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Immobilization of a pectinlyase from Aspergillus niger for application in food technology
Immobilization of a pectinlyase from Aspergillus niger for application in food technology Giovanni Spagna, Pier Giorgio Pifferi, and Edmondo Gilioli School of Specialization in Food Chemistry and Technology, Biotechnological Research, Faculty of Industrial Chemistry,
and Interdepartmental University of Bologna,
Center of Bologna. Italy
The immobilization of a commercial preparation ofpectinlyase (PL, EC 4.2.2.3) derived from Aspeqillus niger was studied in view of its possible application in fruit juice treatments. Pectinlyase is an enzyme that is subject to growing interest for the substitution of other pectic enzymes: e.g., polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1 .I .ll). The PL was immobilized by physical adsorption or by the formation of covalent bonds on organic (cellulose and its derivatives, XAD-amberlites) and inorganic (sulphides, y-alumina, and bentonite) supports. The supports that permitted an effective immobilization with good activity levels and sufficient stabiliry under the operational conditions were found to be the acrylic resin XAD 7 activated with trichlorotriazine and, in particular, bentonite activated with glutaraldehyde. Keywords:
Immobilized
enzymes;
pectic enzymes;
pectinlyase;
Introduction Pectic enzymes are currently used in numerous industrial processes, and in particular in food technology. The main applications include increases in filtration speed and in the amount of juice extracted from fruit pulp, reduction in turbidity, fruit juice clarification, and preparation of CY-Dgalacturonic acid. ’ Among pectic enzymes, pectinlyase (PL, EC 4.2.2.3) is the most interesting, as it is the only one capable of depolymerizing pectins without altering their esterification level.* In fact, PL is an endoenzyme that exerts its action directly on highly esterified pectins through a p-eliminative cleavage of the glycosidic linkages. This leads to the formation of an a,P unsaturated uranic acid residue on the nonreducing side, and to a hemiacetal one, on the reducing side of the glycosidic linkage, with the loss of a water molecule. The present study carries on from previous ones conducted on the immobilization of PL from the Aspergillus niger contained in a commercial enzymic preparation.3,4 The aim of this study was to examine the possibility of
XAD 7; bentonite;
fruit juices
using this enzyme in continuous processes for the treatment of fruit juices. Some of the supports employed for PL immobilization were nylon polyethylene imine copolymer,’ Eudragit L,6 porous glass and DEAE cellulose activated on titanium salt,’ Eupergit C3 y-alumina,’ and nylon 6 and 11.’ More precisely, this article reports the results of PL immobilization on organic (cellulose and its derivatives, XAD-amberlites) and inorganic (sulphides, y-alumina, and bentonite) matrices through simple adsorption or after activation with glutaraldehyde. The acrylic resins have been also activated, after hydrolysis, with carbodiimide, thionyl chloride, and trichlorotriazine. Among the various immobilization procedures adopted, the PL immobilized on XAD 79,‘o and bentonite (BDH)““’ activated were found to be the most active and simultaneously to possess optimum stability over time, in conditions that may potentially be set up for industrial applications.
Materials and methods Materials The enzymic preparation Cytolase PCLS (PCLS, batch 45; Genencar. USA) was employed as the PL source after purification by means of adsorption on BDH (Switzerland).
Address reprint requets to Dr. Giovanni Spagna, School of Specialization in Food Chemistry and Technology, Faculty of Industrial Chemistry. University of Bologna. Viale Risorgimento 4, 40136 Bologna, Italy Received 16 May 1994, revised 30 November 1994; accepted 13 December 1994
Enzyme and Microbial Technology 17:7X&738, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
After esterification, apple pectin (Fluka, Switzerland) was used as the enzyme substrate. For protein determination, bovine serum albumin (BSA; Pentex, USA) was employed as standard and Coomassie Brilliant Blue G-250 (Serva, Germany) was used as a calorimetric agent.
0141-0229/95/$10.00 SSDI 0141-0229(94)01134-D
Papers The following materials were used as supports for immobilization: cellulose (cell; Merck, Germany); cellulose triacetate (TAcell; Merck); polyethylene imine cellulose 300 (PEI-cell; Baker, Holland); XAD-amberlites: aromatics 2, 4, 16, and acrylics 7 and 8 (Rhom and Haas, USA); technical bentonite powder (BDH); y-alumina (130-140 A; Akzo); iron, molybdenum, and stannic sulphides (Strem Chem.), all of which feature a granulometry between 77 and 177 pm. We used 50% glutaraldehyde (GA; Fluka), trichlorotriazine (TCT; Fluka), thionyl chloride (EGA-Chem., Germany), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimmide HCl (EDC; Carlo Erba) as activating agents. Other reagents were 3-methyl-2benzothiazolinone hydrazon chlorohydrate (MBTH; Carlo Erba), N-N-dimethyl formamide (Carlo Erba), and iron trichloride (Merck). All the other reagents not specifically mentioned were supplied by Carlo Erba.
Pectin esterification Two hundred grams of apple pectin were first washed with 60% and 96% ethanol and then with anhydrous methanol to eliminate several impurities such as polyphenols. Pectin was added to 1.5 1 of a cold 0.5 M solution of H,SO, in anhydrous methanol and stored in the refrigerator at 3°C (so as to avoid substrate hydrolysis) for 4 weeks, stirring every now and then. The pectin thus esterified was then filtered and washed fist with 96% ethanol, then with methanol and ethyl ether, and finally allowed to dry overnight in an oven at about 40°C.i3 The pectin esterification level as then determined by means of acid-base titrationi using a mixed indicator consisting of aqueous solutions of 0.4% Bromothymol Blue (1 vol), 0.4% Cresol Red (1 vol), and 0.4% Phenol Red (3 vol) to highlight the color change. We equilibrated 100 ml of a 1% pectin aqueous solution containing two to three drops of the indicator at 3°C overnight and titrated with 0.5 M NaOH. The final titration point was indicated by a red color lasting for at least 30 s (pH 7.5). We then added 20 ml of 0.5 M NaOH and after 30 min titrated excess alkali with 0.5 M HCl until the indicator turned yellow. A blank test was simultaneously performed using distilled water instead of the substrate. The esterification grade was calculated on the basis of the difference between the two titrations given as the amount of soda required for saponification. The average molecular weight (M,) of the esterified pectin was determined by viscosimetty. is Pectin solutions of concentrations from 0.03 to 0.05% were prepared in 0.195 M NaCl and allowed to equilibrate at 3°C. After heating the solution in a thermostated bath to 37°C viscosity was measured by means of the Ostwald capillary viscosimeter. The relationship between viscosity (q) and molecular weight (M) is expressed as: q = K . Ma, where for pectin, K = 1.4 . 10m6 and CL= 1.34.
PL purification The PCLS enzymic preparation was purified according to a method described previously. I6 We added 4 g of bentonite under stirring to 50 ml of 0.07 M citric-phosphate buffer (C-P) at pH 5.0 and sonicated it for about 20 min at 25°C. Subsequently 50 ml of 0.07 M C-P at pH 5.0 and 100 ml of PCLS were added to the suspension thus obtained. After having adjusted the pH value to 5.0 we stirred the suspension for about 2.5 h, and then centrifuged it at 2,600 g for 20 min. The solution was treated again for 2.5 h with 4.0 g of bentonite mixed with 50 ml of 0.07 M C-P at pH 5.0, before being centrifuged once more. The enzymic solution was then diluted twice with water and brought to a pH of 3.2 to 3.4 by adding 1 M HCI, after which it was placed in a refrigerator of 3°C overnight. The precipitate thus obtained, essentially made up of polisacharides, extraneous proteins, and brown pigments, was
730
Enzyme Microb.
Technol.,
1995, vol. 17, August
separated by centrifugation at 5,500 g for 60 min at 3°C and the purified PL containing solution was then used for immobilization. Protein determination was performed according to Bradford methodology.”
Acrylic resin hydrolysis XAD 7 (or XAD 8) was washed five times with water and methanol and then left to dry in an oven at about 55°C after which 20 g of dry resin was suspended in 200 ml of a 2.5 M solution of NaOH containing 10% isopropanol. Hydrolysis was performed by reflux under stirring for about 24 h. The support thus hydrolyzed was then washed several times with water until neutral pH was reached. Finally, it was washed with methanol and once more dried in an oven. To determine the carboxylic groups formed,is we added 10 ml of dimethylformamide to about 0.5 g of hydrolyzed XAD 7 and left the solution to equilibrate for 1 h under stirring at 25°C. Three drops of a 1% Thymol Blue solution in ethanol were then added and titration was finally performed with 0.05 M sodium metoxide dissolved in a benzene-methanol solution (7:3 v/v).
Activation
with glutaraldehyde
We placed 4 ml of a GA solution (2%) dissolved in a 0.05 M buffer at pH 3.5 (citric-phosphate; C-P) or at pH 10 (carbonatebicarbonate) in IO-ml tubes containing 10 mg of the various supports (PEI-cell; Sn sulphide, y-alumina; bentonite and hydrolyzed acrylic resins). After activation in a rotary carousel for 1.5 h at 25°C. the supports were centrifuged, washed at least two or three times in 10 ml of water, and then checked for the eventual presence of GA in the rinse waters by reading absorbance at 235 nm. The amount of GA bound to the support was determined on the basis of the difference between the amount of equilibrated GA and that measured in the solution according to Bersthom’s hydrazone method, without the oxidation step. I9 Two milliliters of standard GA solution (0.01-0.2%) or of an appropriately diluted sample was placed in a 20-ml flask and made to react at 100°C with 2 ml of 0.4% MBTH. After 3 min the mixture was rapidly cooled in an ice bath. We then added 5 ml of 0.4% FeCl, and the solution was brought to volume with acetone. After 15 min at 25°C absorbance of the standard solution or of the sample was read at 635 nm against the blank, which had been obtained by replacing the GA solution with an equal volume of the same solution in which the GA had been dissolved.
Activation
of hydrolyzed acrylic resins
Activation with carbodiimide. Four milliliters of 0.3% EDC in water acidified at pH 4.5 with HCl were placed in lo-ml tubes containing 10 mg of hydrolyzed XAD 7 (or XAD 8). After support activation in a rotary carousel for 2 h at 25”C, the supports were washed with 10 ml water. Activation with thionyl chloride. We converted 5 g of hydrolyzed XAD 7 (or XAD 8) to the corresponding acrylic chloride by reflux treatment under stirring for 6 h with 50 ml of a 2% SOCl, solution in chloroform. Activation with trichlorotriazine. We placed 4 ml of 1.2% TCT solution in dioxane in lo-ml tubes containing 10 mg of XAD 7 (or XAD 8). After activation in a rotary carousel for 20 min at 25”C, the supports were washed twice, first with 10 ml dioxane and then with 10 ml water.
Immobilization of Aspergillus pectinlyase: G. Spagna et al. Immobilization method Between 60 and 100 U of purified PL, brought to the desired pH by the addition of 0.1 M HCl or NaOH, was placed in lo-ml tubes with 10 mg of the various activated or nonactivated supports. After about 16 contact h in a rotary carousel (about 40 rpm) at 4”C, the tubes were centrifuged. The enzymic activity of the supernatants was directly essayed; that of the residues was essayed after being washed with 10 ml of 0.5 M NaCl and 10 ml of 0.5 M C-P at the pH of the enzyme essay. In some cases, PL was equilibrated before immobilization for about 30 min in pectin or albumin solutions (up to 0.5%). In the case of the hydrolyzed XAD 7, the immobilized enzyme was also reticulated at 4°C for 15 min with a 0.1 and 0.01% GA solution in 0.01 M C-P buffer at pH 3.5, after the supports were washed with NaCl and buffer solutions.
Determination ofPL activity We added 3 ml of I. I% esterified pectin, dissolved in 0.05 M C-P buffer at the established pH. under magnetic stirring at 25”C, to 2.0 ml of PL in solution or PL immobilized suspended in the same buffer in a lo-ml tube. After 1 min, to stop the reaction 2 ml of 0.5 M HaSO, was added. For the blank test the order of the reagents was reversed (i.e., the acid was first added to the enzyme, followed by the pectin solution). In the case of the immobilized enzyme, the tubes were centrifuged. The sample solution was read against the blank solution at 235 nm. The increase in absorbance was due to the double conjugate bond of the A 4:5 unsaturated uronide formed during the reaction. One unit of the enzyme is defined as the amount that catalyzes an increase of 0.555 in absorbance at 235 nm, in 1 min at 25°C and at optimal pH.4.20
Storage stability The enzymic activities of the soluble and of the immobilized enzymes were measured in relation to time (up to a maximum of 700 h) at pH 3.5 and 5.0 (0.05 M C-P) and at various temperatures (25-55°C).
Characterization of the free and immobilized enzymes The following parameters were determined for the free enzyme and for the enzyme immobilized on bentonite activated with GA: optimum pH (between 3.5 and 7.5) at 25°C; optimum temperature (between 20 and 90°C) at optimal conditions; adsorption isotherm (by equilibrating up to 250 U of PL); and finally, the and V (with solutions 1%).
PL immobilization Cellulose
by adsorption
and derivatives.
The cellulose showed a suffi(AY) with a poor catalytic response (IY and U,,,. Table 1). Probably, the crystalline structure of this support contributes to reduce adsorption. Moreover, the likeness between cellulose and the pectin may have oriented the enzyme molecule with the active site facing the support,
cient
adsorption
reducing the substrate accessibility (higher steric hindrance). The presence of the substrate during immobilization caused a drop in adsorption (AY) and only a slight increase in activity (v,,,). The long chain of the pectin molecule (about 650 A), by interacting with the enzyme, may have hindered its anchoring on the support, protecting the active site and the conformational structure. With the cellulose triacetate (TA-Cell) AY increased while the activity dropped to zero. The presence of esterified groups reduced hydrophilic structure of this support, making it more similar to the pectin. On the contrary, with PEI-cellulose (PEI-cell) a decrease in AY and an increase in IY and U,,, were observed. The higher hydrophilia of this support and its poor interaction with the enzyme at pH 3.5, resulting from the positive charge of both support and enzyme, may account for this behavior. Inorganic supports. As can be seen in Table 1; sulphide supports are generally characterized by high AY and low IY values. Despite featuring minimum surface areas (< IO m2 g- ‘), these supports feature a very high site density, especially at the fracture zones of superficial faces.22 These
Table 1 PL immobilization on cellulose, its derivatives, and inorganic support by adsorption and covalent attachment with GA at pH 3.5
supports
Pectin
Adsorption Cellulose
of Adsorption follows: UadS units,
with an esterification grade of 93% and an M, of about 28,000; see Materials and methods). Moreover, the active site of the enzyme seemed to contain a carboxylic group and a tyrosine residue. apple pectin
(AY) = the V,,,
+
immobilization ’ IY enzyme active
(IY) defined I/,,, Clads 100, U,, equilibrated enzyme
Results and discussion After purification, the enzyme used had a specific activity of about 20 U mg - ’ of protein. To better interpret the results obtained, the main characteristics of the enzyme*’ are worth bearing in mind. These include: an M, of about 38,000; a dimension of about 30 to 40 A, an isoelectric pH (PI) of 3.6; an optimal pH of about 6.4 (determined using an
TA-cell
+
PEI-cell FE sulphide MO sulphide Sn sulphide y-Alumina Bentonite Covalent (with GA) PEI-cell Sn sulphide V-Alumina
lzyme Microb. Technol.,
f
AY
IY
u act (U g ‘1
25.0 16.7 51.2 27.5 9.0 48.3 54.6 43.0 12.0 7.8 5.6 17.5
0.2 0.6 0 0.2 2.5 0.1 0.1 0.7 3.5 4.3 16.0 4.6
3.0 6.0 0 2.5 15.5 3.0 4.5 17.0 28.0 22.0 65.0 62.0
25.6 2.9 18.5 16.0
0.3 20.5 8.9 5.6
5.5 41.1 115.0 65.0
1995, vol. 17, August
731
Papers strong aspecific interactions may account for the high adsorption, and moreover, being very close, may cause structural distortions of the enzyme molecule and its inactivation. For the stannic sulphide AY values diminished considerably and IY ones increased following the addition of substrate. Although y-alumina has a high density of acidic (Lewis and Bronsted) and basic sites,23 the yield values are quite low. As regards bentonite, AY values were low, whereas the IY and activity values are the highest in Table 1. The presence of pectin caused an increase in AY, which is probably ascribable to the affinity of this support to this compound24 and a proportional decrease in IY, so
XAD-amberlites. XAD-amberlites are resins having a different chemical structure and featuring no functional ionic groups. The XAD 2,4,16 resins are divinylbenzene styrene copolymers (aromatic) at low polarity (p 0.3); the XAD 7 and 8 resins are acrylic ester-based copolymers (acrylic) at a higher polarity (p 1.8), and therefore with a more hydrophobic structure. Moreover, surface area and pore size changed as the degree of reticulation of XAD resins changed. For aromatic resins high AY values are observed (Table 2). They diminished in the presence of pectin (XAD 2 and 4) and remained almost the same, even when the surface area and pore size changed. This seems to suggest that the substrate reduces interactions between enzyme and matrix and, moreover, that the absorption prevalently takes place on the external surface of the matrix, perhaps also because of the great molecular dimensions of the enzyme-substrate complex (molecular exclusion effect). In all cases, however, IY values are low or practically zero. This may be due to (1) the strong hydrophobic interactions of the enzyme with the matrix, thus causing conformational changes in the secondary and tertiary structure of the protein molecule; (2) the interaction between the aromatic rings of the support and the enzyme, which may also involve the tyrosine residue present in its active site. Table 2
PL immobilization
on XAD-amberlites
Acrylic resins generally show less adsorption (AY) associated, in particular for XAD 7, with higher catalytic response (IY and Uact) (Table 2). This behavior is probably due to a lower hydrophobicity of such resin, which modifies the molecular structure of the enzyme to a lesser extent,‘5 as well as its negative dipole moment, which may orientate the active site (carboxylic) of the enzyme outward, increasing the substrate accessibility (lower steric hindrance). Data concerning the pore size and surface area of acrylic resins (Table 2) cannot be directly compared with aromatic ones, as they are related to the dry product and change in an aqueous environment resulting from a marked swelling of the acrylic matrix (about 2.2 times for XAD 7). Moreover, findings reported for XAD 7 were seen to be the same with or without pectin. This result would once again seem to suggest that PL is prevalently adsorbed on the external surface of the matrix. This hypothesis was found to be correct for the adsorption on XAD 7 of the trypsin, an enzyme having dimensions lower than those of PL.9 In fact, the enzyme molecules may also be adsorbed onto the openings of the pore, or close to them. Thus, the penetration of other molecules into the internal surface of the substrate is blocked or reduced; moreover, the diffusion coefficient decreases further for the low temperature (4°C) employed during immobilization. The activity (U,,) of the immobilized PL on XAD 7 as such is maximum at values close to p1 (Figure I), that is, when the enzyme molecules are less susceptible to conformational distortions due to inter- and intramolecular electrostatic repulsion, and therefore more capable of protecting themselves during immobilization. Under these conditions, the molecules take on the optimum configuration at the minimum exposed surface. Instead, the adsorption trend (U,,,) is scarcely affected by pH (Figure 1). As far as XAD 8 support is concerned, AY values are lower, probably because of the lower surface area. Acrylic resins were also hydrolyzed, introducing carboxylic (0.66 mEq g ~ ‘) and oxydrylic groups to increase their reactivity and hydrophilia (Table 2). Moreover, this procedure decreased surface areas and increased pore sizes, at least in the zones (probably superficial ones) where hydrolysis occurred. For both acrylic resins the most outstanding
by adsorption
Surface area
Pore size
supports
(m* g-‘1
(A)
Pectin
AY
IY
Aromatic XAD2
300
90
XAD4
725
40
XAD 16
800
100
_ + + +
38.0 15.0 30.0 14.1 18.5
0.4 0.4 0.1 0 2.2
11.5 7.0 2.0 0 36.0
450
90
_ + _ +
20.0 18.1 10.0 12.1
13.5 12.5 2.6 2.0
188.1 22.0 20.2
-
14.5 13.0
30.0 10.5
275.0 84.0
Acrylic XAD7 XAD8
160
Acrylic hydrolyzed XAD7 XAD8
732
Enzyme Microb.
225
420
Technol.,
1995, vol. 17, August
u act FJ g-‘)
190.1
Immobilization
0
0 2.5
3
3.5
4
4.5
5
5.5
6
6.5
PH Figure 1 Activity (Uact) and adsorption (Uads) of PL immobilized at different pH values on XAD 7 nonactivated (as such, hydrolyzed) and activated with TCT
effect of the hydrolysis was the marked increase in IY values. This may be due to the further increase in hydrophilia and probably in pore size, which increase the accessibility of substrate. Instead, as regards the enzyme adsorption, the hydrolysis process gave contrary effects. Finally, the hydrolysis did not influence the optimum immobilization pH (Figure I ) _ PL immobilization
by covalent attachment
Glutaraldehyde-activated supports. Support activation with GAz6 is one of the most widely used techniques, as it is simple and inexpensive. In our case, it also offered the advantage of not modifying the carboxylic function present in the active site of PL. However, GA presents several drawbacks, such as excessive reactivity and the complex reaction mechanism with the nucleophilic groups, which is not yet fully understood; thus, the effects of GA on enzymic activity cannot be fully predicted. A possible explanation, however, may be attempted. Probably, the nucleophilic groups of the support and of the enzyme, such as the aminic ones, mainly reacted with aldehydic groups (Schiff base) and also with the double bonds originating from the aldolic condensation of the GA (Michael-type adduct). As pH varies, the activation reaction with GA follows a different pattern, causing a different reactivity of the activated support. In an acid environment, GA molecules individually react with the active centers of the support, making a large number of aldehydic groups available to form enzyme bonds. In an alkaline environment, the GA molecules tend to form o-p unsaturated polymers (aldolic condensation), causing a polyaldehyde net to be formed on the support. This net reduces the amount of available aldehydic groups and normally makes the matrix more hydrophobic. The nonactivated supports that exhibited higher activities were activated with glutaraldehyde (Table I). The activation of PEI-cell caused a notable increase in AY and decrease in IY and U,,, values. These findings are probably due to the high density of the GA bound to the
of Aspergillus
pectinlyase:
G. Spagna et al.
aminic groups of the matrix, which may cause a partial unfolding of the enzyme. The activation of inorganic supports with GA is a procedure that has already been adopted to immobilize polygalacturonase and pectinesterase on y-alumina.** It leads, in the case of Sn sulphide, to a decrease in AY and a notable increase in IY. Evidently, the bound GA cannot compensate for the loss of several reactive centers on the support surface, whereas it can reduce its influence on the immobilized enzyme. As regards y-alumina, the GA activation increased both of the yields, allowing good activity. No variations in yields and activity were observed for GA-activated bentonite. (This support shall be examined in greater detail further on.) In all trials, when activation pH was raised from 3.5 to 10, the AY values tended to increase; IY values tended to decrease, showing generally lower activities (data not shown). This seems to confirm the hypothesis on the influence of pH for activation process with GA.
Activated acrylic resins. Acrylic hydrolyzed resins, given the good findings obtained by simple adsorption (Table 2), were activated with GA, EDC, SOCl,, or TCT (Table 3). The XAD 8 is not thawed because after activation, it presents the same relative trends of XAD 7. The activation of XAD 7 has not generally caused substantial variations in AY, probably because of the low number of reactive groups of the hydrolyzed matrix. It would therefore seem that the competition between covalent binding and physical adsorption is decidedly in favor of the latter. On the other hand, higher variations in IY and activity values were observed. No improvement in IY and activity was observed for GA-activated XAD 7 as compared to the hydrolyzed one. This behavior may be accounted for by the fact that the amount of matrix activated with GA was very low. This is an atypical situation, as no aminic groups were available on the support to promote the classical coupling reaction. It might be that the GA interacted with the oxydrylic and carboxylic groups of the matrix via hydrogen bonds. An overall reduction in yields and activities (U,,,) was reported for activation with EDC and SOCl,. On the contrary, as concerns activation with TCT (XAD7-TCT), both yields slightly increased, so that activity increased (from 275 to 335 U g - ‘). The activity (U,,,) and adsorption ( Uads) values for XAD 7-TCT, as pH changed, showed the same trends of the nonactivated ones (Figure I). Moreover, ac-
Table 3
PL immobilization
supports
on XAD 7 by covalent attachment
Activation
AY
IY
(u”;ctI)
GA EDC SOCI, TCT
14.5 16.0 14.0 12.0 15.5
30.0 22.5 17.0 22.0 31.5
275 260 145 165 335
XAD 7 hydrolyzed
Enzyme Microb. Technol.,
1995, vol. 17, August
733
Papers tivation with EDC and SOCl, showed the same behavior (data not shown). This would seem to confirm once again the importance of physical adsorption on the immobilization process. XAD7-TCT binds PL covalently by replacement of only the second chlorine of the triazine ring, so that under immobilization conditions (pH 3.5 and 4”C), the third chlorine probably cannot react. 29 The spacing effect of the triazine ring reduces enzyme-matrix interactions and the steric hindrance of the biocatalyst toward the substrate. This fact may explain the high activity observed in this case as compared to activation with EDC and SOCI,. Finally, for XAD 7 activated or not, the yields were scarcely affected by the presence of the substrate (pectin up to 1 mg ml-‘) and by the increase in ionic force (NaCl up to 1 mg ml-‘).
Stabilization of immobilized PL On XAD 7. As can be seen in Table 4, the immobilization of PL on hydrolyzed or activated XAD 7 (tests 1 and 6), albeit ensuring high activity values ((I,,,), was associated with low stability over time, so that its use was curtailed. The stabilization of the immobilized enzyme by other methods, widely reported in the literature and entailing the use of additives (substrate, proteins, etc.) and/or crosslinking with was therefore investigated. Treatbifunctional agents3’ ment with additives, namely pectin, albumin and the enzyme itself, did not lead to any improvement in the stability of the immobilized enzyme; only the results obtained with the addition of albumin are reported as an example (tests 2 and 7). Cross-linking with GA was found to be an extremely delicate reaction to perform. In fact, in bland reaction conditions, pH 3.5 (at which value GA reactivity was low), low temperature, limited reaction times, and low GA concentration (0. l%), the enzyme was completely inactivated (test 3). Although it doubled the stability, the reduction in GA concentration (0.01%) always led to a considerable decrease in activity (test 4). The action of GA and of other cross-linking agents cause the molecule of the enzyme to assume a less free structure (“rigidification”), which leads to a lower unfolding rate and an increase in stability.3’ However, the resulting conformational changes and chemical alterations undergone by the enzyme cause a contem-
Table 4
Test 1 2 3 4 5 6 7 8
Stabilization
trials of PL immobilized
supports
on XAD7 hydrolyzed
porary reduction in activity. This negative effect was partially reduced by adding albumin before reticulation (tests 5 and 8). The immobilization of PL on TCT-activated XAD 7 (XAD7-TCT) was associated in all cases with an increase in activity and in stability (compare tests 6-8 with test l-5). This higher stability was probably due to a further increase in the rigidity of the enzyme molecule caused by the settingup of covalent enzyme-matrix bonds.
On inorganic support. The stability of Sn sulphide and y-alumina were low (about 1.2) in all immobilization methods examined. Instead, it was good for bentonite as such (2.4) and better for an activated one (4.7). We tried to increase such results. Bentonite is an aluminum silicate, the main component of which is montmorillonite. The elementary unit layer of montmorillonite consists of three sheets: an octahedral sheet of hydragillite-brucite included between two tetrahedral sheets of silicon and oxygen, which contain exchangeable cations. 32 Polar organic molecules, such as those of enzymes, can therefore be adsorbed mainly by electrostatic interaction, even though interactions by hydrogen and Van der Waals bonds are also important. The results of the various tests are shown in Table 5. AY values for the PL immobilized on bentonite by simple adsorption (test 1) were low, perhaps because the adsorption almost exclusively took place on the external surface of the support, which was characterized by a low surface area (about 15 m2 g - ’ 1. The mechanism of intralamellar enzyme intercalation reported for other enzymes” was probably prevented by the dimensions of PL (30-40 A), and low immobilization temperature. This did not permit the enzyme to penetrate between the unit silica layers (about 10 A) of a bentonite not previously swollen. On the contrary, IY values were fairly high. This is presumably because interactions between the protonate aminic groups of the enzyme and the nucleophilic centers of the bentonite led to a statistical orientation of the enzyme molecules with the active site facing outward, whereas, as a result of the low AY value, no competition of the enzyme molecules to the substrate seemed to be possible. Finally, as can be noted, the stability was high (test 1). By allowing the albumin to be preadsorbed onto the matrix before contact with the enzyme (test 2), AY increased considerably while IY markedly decreased. This would
or activated with TCT
XAD 7 hydrolyzed
XAD 7-TCT
Enzyme Microb.
Technol.,
275 225 0 170 210 335 300 270
;:L + AlbIb PL + GA (0.1%) PL + GA (E.Ol%) + Alb) + GA (0.01%) bpL’ (PL + AlbIb (PL + Albjb + GA (0.01%)
“Stability is defined as the ratio between the half-life times (t,,,) of immobilized bPL was pre-equilibrated with albumin (Alb) prior to immobilization
734
Stability” (pH 3.5, 25°C)
Sequence of immobilization
1995, vol. 17, August
and free PL
1.1 1.1 2.0 2.2 1.3 1.3 2.5
Immobilization of Aspergillus Table 5
Test 1 2 3 4 5 6 7
Stabilization
trials of PL immobilized
Bentonite
‘See Table 4 bPL was pre-equilibrated
PL Alb (PL (PL GA GA GA
with albumin
G. Spagna et al.
on bentonite and activated with GA
Sequent of immobilization
support
pectinlyase:
+ PL + AlbIb + pectinlb + (PL + AlbIb + (PL + pectinIb
AY 5.6 33.0 14.1 17.5 18.0 14.0 16.0
IY
u act (U 9-l)
Stability’ (pH 3.5, 25°C)
67 49 60 62 42 65 65
2.4
16.0 2.0 5.7 4.6 3.0 6.0 5.6
3.5 2.4 4.7 7.6 4.7
(Alb) or with pectin prior to immobilization
seem to indicate that albumin (PI 4.9) has a strong affinity with the support and that it favors enzyme adsorption. The drastic drop in IY values may be due to a number of factors, including (1) deformation of the PL molecule structure during binding with the preadsorbed albumin; (2) possible interaction of the enzyme with albumin via the dissociated carboxyl group of the active site; and (3) steric hindrance of the substrate. This fact is not surprising, because albumin (1 mg ml-‘) causes a slight decrease in activity (about 18%) even for the free enzyme. When, on the other hand, the enzyme was preequilibrated with albumin before coming into contact with the support (test 3), yield values were intermediate between the previous ones (tests 1 and 2), and stability increased further. The replacement of the albumin with pectin during the immobilization on bentonite did not affect the initial stability (test 4). In the immobilization of PL on nonactivated bentonite (tests 14), washing of the biocatalyst with NaCl solution (1 .O M for 1 h) caused a partial leakage of the enzyme in the solution (about 25% of the total amount adsorbed), as a result of the increase in the ionic force and the cationic substitution of the sodium on the surface of the matrix. The activation of bentonite with GA (test 5, as compared to the simple adsorption (test l), caused a considerable increase in AY values accompanied by a strong inactivation (IY and U,,, were both low), and a redoubling of stability. The establishment of a high density of bonds for each single molecule of adsorbed enzyme may cause (1) chemical modification of its surface; (2) distortion of its secondary and tertiary structure, reducing the conforrnational adaptability (decrease in IY and U,,,); and (3) “rigidification” of its protein conformation, reducing the unfolding rate (increase in stability). 3o The albumin (test 6) reduced GA-inactivation, permitting, moreover, an increase in stability. The best results were obtained with an albumin concentration of 0.25 mg ml-‘. A model is proposed according to which the GA covalently binds both the enzyme and the albumin to the support, and also forms bridges between them. The amount of GA bound to the support (160 mEq g - ‘) was overabundant with respect to the total protein enzyme adsorbed. It is therefore probable that as a result of its small size, the GA
molecules penetrated between the intralamellar spaces of the bentonite and diffused themselves outwardly (at least the molecules not directly bound to the support), reticulating the proteins bound to the surface. Pectin (test 7), like albumin, increased activity; however, it did not affect stability. Probably this temporary protective effect depended on its incapacity to react with GA like the albumin. Finally, in the activation of bentonite (tests 5-7), a higher stability together with a lower leakage of the immobilized enzyme (which dropped from 25 to 3%) may be noted, as compared to the nonactivated one (tests l-4). This light leakage is related to the enzyme molecules weakly bound to the support mainly through electrostatic interactions. As concerns the GA-activated y-alumina and Sn sulphide, the same stabilization trials of bentonite gave an insufficient increase in stability (about 1.4).
Characterization of PL immobilized on GA-activated bentonite Given the good findings of test 6 of Table 6, this methodology was chosen for its chemicophysical characterization. All the results of immobilized enzyme were compared to the free one.
Optimal pH. The immobilized
enzyme exhibited an optimal pH shift of about 0.4 toward the acid zone (Figure 2). This effect was probably due to the presence of protonated groups of the albumin which, according to the policationic support theory,33 favor an unequal distribution of hydrogen ions and oxydryls in the solution, with a greater concentration of oxydryl ions close to the microenvironment of the immobilized enzyme.
Optimal temperature. The immobilized wider temperature range in maximum (Figure 3). At temperatures above immobilized enzyme curve tion energy required for the due to the establishment of the matrix and the enzyme Enzyme Microb.
Technol.,
enzyme showed a which catalytic activity was at a 4O”C, the greater slope of the suggested an increase in activareaction to take place. This was bonds between the enzyme and and the albumin.30 1995, vol. 17, August
735
Papers 100
l Immobilized *
Free
PL
PL
ot
,~-,
:
4
3
0
Od
5
6
8
7
0
20
40
60
80
100
120
140
160
Usup (U ml-‘)
PH Figure 2 Activity (Uact) of free and immobilized PL on bentonite activated with GA (bentonite-GA) as a function of pH
Figure 4 GA
Adsorption isotherm. The adsorption isotherm of immobilized enzyme showed a sigmoidal trend (Figure 4). This would seem to suggest a cooperative mechanism of adsorption,34 in which the enzyme molecules already bound to the support favor further adsorption. This trend may be due to the proximity of the immobilization pH (3.5) to the enzyme p1, which minimized the lateral repulsion interactions among the adsorbed enzyme molecules. At high enzyme concentration (USup), Uads values were seen to increase further, whereas U,,, values simultaneously decreased. This behavior was the result of the formation of a molecular enzyme multilayer on the surface of the support (“overcrowding”), which caused a partial deactivation of the biocatalyst, perhaps ascribable to the presence of albumin.
cules bound to the support took on a structural conformation with a minimum exposed surface and an almost zero total charge. Under these conditions, electrostatic intermolecular repulsions were at a minimum and enzyme stability over time was therefore greater. Nevertheless, as can be noted in Figure 5, temperature was the most important parameter involved in the enzyme inactivation process. The unfolding rate and dissociation of the functions involved in the protein-support bonds increased as temperature increased. It may be reasonably assumed that the negative charge of the matrix increased because of the dissociation of the silan groups. PL also tended to become negatively charged, as the dissociation of the carboxylic groups could not be compensated for by the protonation of the aminic groups, which were present in lower numbers and, moreover, partially involved in the bonds with the GA and the albumin. This electrostatic repulsion effect was more obviously marked at pH 5. The other destabilizing factor associated with temperature increase was the destructuring of the coordination solvent due to the increase in molecular agitation. The pattern of residual activity as a function of time
Storage stability. The immobilized
enzyme exhibited considerable stability, with half-life times on average greater at pH 3.5 than at pH 5.0 (Figure 5). At a pH storage of 3.5 (close to PI), the enzyme mole-
100
1* e
Free
Adsorption
isotherm of PL immobilized
3
PL
Storage
600
Immobilized
pH
l
‘j 500
0 ~+ 10
pH:
pH 3.5
v pH 5.0
0 20
30
40
50
60
Temperature
70
Enzyme Microb.
Technol.,
80
90
100
(“C)
Figure 2 Activity (Uact) of free and immobilized nite-GA as a function of temperature
736
on bentonite-
20
30
40 T
PL on bento-
1995, vol. 17, August
50
60
(“C)
Figure 5 Half-life times (t,,, of PL immobilized on bentonite-GA, under different storage pH as a function of temperature
Immobilization (Figure 6) could provide information as to the enzyme inactivation mechanism. The linear trend observed at 25°C suggests that inactivation followed a kinetic curve of the first order, whereas the segments relating to inactivation at 40 and 55°C suggest a more complex mechanism involving at least two phases, with two inactivation constants, the second of which was slower than the first one. Deviation from the first order in the inactivation rate of the immobilized PL, which has also been reported for other immobilized enzymes, 35 is described as the sum of at least two exponential terms: residual activity = 4 exp (-kit) + (1 - +) exp ( - k.$), where 4 is the frequency factor, k, and k, are the two kinetic constants, and t is the time. The equation describes two possible basic inactivation mechanisms, one of which was a consecutive reaction in which the immobilized enzyme lost activity via two steps, and the other a competitive reaction in which at least two immobilized enzyme populations with different stability lost activity at different rates. This latter hypothesis, which seems to be the most probable,j5 suggests that the covalent binding transforms a homogeneous enzyme to several heterogeneous ones, with different kinds and number of bonds. The apparent K,,, of the immobilized enzyme was found to be almost equal to that of the free enzyme (0.2 mM). This suggests that the affinity of the active site of the immobilized enzyme to the substrate was the same as that of the free one, as probably its conformation had not undergone a substantial change.
Calculation of K,.
Conclusions
40°C
*
55°C
References 1.
3.
4.
5.
7.
8.
9. 10.
11.
4 2 0
100
200
300
400
600
600
700
12.
time (h)
13. Figure 6 Relative activity of PL immobilized under different storage temperatures
on bentonite-GA,
et al.
This research was supported by the National Research Council of Italy, Special Project RAISA, Subproject no. 4, paper no. 1844.
6. 25°C
G. Spagna
Acknowledgments
Storage temperature: v
pectinlyase:
appreciable increase in stability (2.5). Despite this improvement, however, half-life time (213 h) was still too low to permit industrial applications. In immobilization trials of PL on inorganic support, that on glutaraldehyde-activated bentonite in the presence of albumin, albeit exhibiting lower activity (65 U g - I), was seen to possess considerable stability (7.6)) corresponding to a fairly high half-life time (650 h). With respect to the one in solution, a further advantage of the enzyme thus immobilized was given by the slight shift in the optimum pH toward acidic values and by its greater relative activity at lower temperatures. Furthermore, bentonite features a number of other characteristics that make it particularly suitable as a support for enzyme immobilization in the food and pharmaceutical industries, namely nontoxicity (food grade), easy availability, low cost, microbiologic stability, good thermal and mechanical resistance, and, finally, the possibility of being activated by a simple and inexpensive method.
2.
Cellulose and its derivatives gave poor results, whether concerning catalytic response or stability. Among XADamberlites the XAD 7 activated with trichlorotriazine permitted PL immobilization with fairly good adsorption (AY) and immobilization (IY) yields as well as good catalytic activity (335 U g-i). However, the stability of the immobilized enzyme was found to be low (1.3). Attempts made to improve stability by means of glutaraldehyde led to an
of Aspergillus
Rombouts. F. M., and Pilnik, W. Pectic enzymes: Economic microbiology. In: Microbial Enzymes and Bioconversion (Pilnik, W., ed.). Academic Press. London, 1980, 227-282 Withaker, J. R. Microbial pectolytic enzymes. In Microbial Enzymes and Biorechnology (Fogart, W. G., and C. T. Kelly. eds.). Elsevier Applied Science, London, 1992, 133-175 Spagna. G.. Pifferi, P. G., and Martino, A. Pectinlyase immobilization on epoxy supports for application in the food processing industry. J. Chem. Tech. Biorech. 1993. 51, 37%385 Spagna, G.. Pifferi, P. G., Tramontini, M., and Albertini, A. Pectinlyase immobilization on polyamides for application in the food processing industry. J. Chem. Tech. Biorechno/. 1994. 59, 341348 Lozano, P., Majon, A., RomoJaro. F.. and Iborra, J. L. Properties of pectolytic enzymes covalently bound to nylon for apricot juice clarification. Proc. Biochem. 1988, 3. 75-78 Dinnella, C., Lanzarini, G.. and Palleschi, C. A model system for biocatalysis involving macromolecular substrates. Cerev. Biotech. 1992, 17, 32-35 Hanish, W. H.. Richard, P. A. D., and Nyo, S. Poly(methoxy galacturonide) lyase immobilized via titanium onto solid supports. Biotech. Bioeng. 1978, 20, 95-106 Dinella, C.. Lanzarini, G., Stagni, A.. and Palleschi, C. Immobilization of an endo-pectinlyase on y-alumina. J. Chem. Tech. Biotech. 1994.59, 237-241 Ampon. K. Distribution of an enzyme in porous polymer beads. J. Chem. Tech. Biotech. 1992. 55, 185-190 Basri, M.. Ampon, K.. Zimwan Yunus. W. M., Razak, C. N. A.. and Salleh, A. B. Immobilization of hydrophobic lipase derivatives on the organic polymer beads. J. Chem. Tech. Biotech. 1994. 59, 3744 Garwood, G. A., Mortland. M. M., and Pinnavaia, T. J. Immobilization of glucose oxidase on montmotillonite clay: Hydrophobic and ionic modes of binding. J. Mol. Catal. 1983, 22, 153-163 Tomar. M., and Probhu. K. A. Immobilization of cane invertase on bentonite. Enzyme Microb. Technol. 1985, I, 454-458 Kohn, R., Marcovic, O., and Machova. R. Deesterification mode of pectin by pectin esterases of Aspergillus foetidus, tomatoes and alfalfa. Cal. Czech. Chem. Comm. 1983, 48, 791-797
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18. 19.
20. 21.
22. 23. 24. 25.
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The Pectic Substances (Kertesz, 2. I., eds.). New York, 1951, 215-230 Owens. H. S.. Lotzkar, H., Schultz, T. H., and Maclav, W. D. Shape and size of pectinic acid molecules deduced from viscometric measurement. J. Amer. Chem. Sot. 1946, 68, 1628-1632 Spagna, G., and Pifferi, P. G. The purification of a commercial pectinlyase. Food Chem. 1994, 50, 343-349 Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254 Patchomik, A., and Rogozinski. E. S. Determination of ending groups, Anal. Chem. 1961, 33, 803-807 Pifferi, P. G.. Malacarne, A., Lanzarini, G., and Casoli. U. Spectrophotometric method for the determination of pectin methylesterase activity of bestom’s hydrazone. Chem. Mikrob. Tech. Lebensm. 1985, 9, 65-69 Albersheim, P. Pectin lyase from fungi. Methods Enzymol. 1968. 8, 628-635 Okai, A. A. E., and Geirschener, K. Size and charge properties of the pectic and cellulolytic enzymes in a commercial enzyme preparation. Z. Lebensm. Unters. Forsch. 1991, 192, 244-248 Kasztelan, S. A descriptive model of surfaces sites on MoS2 (WS2) particles. Langumir 1990. 6, 59c595 Goodboy, K. P., and Fleming. H. L. Trends in adsorption with aluminas. CEP November. 1984. 63-68 Weetall, H. H. Covalent coupling for inorganic support materials. Methods Emymol. 1976, 44, 134-148 Wahlgreen, M., and Amebrant, T. Protein adsorption to solid surfaces. Tibtech. 1991, 9, 201-208
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Monsan, P. Optimization of glutaraldehyde activation of a support for enzyme immobilization. J. Molec. Cafal. 1977178. 3, 371-384
27.
Richards, F. M., and Knowles, J. R. Glutaraldebyde as a protein cross-linking reagent. J. Mol. Biol. 1968, 37, 231-233
28.
Pifferi, P. G., Spagna, G., Nava Rincon, R., and Setti, L. A new method of immobilization of pectic enzymes. Biotech. Tech. 1993. 7,457-460
29.
Marikiwa, Y.. Tezuka, T.. Teranishi. M., Kimura, K.. Fujimoto. Y., and Samejima, H. Dichloro-s-triazinyl resin as carrier of immobilized enzyme. Agr. Biol. Chem. 1976, 40, 1137-l 142
30.
Mozhaev, V. Mechanism based strategies lization. Tibtech. 1993, 11, 88-95
31.
Guisan, J. M., Alvaro, G., Lafuente, R. F., Resell. M. C., Garcia, J. L., and Tagliani. A. Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila. Biotech. Bioeng. 1992, 42, 4551164
32.
Kirk. R. E.. and Othmer. D. F. Bentonite. In: Encyclopedia of Chemical Technology. Vol. 4. Interscience, New York, 1964, 337360
33.
Chibata, I. Properties of immobilized enzymes and microbial cells. In: immobilized Enzymes. (Chibata, 1. ed.). Halsted. New York. 1978. 108-147
34.
Giles, G. H., D’Silva, A. D.. and Easton, I. A. A general treatment and classification of the solute adsorption isotherm. J. Colloid Interjac. Sri. 1974, 47, 766778
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Ulbrich. R.. and Schelleriberger, A. Studies on thermal inactivation of immobilized enzymes. Biorech. Bioeng. 1983. 28, 5 1I-522
for protein thermostabi-
and Interdepartmental University of Bologna,
Center of Bologna. Italy
The immobilization of a commercial preparation ofpectinlyase (PL, EC 4.2.2.3) derived from Aspeqillus niger was studied in view of its possible application in fruit juice treatments. Pectinlyase is an enzyme that is subject to growing interest for the substitution of other pectic enzymes: e.g., polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1 .I .ll). The PL was immobilized by physical adsorption or by the formation of covalent bonds on organic (cellulose and its derivatives, XAD-amberlites) and inorganic (sulphides, y-alumina, and bentonite) supports. The supports that permitted an effective immobilization with good activity levels and sufficient stabiliry under the operational conditions were found to be the acrylic resin XAD 7 activated with trichlorotriazine and, in particular, bentonite activated with glutaraldehyde. Keywords:
Immobilized
enzymes;
pectic enzymes;
pectinlyase;
Introduction Pectic enzymes are currently used in numerous industrial processes, and in particular in food technology. The main applications include increases in filtration speed and in the amount of juice extracted from fruit pulp, reduction in turbidity, fruit juice clarification, and preparation of CY-Dgalacturonic acid. ’ Among pectic enzymes, pectinlyase (PL, EC 4.2.2.3) is the most interesting, as it is the only one capable of depolymerizing pectins without altering their esterification level.* In fact, PL is an endoenzyme that exerts its action directly on highly esterified pectins through a p-eliminative cleavage of the glycosidic linkages. This leads to the formation of an a,P unsaturated uranic acid residue on the nonreducing side, and to a hemiacetal one, on the reducing side of the glycosidic linkage, with the loss of a water molecule. The present study carries on from previous ones conducted on the immobilization of PL from the Aspergillus niger contained in a commercial enzymic preparation.3,4 The aim of this study was to examine the possibility of
XAD 7; bentonite;
fruit juices
using this enzyme in continuous processes for the treatment of fruit juices. Some of the supports employed for PL immobilization were nylon polyethylene imine copolymer,’ Eudragit L,6 porous glass and DEAE cellulose activated on titanium salt,’ Eupergit C3 y-alumina,’ and nylon 6 and 11.’ More precisely, this article reports the results of PL immobilization on organic (cellulose and its derivatives, XAD-amberlites) and inorganic (sulphides, y-alumina, and bentonite) matrices through simple adsorption or after activation with glutaraldehyde. The acrylic resins have been also activated, after hydrolysis, with carbodiimide, thionyl chloride, and trichlorotriazine. Among the various immobilization procedures adopted, the PL immobilized on XAD 79,‘o and bentonite (BDH)““’ activated were found to be the most active and simultaneously to possess optimum stability over time, in conditions that may potentially be set up for industrial applications.
Materials and methods Materials The enzymic preparation Cytolase PCLS (PCLS, batch 45; Genencar. USA) was employed as the PL source after purification by means of adsorption on BDH (Switzerland).
Address reprint requets to Dr. Giovanni Spagna, School of Specialization in Food Chemistry and Technology, Faculty of Industrial Chemistry. University of Bologna. Viale Risorgimento 4, 40136 Bologna, Italy Received 16 May 1994, revised 30 November 1994; accepted 13 December 1994
Enzyme and Microbial Technology 17:7X&738, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
After esterification, apple pectin (Fluka, Switzerland) was used as the enzyme substrate. For protein determination, bovine serum albumin (BSA; Pentex, USA) was employed as standard and Coomassie Brilliant Blue G-250 (Serva, Germany) was used as a calorimetric agent.
0141-0229/95/$10.00 SSDI 0141-0229(94)01134-D
Papers The following materials were used as supports for immobilization: cellulose (cell; Merck, Germany); cellulose triacetate (TAcell; Merck); polyethylene imine cellulose 300 (PEI-cell; Baker, Holland); XAD-amberlites: aromatics 2, 4, 16, and acrylics 7 and 8 (Rhom and Haas, USA); technical bentonite powder (BDH); y-alumina (130-140 A; Akzo); iron, molybdenum, and stannic sulphides (Strem Chem.), all of which feature a granulometry between 77 and 177 pm. We used 50% glutaraldehyde (GA; Fluka), trichlorotriazine (TCT; Fluka), thionyl chloride (EGA-Chem., Germany), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimmide HCl (EDC; Carlo Erba) as activating agents. Other reagents were 3-methyl-2benzothiazolinone hydrazon chlorohydrate (MBTH; Carlo Erba), N-N-dimethyl formamide (Carlo Erba), and iron trichloride (Merck). All the other reagents not specifically mentioned were supplied by Carlo Erba.
Pectin esterification Two hundred grams of apple pectin were first washed with 60% and 96% ethanol and then with anhydrous methanol to eliminate several impurities such as polyphenols. Pectin was added to 1.5 1 of a cold 0.5 M solution of H,SO, in anhydrous methanol and stored in the refrigerator at 3°C (so as to avoid substrate hydrolysis) for 4 weeks, stirring every now and then. The pectin thus esterified was then filtered and washed fist with 96% ethanol, then with methanol and ethyl ether, and finally allowed to dry overnight in an oven at about 40°C.i3 The pectin esterification level as then determined by means of acid-base titrationi using a mixed indicator consisting of aqueous solutions of 0.4% Bromothymol Blue (1 vol), 0.4% Cresol Red (1 vol), and 0.4% Phenol Red (3 vol) to highlight the color change. We equilibrated 100 ml of a 1% pectin aqueous solution containing two to three drops of the indicator at 3°C overnight and titrated with 0.5 M NaOH. The final titration point was indicated by a red color lasting for at least 30 s (pH 7.5). We then added 20 ml of 0.5 M NaOH and after 30 min titrated excess alkali with 0.5 M HCl until the indicator turned yellow. A blank test was simultaneously performed using distilled water instead of the substrate. The esterification grade was calculated on the basis of the difference between the two titrations given as the amount of soda required for saponification. The average molecular weight (M,) of the esterified pectin was determined by viscosimetty. is Pectin solutions of concentrations from 0.03 to 0.05% were prepared in 0.195 M NaCl and allowed to equilibrate at 3°C. After heating the solution in a thermostated bath to 37°C viscosity was measured by means of the Ostwald capillary viscosimeter. The relationship between viscosity (q) and molecular weight (M) is expressed as: q = K . Ma, where for pectin, K = 1.4 . 10m6 and CL= 1.34.
PL purification The PCLS enzymic preparation was purified according to a method described previously. I6 We added 4 g of bentonite under stirring to 50 ml of 0.07 M citric-phosphate buffer (C-P) at pH 5.0 and sonicated it for about 20 min at 25°C. Subsequently 50 ml of 0.07 M C-P at pH 5.0 and 100 ml of PCLS were added to the suspension thus obtained. After having adjusted the pH value to 5.0 we stirred the suspension for about 2.5 h, and then centrifuged it at 2,600 g for 20 min. The solution was treated again for 2.5 h with 4.0 g of bentonite mixed with 50 ml of 0.07 M C-P at pH 5.0, before being centrifuged once more. The enzymic solution was then diluted twice with water and brought to a pH of 3.2 to 3.4 by adding 1 M HCI, after which it was placed in a refrigerator of 3°C overnight. The precipitate thus obtained, essentially made up of polisacharides, extraneous proteins, and brown pigments, was
730
Enzyme Microb.
Technol.,
1995, vol. 17, August
separated by centrifugation at 5,500 g for 60 min at 3°C and the purified PL containing solution was then used for immobilization. Protein determination was performed according to Bradford methodology.”
Acrylic resin hydrolysis XAD 7 (or XAD 8) was washed five times with water and methanol and then left to dry in an oven at about 55°C after which 20 g of dry resin was suspended in 200 ml of a 2.5 M solution of NaOH containing 10% isopropanol. Hydrolysis was performed by reflux under stirring for about 24 h. The support thus hydrolyzed was then washed several times with water until neutral pH was reached. Finally, it was washed with methanol and once more dried in an oven. To determine the carboxylic groups formed,is we added 10 ml of dimethylformamide to about 0.5 g of hydrolyzed XAD 7 and left the solution to equilibrate for 1 h under stirring at 25°C. Three drops of a 1% Thymol Blue solution in ethanol were then added and titration was finally performed with 0.05 M sodium metoxide dissolved in a benzene-methanol solution (7:3 v/v).
Activation
with glutaraldehyde
We placed 4 ml of a GA solution (2%) dissolved in a 0.05 M buffer at pH 3.5 (citric-phosphate; C-P) or at pH 10 (carbonatebicarbonate) in IO-ml tubes containing 10 mg of the various supports (PEI-cell; Sn sulphide, y-alumina; bentonite and hydrolyzed acrylic resins). After activation in a rotary carousel for 1.5 h at 25°C. the supports were centrifuged, washed at least two or three times in 10 ml of water, and then checked for the eventual presence of GA in the rinse waters by reading absorbance at 235 nm. The amount of GA bound to the support was determined on the basis of the difference between the amount of equilibrated GA and that measured in the solution according to Bersthom’s hydrazone method, without the oxidation step. I9 Two milliliters of standard GA solution (0.01-0.2%) or of an appropriately diluted sample was placed in a 20-ml flask and made to react at 100°C with 2 ml of 0.4% MBTH. After 3 min the mixture was rapidly cooled in an ice bath. We then added 5 ml of 0.4% FeCl, and the solution was brought to volume with acetone. After 15 min at 25°C absorbance of the standard solution or of the sample was read at 635 nm against the blank, which had been obtained by replacing the GA solution with an equal volume of the same solution in which the GA had been dissolved.
Activation
of hydrolyzed acrylic resins
Activation with carbodiimide. Four milliliters of 0.3% EDC in water acidified at pH 4.5 with HCl were placed in lo-ml tubes containing 10 mg of hydrolyzed XAD 7 (or XAD 8). After support activation in a rotary carousel for 2 h at 25”C, the supports were washed with 10 ml water. Activation with thionyl chloride. We converted 5 g of hydrolyzed XAD 7 (or XAD 8) to the corresponding acrylic chloride by reflux treatment under stirring for 6 h with 50 ml of a 2% SOCl, solution in chloroform. Activation with trichlorotriazine. We placed 4 ml of 1.2% TCT solution in dioxane in lo-ml tubes containing 10 mg of XAD 7 (or XAD 8). After activation in a rotary carousel for 20 min at 25”C, the supports were washed twice, first with 10 ml dioxane and then with 10 ml water.
Immobilization of Aspergillus pectinlyase: G. Spagna et al. Immobilization method Between 60 and 100 U of purified PL, brought to the desired pH by the addition of 0.1 M HCl or NaOH, was placed in lo-ml tubes with 10 mg of the various activated or nonactivated supports. After about 16 contact h in a rotary carousel (about 40 rpm) at 4”C, the tubes were centrifuged. The enzymic activity of the supernatants was directly essayed; that of the residues was essayed after being washed with 10 ml of 0.5 M NaCl and 10 ml of 0.5 M C-P at the pH of the enzyme essay. In some cases, PL was equilibrated before immobilization for about 30 min in pectin or albumin solutions (up to 0.5%). In the case of the hydrolyzed XAD 7, the immobilized enzyme was also reticulated at 4°C for 15 min with a 0.1 and 0.01% GA solution in 0.01 M C-P buffer at pH 3.5, after the supports were washed with NaCl and buffer solutions.
Determination ofPL activity We added 3 ml of I. I% esterified pectin, dissolved in 0.05 M C-P buffer at the established pH. under magnetic stirring at 25”C, to 2.0 ml of PL in solution or PL immobilized suspended in the same buffer in a lo-ml tube. After 1 min, to stop the reaction 2 ml of 0.5 M HaSO, was added. For the blank test the order of the reagents was reversed (i.e., the acid was first added to the enzyme, followed by the pectin solution). In the case of the immobilized enzyme, the tubes were centrifuged. The sample solution was read against the blank solution at 235 nm. The increase in absorbance was due to the double conjugate bond of the A 4:5 unsaturated uronide formed during the reaction. One unit of the enzyme is defined as the amount that catalyzes an increase of 0.555 in absorbance at 235 nm, in 1 min at 25°C and at optimal pH.4.20
Storage stability The enzymic activities of the soluble and of the immobilized enzymes were measured in relation to time (up to a maximum of 700 h) at pH 3.5 and 5.0 (0.05 M C-P) and at various temperatures (25-55°C).
Characterization of the free and immobilized enzymes The following parameters were determined for the free enzyme and for the enzyme immobilized on bentonite activated with GA: optimum pH (between 3.5 and 7.5) at 25°C; optimum temperature (between 20 and 90°C) at optimal conditions; adsorption isotherm (by equilibrating up to 250 U of PL); and finally, the and V (with solutions 1%).
PL immobilization Cellulose
by adsorption
and derivatives.
The cellulose showed a suffi(AY) with a poor catalytic response (IY and U,,,. Table 1). Probably, the crystalline structure of this support contributes to reduce adsorption. Moreover, the likeness between cellulose and the pectin may have oriented the enzyme molecule with the active site facing the support,
cient
adsorption
reducing the substrate accessibility (higher steric hindrance). The presence of the substrate during immobilization caused a drop in adsorption (AY) and only a slight increase in activity (v,,,). The long chain of the pectin molecule (about 650 A), by interacting with the enzyme, may have hindered its anchoring on the support, protecting the active site and the conformational structure. With the cellulose triacetate (TA-Cell) AY increased while the activity dropped to zero. The presence of esterified groups reduced hydrophilic structure of this support, making it more similar to the pectin. On the contrary, with PEI-cellulose (PEI-cell) a decrease in AY and an increase in IY and U,,, were observed. The higher hydrophilia of this support and its poor interaction with the enzyme at pH 3.5, resulting from the positive charge of both support and enzyme, may account for this behavior. Inorganic supports. As can be seen in Table 1; sulphide supports are generally characterized by high AY and low IY values. Despite featuring minimum surface areas (< IO m2 g- ‘), these supports feature a very high site density, especially at the fracture zones of superficial faces.22 These
Table 1 PL immobilization on cellulose, its derivatives, and inorganic support by adsorption and covalent attachment with GA at pH 3.5
supports
Pectin
Adsorption Cellulose
of Adsorption follows: UadS units,
with an esterification grade of 93% and an M, of about 28,000; see Materials and methods). Moreover, the active site of the enzyme seemed to contain a carboxylic group and a tyrosine residue. apple pectin
(AY) = the V,,,
+
immobilization ’ IY enzyme active
(IY) defined I/,,, Clads 100, U,, equilibrated enzyme
Results and discussion After purification, the enzyme used had a specific activity of about 20 U mg - ’ of protein. To better interpret the results obtained, the main characteristics of the enzyme*’ are worth bearing in mind. These include: an M, of about 38,000; a dimension of about 30 to 40 A, an isoelectric pH (PI) of 3.6; an optimal pH of about 6.4 (determined using an
TA-cell
+
PEI-cell FE sulphide MO sulphide Sn sulphide y-Alumina Bentonite Covalent (with GA) PEI-cell Sn sulphide V-Alumina
lzyme Microb. Technol.,
f
AY
IY
u act (U g ‘1
25.0 16.7 51.2 27.5 9.0 48.3 54.6 43.0 12.0 7.8 5.6 17.5
0.2 0.6 0 0.2 2.5 0.1 0.1 0.7 3.5 4.3 16.0 4.6
3.0 6.0 0 2.5 15.5 3.0 4.5 17.0 28.0 22.0 65.0 62.0
25.6 2.9 18.5 16.0
0.3 20.5 8.9 5.6
5.5 41.1 115.0 65.0
1995, vol. 17, August
731
Papers strong aspecific interactions may account for the high adsorption, and moreover, being very close, may cause structural distortions of the enzyme molecule and its inactivation. For the stannic sulphide AY values diminished considerably and IY ones increased following the addition of substrate. Although y-alumina has a high density of acidic (Lewis and Bronsted) and basic sites,23 the yield values are quite low. As regards bentonite, AY values were low, whereas the IY and activity values are the highest in Table 1. The presence of pectin caused an increase in AY, which is probably ascribable to the affinity of this support to this compound24 and a proportional decrease in IY, so
XAD-amberlites. XAD-amberlites are resins having a different chemical structure and featuring no functional ionic groups. The XAD 2,4,16 resins are divinylbenzene styrene copolymers (aromatic) at low polarity (p 0.3); the XAD 7 and 8 resins are acrylic ester-based copolymers (acrylic) at a higher polarity (p 1.8), and therefore with a more hydrophobic structure. Moreover, surface area and pore size changed as the degree of reticulation of XAD resins changed. For aromatic resins high AY values are observed (Table 2). They diminished in the presence of pectin (XAD 2 and 4) and remained almost the same, even when the surface area and pore size changed. This seems to suggest that the substrate reduces interactions between enzyme and matrix and, moreover, that the absorption prevalently takes place on the external surface of the matrix, perhaps also because of the great molecular dimensions of the enzyme-substrate complex (molecular exclusion effect). In all cases, however, IY values are low or practically zero. This may be due to (1) the strong hydrophobic interactions of the enzyme with the matrix, thus causing conformational changes in the secondary and tertiary structure of the protein molecule; (2) the interaction between the aromatic rings of the support and the enzyme, which may also involve the tyrosine residue present in its active site. Table 2
PL immobilization
on XAD-amberlites
Acrylic resins generally show less adsorption (AY) associated, in particular for XAD 7, with higher catalytic response (IY and Uact) (Table 2). This behavior is probably due to a lower hydrophobicity of such resin, which modifies the molecular structure of the enzyme to a lesser extent,‘5 as well as its negative dipole moment, which may orientate the active site (carboxylic) of the enzyme outward, increasing the substrate accessibility (lower steric hindrance). Data concerning the pore size and surface area of acrylic resins (Table 2) cannot be directly compared with aromatic ones, as they are related to the dry product and change in an aqueous environment resulting from a marked swelling of the acrylic matrix (about 2.2 times for XAD 7). Moreover, findings reported for XAD 7 were seen to be the same with or without pectin. This result would once again seem to suggest that PL is prevalently adsorbed on the external surface of the matrix. This hypothesis was found to be correct for the adsorption on XAD 7 of the trypsin, an enzyme having dimensions lower than those of PL.9 In fact, the enzyme molecules may also be adsorbed onto the openings of the pore, or close to them. Thus, the penetration of other molecules into the internal surface of the substrate is blocked or reduced; moreover, the diffusion coefficient decreases further for the low temperature (4°C) employed during immobilization. The activity (U,,) of the immobilized PL on XAD 7 as such is maximum at values close to p1 (Figure I), that is, when the enzyme molecules are less susceptible to conformational distortions due to inter- and intramolecular electrostatic repulsion, and therefore more capable of protecting themselves during immobilization. Under these conditions, the molecules take on the optimum configuration at the minimum exposed surface. Instead, the adsorption trend (U,,,) is scarcely affected by pH (Figure 1). As far as XAD 8 support is concerned, AY values are lower, probably because of the lower surface area. Acrylic resins were also hydrolyzed, introducing carboxylic (0.66 mEq g ~ ‘) and oxydrylic groups to increase their reactivity and hydrophilia (Table 2). Moreover, this procedure decreased surface areas and increased pore sizes, at least in the zones (probably superficial ones) where hydrolysis occurred. For both acrylic resins the most outstanding
by adsorption
Surface area
Pore size
supports
(m* g-‘1
(A)
Pectin
AY
IY
Aromatic XAD2
300
90
XAD4
725
40
XAD 16
800
100
_ + + +
38.0 15.0 30.0 14.1 18.5
0.4 0.4 0.1 0 2.2
11.5 7.0 2.0 0 36.0
450
90
_ + _ +
20.0 18.1 10.0 12.1
13.5 12.5 2.6 2.0
188.1 22.0 20.2
-
14.5 13.0
30.0 10.5
275.0 84.0
Acrylic XAD7 XAD8
160
Acrylic hydrolyzed XAD7 XAD8
732
Enzyme Microb.
225
420
Technol.,
1995, vol. 17, August
u act FJ g-‘)
190.1
Immobilization
0
0 2.5
3
3.5
4
4.5
5
5.5
6
6.5
PH Figure 1 Activity (Uact) and adsorption (Uads) of PL immobilized at different pH values on XAD 7 nonactivated (as such, hydrolyzed) and activated with TCT
effect of the hydrolysis was the marked increase in IY values. This may be due to the further increase in hydrophilia and probably in pore size, which increase the accessibility of substrate. Instead, as regards the enzyme adsorption, the hydrolysis process gave contrary effects. Finally, the hydrolysis did not influence the optimum immobilization pH (Figure I ) _ PL immobilization
by covalent attachment
Glutaraldehyde-activated supports. Support activation with GAz6 is one of the most widely used techniques, as it is simple and inexpensive. In our case, it also offered the advantage of not modifying the carboxylic function present in the active site of PL. However, GA presents several drawbacks, such as excessive reactivity and the complex reaction mechanism with the nucleophilic groups, which is not yet fully understood; thus, the effects of GA on enzymic activity cannot be fully predicted. A possible explanation, however, may be attempted. Probably, the nucleophilic groups of the support and of the enzyme, such as the aminic ones, mainly reacted with aldehydic groups (Schiff base) and also with the double bonds originating from the aldolic condensation of the GA (Michael-type adduct). As pH varies, the activation reaction with GA follows a different pattern, causing a different reactivity of the activated support. In an acid environment, GA molecules individually react with the active centers of the support, making a large number of aldehydic groups available to form enzyme bonds. In an alkaline environment, the GA molecules tend to form o-p unsaturated polymers (aldolic condensation), causing a polyaldehyde net to be formed on the support. This net reduces the amount of available aldehydic groups and normally makes the matrix more hydrophobic. The nonactivated supports that exhibited higher activities were activated with glutaraldehyde (Table I). The activation of PEI-cell caused a notable increase in AY and decrease in IY and U,,, values. These findings are probably due to the high density of the GA bound to the
of Aspergillus
pectinlyase:
G. Spagna et al.
aminic groups of the matrix, which may cause a partial unfolding of the enzyme. The activation of inorganic supports with GA is a procedure that has already been adopted to immobilize polygalacturonase and pectinesterase on y-alumina.** It leads, in the case of Sn sulphide, to a decrease in AY and a notable increase in IY. Evidently, the bound GA cannot compensate for the loss of several reactive centers on the support surface, whereas it can reduce its influence on the immobilized enzyme. As regards y-alumina, the GA activation increased both of the yields, allowing good activity. No variations in yields and activity were observed for GA-activated bentonite. (This support shall be examined in greater detail further on.) In all trials, when activation pH was raised from 3.5 to 10, the AY values tended to increase; IY values tended to decrease, showing generally lower activities (data not shown). This seems to confirm the hypothesis on the influence of pH for activation process with GA.
Activated acrylic resins. Acrylic hydrolyzed resins, given the good findings obtained by simple adsorption (Table 2), were activated with GA, EDC, SOCl,, or TCT (Table 3). The XAD 8 is not thawed because after activation, it presents the same relative trends of XAD 7. The activation of XAD 7 has not generally caused substantial variations in AY, probably because of the low number of reactive groups of the hydrolyzed matrix. It would therefore seem that the competition between covalent binding and physical adsorption is decidedly in favor of the latter. On the other hand, higher variations in IY and activity values were observed. No improvement in IY and activity was observed for GA-activated XAD 7 as compared to the hydrolyzed one. This behavior may be accounted for by the fact that the amount of matrix activated with GA was very low. This is an atypical situation, as no aminic groups were available on the support to promote the classical coupling reaction. It might be that the GA interacted with the oxydrylic and carboxylic groups of the matrix via hydrogen bonds. An overall reduction in yields and activities (U,,,) was reported for activation with EDC and SOCl,. On the contrary, as concerns activation with TCT (XAD7-TCT), both yields slightly increased, so that activity increased (from 275 to 335 U g - ‘). The activity (U,,,) and adsorption ( Uads) values for XAD 7-TCT, as pH changed, showed the same trends of the nonactivated ones (Figure I). Moreover, ac-
Table 3
PL immobilization
supports
on XAD 7 by covalent attachment
Activation
AY
IY
(u”;ctI)
GA EDC SOCI, TCT
14.5 16.0 14.0 12.0 15.5
30.0 22.5 17.0 22.0 31.5
275 260 145 165 335
XAD 7 hydrolyzed
Enzyme Microb. Technol.,
1995, vol. 17, August
733
Papers tivation with EDC and SOCl, showed the same behavior (data not shown). This would seem to confirm once again the importance of physical adsorption on the immobilization process. XAD7-TCT binds PL covalently by replacement of only the second chlorine of the triazine ring, so that under immobilization conditions (pH 3.5 and 4”C), the third chlorine probably cannot react. 29 The spacing effect of the triazine ring reduces enzyme-matrix interactions and the steric hindrance of the biocatalyst toward the substrate. This fact may explain the high activity observed in this case as compared to activation with EDC and SOCI,. Finally, for XAD 7 activated or not, the yields were scarcely affected by the presence of the substrate (pectin up to 1 mg ml-‘) and by the increase in ionic force (NaCl up to 1 mg ml-‘).
Stabilization of immobilized PL On XAD 7. As can be seen in Table 4, the immobilization of PL on hydrolyzed or activated XAD 7 (tests 1 and 6), albeit ensuring high activity values ((I,,,), was associated with low stability over time, so that its use was curtailed. The stabilization of the immobilized enzyme by other methods, widely reported in the literature and entailing the use of additives (substrate, proteins, etc.) and/or crosslinking with was therefore investigated. Treatbifunctional agents3’ ment with additives, namely pectin, albumin and the enzyme itself, did not lead to any improvement in the stability of the immobilized enzyme; only the results obtained with the addition of albumin are reported as an example (tests 2 and 7). Cross-linking with GA was found to be an extremely delicate reaction to perform. In fact, in bland reaction conditions, pH 3.5 (at which value GA reactivity was low), low temperature, limited reaction times, and low GA concentration (0. l%), the enzyme was completely inactivated (test 3). Although it doubled the stability, the reduction in GA concentration (0.01%) always led to a considerable decrease in activity (test 4). The action of GA and of other cross-linking agents cause the molecule of the enzyme to assume a less free structure (“rigidification”), which leads to a lower unfolding rate and an increase in stability.3’ However, the resulting conformational changes and chemical alterations undergone by the enzyme cause a contem-
Table 4
Test 1 2 3 4 5 6 7 8
Stabilization
trials of PL immobilized
supports
on XAD7 hydrolyzed
porary reduction in activity. This negative effect was partially reduced by adding albumin before reticulation (tests 5 and 8). The immobilization of PL on TCT-activated XAD 7 (XAD7-TCT) was associated in all cases with an increase in activity and in stability (compare tests 6-8 with test l-5). This higher stability was probably due to a further increase in the rigidity of the enzyme molecule caused by the settingup of covalent enzyme-matrix bonds.
On inorganic support. The stability of Sn sulphide and y-alumina were low (about 1.2) in all immobilization methods examined. Instead, it was good for bentonite as such (2.4) and better for an activated one (4.7). We tried to increase such results. Bentonite is an aluminum silicate, the main component of which is montmorillonite. The elementary unit layer of montmorillonite consists of three sheets: an octahedral sheet of hydragillite-brucite included between two tetrahedral sheets of silicon and oxygen, which contain exchangeable cations. 32 Polar organic molecules, such as those of enzymes, can therefore be adsorbed mainly by electrostatic interaction, even though interactions by hydrogen and Van der Waals bonds are also important. The results of the various tests are shown in Table 5. AY values for the PL immobilized on bentonite by simple adsorption (test 1) were low, perhaps because the adsorption almost exclusively took place on the external surface of the support, which was characterized by a low surface area (about 15 m2 g - ’ 1. The mechanism of intralamellar enzyme intercalation reported for other enzymes” was probably prevented by the dimensions of PL (30-40 A), and low immobilization temperature. This did not permit the enzyme to penetrate between the unit silica layers (about 10 A) of a bentonite not previously swollen. On the contrary, IY values were fairly high. This is presumably because interactions between the protonate aminic groups of the enzyme and the nucleophilic centers of the bentonite led to a statistical orientation of the enzyme molecules with the active site facing outward, whereas, as a result of the low AY value, no competition of the enzyme molecules to the substrate seemed to be possible. Finally, as can be noted, the stability was high (test 1). By allowing the albumin to be preadsorbed onto the matrix before contact with the enzyme (test 2), AY increased considerably while IY markedly decreased. This would
or activated with TCT
XAD 7 hydrolyzed
XAD 7-TCT
Enzyme Microb.
Technol.,
275 225 0 170 210 335 300 270
;:L + AlbIb PL + GA (0.1%) PL + GA (E.Ol%) + Alb) + GA (0.01%) bpL’ (PL + AlbIb (PL + Albjb + GA (0.01%)
“Stability is defined as the ratio between the half-life times (t,,,) of immobilized bPL was pre-equilibrated with albumin (Alb) prior to immobilization
734
Stability” (pH 3.5, 25°C)
Sequence of immobilization
1995, vol. 17, August
and free PL
1.1 1.1 2.0 2.2 1.3 1.3 2.5
Immobilization of Aspergillus Table 5
Test 1 2 3 4 5 6 7
Stabilization
trials of PL immobilized
Bentonite
‘See Table 4 bPL was pre-equilibrated
PL Alb (PL (PL GA GA GA
with albumin
G. Spagna et al.
on bentonite and activated with GA
Sequent of immobilization
support
pectinlyase:
+ PL + AlbIb + pectinlb + (PL + AlbIb + (PL + pectinIb
AY 5.6 33.0 14.1 17.5 18.0 14.0 16.0
IY
u act (U 9-l)
Stability’ (pH 3.5, 25°C)
67 49 60 62 42 65 65
2.4
16.0 2.0 5.7 4.6 3.0 6.0 5.6
3.5 2.4 4.7 7.6 4.7
(Alb) or with pectin prior to immobilization
seem to indicate that albumin (PI 4.9) has a strong affinity with the support and that it favors enzyme adsorption. The drastic drop in IY values may be due to a number of factors, including (1) deformation of the PL molecule structure during binding with the preadsorbed albumin; (2) possible interaction of the enzyme with albumin via the dissociated carboxyl group of the active site; and (3) steric hindrance of the substrate. This fact is not surprising, because albumin (1 mg ml-‘) causes a slight decrease in activity (about 18%) even for the free enzyme. When, on the other hand, the enzyme was preequilibrated with albumin before coming into contact with the support (test 3), yield values were intermediate between the previous ones (tests 1 and 2), and stability increased further. The replacement of the albumin with pectin during the immobilization on bentonite did not affect the initial stability (test 4). In the immobilization of PL on nonactivated bentonite (tests 14), washing of the biocatalyst with NaCl solution (1 .O M for 1 h) caused a partial leakage of the enzyme in the solution (about 25% of the total amount adsorbed), as a result of the increase in the ionic force and the cationic substitution of the sodium on the surface of the matrix. The activation of bentonite with GA (test 5, as compared to the simple adsorption (test l), caused a considerable increase in AY values accompanied by a strong inactivation (IY and U,,, were both low), and a redoubling of stability. The establishment of a high density of bonds for each single molecule of adsorbed enzyme may cause (1) chemical modification of its surface; (2) distortion of its secondary and tertiary structure, reducing the conforrnational adaptability (decrease in IY and U,,,); and (3) “rigidification” of its protein conformation, reducing the unfolding rate (increase in stability). 3o The albumin (test 6) reduced GA-inactivation, permitting, moreover, an increase in stability. The best results were obtained with an albumin concentration of 0.25 mg ml-‘. A model is proposed according to which the GA covalently binds both the enzyme and the albumin to the support, and also forms bridges between them. The amount of GA bound to the support (160 mEq g - ‘) was overabundant with respect to the total protein enzyme adsorbed. It is therefore probable that as a result of its small size, the GA
molecules penetrated between the intralamellar spaces of the bentonite and diffused themselves outwardly (at least the molecules not directly bound to the support), reticulating the proteins bound to the surface. Pectin (test 7), like albumin, increased activity; however, it did not affect stability. Probably this temporary protective effect depended on its incapacity to react with GA like the albumin. Finally, in the activation of bentonite (tests 5-7), a higher stability together with a lower leakage of the immobilized enzyme (which dropped from 25 to 3%) may be noted, as compared to the nonactivated one (tests l-4). This light leakage is related to the enzyme molecules weakly bound to the support mainly through electrostatic interactions. As concerns the GA-activated y-alumina and Sn sulphide, the same stabilization trials of bentonite gave an insufficient increase in stability (about 1.4).
Characterization of PL immobilized on GA-activated bentonite Given the good findings of test 6 of Table 6, this methodology was chosen for its chemicophysical characterization. All the results of immobilized enzyme were compared to the free one.
Optimal pH. The immobilized
enzyme exhibited an optimal pH shift of about 0.4 toward the acid zone (Figure 2). This effect was probably due to the presence of protonated groups of the albumin which, according to the policationic support theory,33 favor an unequal distribution of hydrogen ions and oxydryls in the solution, with a greater concentration of oxydryl ions close to the microenvironment of the immobilized enzyme.
Optimal temperature. The immobilized wider temperature range in maximum (Figure 3). At temperatures above immobilized enzyme curve tion energy required for the due to the establishment of the matrix and the enzyme Enzyme Microb.
Technol.,
enzyme showed a which catalytic activity was at a 4O”C, the greater slope of the suggested an increase in activareaction to take place. This was bonds between the enzyme and and the albumin.30 1995, vol. 17, August
735
Papers 100
l Immobilized *
Free
PL
PL
ot
,~-,
:
4
3
0
Od
5
6
8
7
0
20
40
60
80
100
120
140
160
Usup (U ml-‘)
PH Figure 2 Activity (Uact) of free and immobilized PL on bentonite activated with GA (bentonite-GA) as a function of pH
Figure 4 GA
Adsorption isotherm. The adsorption isotherm of immobilized enzyme showed a sigmoidal trend (Figure 4). This would seem to suggest a cooperative mechanism of adsorption,34 in which the enzyme molecules already bound to the support favor further adsorption. This trend may be due to the proximity of the immobilization pH (3.5) to the enzyme p1, which minimized the lateral repulsion interactions among the adsorbed enzyme molecules. At high enzyme concentration (USup), Uads values were seen to increase further, whereas U,,, values simultaneously decreased. This behavior was the result of the formation of a molecular enzyme multilayer on the surface of the support (“overcrowding”), which caused a partial deactivation of the biocatalyst, perhaps ascribable to the presence of albumin.
cules bound to the support took on a structural conformation with a minimum exposed surface and an almost zero total charge. Under these conditions, electrostatic intermolecular repulsions were at a minimum and enzyme stability over time was therefore greater. Nevertheless, as can be noted in Figure 5, temperature was the most important parameter involved in the enzyme inactivation process. The unfolding rate and dissociation of the functions involved in the protein-support bonds increased as temperature increased. It may be reasonably assumed that the negative charge of the matrix increased because of the dissociation of the silan groups. PL also tended to become negatively charged, as the dissociation of the carboxylic groups could not be compensated for by the protonation of the aminic groups, which were present in lower numbers and, moreover, partially involved in the bonds with the GA and the albumin. This electrostatic repulsion effect was more obviously marked at pH 5. The other destabilizing factor associated with temperature increase was the destructuring of the coordination solvent due to the increase in molecular agitation. The pattern of residual activity as a function of time
Storage stability. The immobilized
enzyme exhibited considerable stability, with half-life times on average greater at pH 3.5 than at pH 5.0 (Figure 5). At a pH storage of 3.5 (close to PI), the enzyme mole-
100
1* e
Free
Adsorption
isotherm of PL immobilized
3
PL
Storage
600
Immobilized
pH
l
‘j 500
0 ~+ 10
pH:
pH 3.5
v pH 5.0
0 20
30
40
50
60
Temperature
70
Enzyme Microb.
Technol.,
80
90
100
(“C)
Figure 2 Activity (Uact) of free and immobilized nite-GA as a function of temperature
736
on bentonite-
20
30
40 T
PL on bento-
1995, vol. 17, August
50
60
(“C)
Figure 5 Half-life times (t,,, of PL immobilized on bentonite-GA, under different storage pH as a function of temperature
Immobilization (Figure 6) could provide information as to the enzyme inactivation mechanism. The linear trend observed at 25°C suggests that inactivation followed a kinetic curve of the first order, whereas the segments relating to inactivation at 40 and 55°C suggest a more complex mechanism involving at least two phases, with two inactivation constants, the second of which was slower than the first one. Deviation from the first order in the inactivation rate of the immobilized PL, which has also been reported for other immobilized enzymes, 35 is described as the sum of at least two exponential terms: residual activity = 4 exp (-kit) + (1 - +) exp ( - k.$), where 4 is the frequency factor, k, and k, are the two kinetic constants, and t is the time. The equation describes two possible basic inactivation mechanisms, one of which was a consecutive reaction in which the immobilized enzyme lost activity via two steps, and the other a competitive reaction in which at least two immobilized enzyme populations with different stability lost activity at different rates. This latter hypothesis, which seems to be the most probable,j5 suggests that the covalent binding transforms a homogeneous enzyme to several heterogeneous ones, with different kinds and number of bonds. The apparent K,,, of the immobilized enzyme was found to be almost equal to that of the free enzyme (0.2 mM). This suggests that the affinity of the active site of the immobilized enzyme to the substrate was the same as that of the free one, as probably its conformation had not undergone a substantial change.
Calculation of K,.
Conclusions
40°C
*
55°C
References 1.
3.
4.
5.
7.
8.
9. 10.
11.
4 2 0
100
200
300
400
600
600
700
12.
time (h)
13. Figure 6 Relative activity of PL immobilized under different storage temperatures
on bentonite-GA,
et al.
This research was supported by the National Research Council of Italy, Special Project RAISA, Subproject no. 4, paper no. 1844.
6. 25°C
G. Spagna
Acknowledgments
Storage temperature: v
pectinlyase:
appreciable increase in stability (2.5). Despite this improvement, however, half-life time (213 h) was still too low to permit industrial applications. In immobilization trials of PL on inorganic support, that on glutaraldehyde-activated bentonite in the presence of albumin, albeit exhibiting lower activity (65 U g - I), was seen to possess considerable stability (7.6)) corresponding to a fairly high half-life time (650 h). With respect to the one in solution, a further advantage of the enzyme thus immobilized was given by the slight shift in the optimum pH toward acidic values and by its greater relative activity at lower temperatures. Furthermore, bentonite features a number of other characteristics that make it particularly suitable as a support for enzyme immobilization in the food and pharmaceutical industries, namely nontoxicity (food grade), easy availability, low cost, microbiologic stability, good thermal and mechanical resistance, and, finally, the possibility of being activated by a simple and inexpensive method.
2.
Cellulose and its derivatives gave poor results, whether concerning catalytic response or stability. Among XADamberlites the XAD 7 activated with trichlorotriazine permitted PL immobilization with fairly good adsorption (AY) and immobilization (IY) yields as well as good catalytic activity (335 U g-i). However, the stability of the immobilized enzyme was found to be low (1.3). Attempts made to improve stability by means of glutaraldehyde led to an
of Aspergillus
Rombouts. F. M., and Pilnik, W. Pectic enzymes: Economic microbiology. In: Microbial Enzymes and Bioconversion (Pilnik, W., ed.). Academic Press. London, 1980, 227-282 Withaker, J. R. Microbial pectolytic enzymes. In Microbial Enzymes and Biorechnology (Fogart, W. G., and C. T. Kelly. eds.). Elsevier Applied Science, London, 1992, 133-175 Spagna. G.. Pifferi, P. G., and Martino, A. Pectinlyase immobilization on epoxy supports for application in the food processing industry. J. Chem. Tech. Biorech. 1993. 51, 37%385 Spagna, G.. Pifferi, P. G., Tramontini, M., and Albertini, A. Pectinlyase immobilization on polyamides for application in the food processing industry. J. Chem. Tech. Biorechno/. 1994. 59, 341348 Lozano, P., Majon, A., RomoJaro. F.. and Iborra, J. L. Properties of pectolytic enzymes covalently bound to nylon for apricot juice clarification. Proc. Biochem. 1988, 3. 75-78 Dinnella, C., Lanzarini, G.. and Palleschi, C. A model system for biocatalysis involving macromolecular substrates. Cerev. Biotech. 1992, 17, 32-35 Hanish, W. H.. Richard, P. A. D., and Nyo, S. Poly(methoxy galacturonide) lyase immobilized via titanium onto solid supports. Biotech. Bioeng. 1978, 20, 95-106 Dinella, C.. Lanzarini, G., Stagni, A.. and Palleschi, C. Immobilization of an endo-pectinlyase on y-alumina. J. Chem. Tech. Biotech. 1994.59, 237-241 Ampon. K. Distribution of an enzyme in porous polymer beads. J. Chem. Tech. Biotech. 1992. 55, 185-190 Basri, M.. Ampon, K.. Zimwan Yunus. W. M., Razak, C. N. A.. and Salleh, A. B. Immobilization of hydrophobic lipase derivatives on the organic polymer beads. J. Chem. Tech. Biotech. 1994. 59, 3744 Garwood, G. A., Mortland. M. M., and Pinnavaia, T. J. Immobilization of glucose oxidase on montmotillonite clay: Hydrophobic and ionic modes of binding. J. Mol. Catal. 1983, 22, 153-163 Tomar. M., and Probhu. K. A. Immobilization of cane invertase on bentonite. Enzyme Microb. Technol. 1985, I, 454-458 Kohn, R., Marcovic, O., and Machova. R. Deesterification mode of pectin by pectin esterases of Aspergillus foetidus, tomatoes and alfalfa. Cal. Czech. Chem. Comm. 1983, 48, 791-797
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16. 17.
18. 19.
20. 21.
22. 23. 24. 25.
738
The Pectic Substances (Kertesz, 2. I., eds.). New York, 1951, 215-230 Owens. H. S.. Lotzkar, H., Schultz, T. H., and Maclav, W. D. Shape and size of pectinic acid molecules deduced from viscometric measurement. J. Amer. Chem. Sot. 1946, 68, 1628-1632 Spagna, G., and Pifferi, P. G. The purification of a commercial pectinlyase. Food Chem. 1994, 50, 343-349 Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254 Patchomik, A., and Rogozinski. E. S. Determination of ending groups, Anal. Chem. 1961, 33, 803-807 Pifferi, P. G.. Malacarne, A., Lanzarini, G., and Casoli. U. Spectrophotometric method for the determination of pectin methylesterase activity of bestom’s hydrazone. Chem. Mikrob. Tech. Lebensm. 1985, 9, 65-69 Albersheim, P. Pectin lyase from fungi. Methods Enzymol. 1968. 8, 628-635 Okai, A. A. E., and Geirschener, K. Size and charge properties of the pectic and cellulolytic enzymes in a commercial enzyme preparation. Z. Lebensm. Unters. Forsch. 1991, 192, 244-248 Kasztelan, S. A descriptive model of surfaces sites on MoS2 (WS2) particles. Langumir 1990. 6, 59c595 Goodboy, K. P., and Fleming. H. L. Trends in adsorption with aluminas. CEP November. 1984. 63-68 Weetall, H. H. Covalent coupling for inorganic support materials. Methods Emymol. 1976, 44, 134-148 Wahlgreen, M., and Amebrant, T. Protein adsorption to solid surfaces. Tibtech. 1991, 9, 201-208
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