyticactivities of the proteases effectively inactivated all tested fungalphytases. However, the effect was more pronounced on Peniophoraand Aspergillus T phytases compared to Aspergillus A and R phytases.The Bacillus phytase was susceptible to pepsin inactivation at the samedegree as the fungal phytases but was rather resistant in the presenceof pancreatin. Under conditions of this study, both pepsin andpancreatin had no apparent effect on the enzymatic activity of E. coliphytase.

The results obtained under the conditions of this experiment suggestthat exposure of phytate hydrolysing enzymes to endogenouslysecreted protease activities in the host animals would limit theirpotential activities. The only exception was the E. coli phytase whichwas found to be resistant to both pepsin and pancreatin inactivation.Also one may expect that this enzyme will be transferred into thesmall intestine in a more active form compared to other enzymesfurther phytate hydrolysing effects in the lower part of the alimentarytract will be of little importance due to the neutral or mild alkalineconditions in those segments. Furthermore, residual phytase activitiescould only act in case of the Bacillus phytase due to its pH optimum atpH 7.0, while other phytases are not active at these pH conditions (seeFigs. 1A and B). The resistance of E. coli and Bacillus (especially topancreatin) phytases to protease inactivation cannot be explained

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TABLE IV Residual phytase activities after incubation at 40°C in a solution con-taining porcine proteases

Phytase Pepsin Pancreatin

Aspergillus A 25.9 22.6AspergillusR 32.2 26.7Peniophora 1.8 0Aspergillus T 8.1 0E. coli 94.6 95.9Bacillus 19.3 91.1

The data are expressed as percentages of activity determined at 40° C for 60 min incubation afterOmin preincubation with the respective protease.

based on the fact that these enzymes are described as 6-phytasesbecause Peniophora, also a 6-phytase, was susceptible to proteaseactivities. The resistance can be attributed to molecular structure.

3.4. Stability in Digesta Supernatants

The results of incubating the phytases in digesta supernatants fromdifferent segments of the GIT of chickens are shown in Table V. Itshould be kept in mind that the results represent the stability in digestaof each segment of the GIT independent of the results in thepreceeding segment incubated in fresh or frozen digesta supernatants.The most pronounced effects on the activities of enzymes wereobserved in supernatants from stomach. Again only the E. coli phytasewas found to be very stable in digesta supernatant from the stomach.Stable activities of all enzymes were observed in the supernatants ofcrop and duodenum contents. Furthermore, the Bacillus phytase,besides the E. coli enzyme, displayed high stability in the digestasupernatants from various segments of the small intestine. When

TABLE V Residual phytase activities [%] after 60 min incubation at 40°C in digestasupernatants from various segments of the digestive tract

Name of phytase

Aspergillus AAspergillus RPeniophoraAspergillus TE. coliBacillus

Crop

98.597.496.892.696.993.5

Stomach

60.467.859.256.892.870.8

Duodenum

93.696.494.890.396.895.3

Jejunum

60.290.091.142.986.791.5

Ileum

54.581.384.856.480.497.3

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Aspergillus phytase was expressed in rape, it seems to be more stableagainst proteolytic inactivation than the enzyme in its homologoushost system.

These results confirm the results obtained for resistance to proteaseinactivation as well as pH behaviour. Since temperature conditionsremain fairly constant within the GIT, endogenous proteolyticactivities and considerable pH variations within the GIT could beresponsible for the variations observed in the activities of the enzymes.The enzymes suffer considerable loss of activities in the stomach due tolow pH and the action of secreted proteolytic enzymes present in thisregion. The high activity of the E. coli phytase in the stomach could beattributed to its ability to maintain its activity in the presence ofproteolytic activities and low pH. Most enzymes show appreciablestability in conditions of the small intestine. This is especially valid forBacillus phytase in those segments where the pH values reach nearlyneutrality. However, in in vivo application, it is doubtful if this enzymewould be able to reach the lower gut before inactivation, particularlyin the gastric region. Quantitative in vivo measurements are necessaryto ascertain the results of this study.

4. CONCLUSIONS

All phytase preparations used as feed additives today are of fungalorigin. For these enzyme preparations the efficiency in improvingphosphorus availability from feed of plant origin is well documented.However, fungal phytases have some shortcomings, i.e., theirsensitivity to heat treatment and their inactivation under theconditions present in the stomach. These seem to be general featuresof fungal enzymes, although different properties depending on thesource of phytase gene and production organisms can be considered.Therefore, for further developments, it is of interest to look for othersources of phytases. The existence of bacterial phytases is well knownand the molecular structure of an E. coli phytase (Lim et al., 2000) anda Bacillus phytase (Ha et al., 2000) were discovered recently. Bothenzymes do not exhibit any sequence or structural similarity and havebeen evolved independently from different families of acid or alkalinephosphatases. However, as shown in the present study, among

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bacterial phytases, an enzyme with high temperature stability {Bacillusphytase) or high proteolytic stability (E. coli phytase) do exist, waitingfor exploiting for their favourable properties as feed additives. Forfurther developments it seems advisable to consider these aspects.

Acknowledgments

Research stay of Dr. F. A. Igbasan in Germany is being supported byAlexander von Humboldt Research Foundation. Technical assistanceby Dr. K. Schäfer, Mrs. Anneliese Lenke and Mrs. Sybille Striegel isgratefully acknowledged.

References

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