Kinetic model of the hydrolysis of polypeptides catalyzed by Alcalase® immobilized on 10% glyoxyl-agarose

Enzyme and Microbial Technology 36 (2005) 555–564

Kinetic model of the hydrolysis of polypeptides catalyzed by Alcalase® immobilized on 10% glyoxyl-agarose Paulo W. Tardioli, Ruy Sousa Jr., Roberto C. Giordano, Raquel L.C. Giordano∗ Departamento de Engenharia Qu´ımica, Universidade Federal de S˜ao Carlos, Rodovia Washington Luiz, km 235, 13565-905, S˜ao Carlos, SP, Brazil Received 4 March 2004; accepted 7 December 2004

Abstract The sequential hydrolysis of cheese whey proteins can improve physical, chemical and organoleptic properties of this dairy by-product, increasing its applications in the food and pharmaceutical industry. The hydrolysis of polypeptides (50 ◦ C, pH 9.5), catalyzed by Alcalase® immobilized on 10% agarose (weight basis), activated with linear aliphatic aldehyde groups (glyoxyl-agarose), is studied here. The reaction substrate (polypeptides) is the product of a previous, sequential hydrolyses of cheese whey proteins by trypsin, chymotrypsin and carboxypeptidase A. A Michaelis–Menten model with product inhibition was fitted to the experimental data after long-term batch assays. Kinetic parameters k, KM and KI were correlated with respect to the degree of hydrolysis of the substrate in the upstream proteolyses, thus providing a general, semi-empirical rate equation. With this approach, the kinetic model may be included in process optimization algorithms, which may span different regions of operation for the proteolytic reactors. Parameters k, KM and KI ranged from 0.005 to 0.029 mmol/min/UBAEE , from 4.0 to 13.7 mM, and from 0.19 to 1.56 mM, respectively, when the previous degree of proteolysis (pre-hydrolysis) changed from 20 to 2%. © 2004 Elsevier Inc. All rights reserved. Keywords: Cheese whey; Polypeptide hydrolysis; Alcalase® -glyoxyl; Michaelis–Menten model; Competitive inhibition

1. Introduction The sequential-controlled enzymatic hydrolysis of cheese whey proteins (mainly ␤-lactoglobulin, ␣-lactoalbumin and serum albumin), using the immobilized proteases trypsin, chymotrypsin, carboxypeptidase A (CPA) and Alcalase® (commercial preparation of subtilisin), as shown in Fig. 1, may generate several final products of interest. Moreover, this process may aggregate economic value to an otherwise highly-pollutant residue (DBO ∼30,000 mg O2 /l) that is frequently discarded in natura, particularly in Brazil [1]. The reduction of the contents of hydrophobic amino acids in, mainly the aromatic ones [2], will solve one of the main problems of the protein hydrolysates for human feeding, i.e. the bitter taste [3–6]. Eliminating or reducing the contents of phenylalanine, one could generate a final product ∗

Corresponding author. Tel.: +55 16 3351 8707; fax: +55 16 3351 8266. E-mail addresses: [email protected] (R.C. Giordano), [email protected] (R.L.C. Giordano). 0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.12.002

to be ingested by phenylketonuria (PKU) patients, preventing the outcome of brain injuries [7]. Aromatic amino acids are mainly liberated by action of CPA [2,8,9], thus providing an adequate source of protein for PKU patients, after the proper downstream purification. Besides, after separation of the free amino acids from the reaction medium and under the action of Alcalase® , the final product would be composed by small peptides [10]. A product with these characteristics would be more acceptable than a mixture of free amino acids, typical product of acid proteolysis, which render a hyperosmotic diet, causing intestinal secretion and consequentially diarrhea [11]. Moreover, a protein hydrolysate composed by small peptides, without bitter taste, is very attractive for use by food industries [12,13]. To prepare a hydrolysate by an enzymatic route in an industrial scale, it would be important to use enzymes immobilized on insoluble supports, to reduce the process costs: recovery of soluble enzymes could be economically unfeasible. Furthermore, the utilization of high temperatures would be also very important to prevent microbial contamination.

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Fig. 1. Steps involved in the preparation of a protein hydrolysate composed by small peptides with low concentration of aromatic amino acids.

However, that condition could inactivate the free enzyme, a highly undesirable characteristic in a process that uses expensive enzymes to obtain products of medium value. Blanco and Guis´an [14], Guis´an et al. [15], Tardioli et al. [2,10] describe adequate conditions to obtain highly-stabilized derivatives of trypsin, chymotrypsin, CPA and Alcalase® , respectively. The knowledge of the kinetics of the reactions catalyzed by those biocatalysts is essential to optimize the economics of the process. Particularly for step 6 (Fig. 1), it would be very important to establish a kinetic model that would take into account the degree of hydrolysis of the substrate in the upstream proteolyses (steps 2–4). This rate equation could then be included in searching algorithms for process optimization, which may span different operational regions for the proteolytic reactors. This work puts forth such a kinetic model, for the hydrolysis of polypeptides catalyzed by Alcalase® immobilized on agarose activated with linear-aliphatic aldehyde groups (glyoxylagarose). 1.1. Kinetic model According to Svendsen [16] and Adler-Nissen [3], it is generally accepted that serine proteases like Alcalase® , when acting on peptide bonds, generally follow Michaelis–Menten kinetic. Furthermore, it is generally accepted that, in the hydrolysis of proteins catalyzed by Alcalase® , the reaction products (i.e. peptides which are continuously formed during the hydrolysis) competitively inhibit the hydrolytic reaction [3,12,17–19]. According to Adler-Nissen [3], the com-

petition for the active sites in the enzyme occurs between two nucleophilic agents: water, favoring the hydrolysis, and the free amino groups, favoring the transpeptidation reaction (Fig. 2 ). In a previous work, Souza Jr. et al. [19] studied the kinetics of the hydrolysis of whole cheese whey proteins, catalyzed by Alcalase® immobilized on 6% glyoxyl-agarose, and verified that a Michaelis–Menten model with product competitive inhibition represented well the system behavior. In that model, the molar concentrations of the complex substrate were lumped into a single variable, i.e. the number of peptide bonds available to be cleaved by Alcalase® , N (mM). The concentration of inhibitor (I) was considered equal to the concentration of cleaved peptide bonds (N0 −N), i.e. equal to the concentration of N-terminal groups that can link to the enzyme, inhibiting its hydrolase activity. This is actually a usual procedure for this kind of reaction: in fact, it would not be feasible to deal explicitly with each molecule, among the huge number of proteins, polypeptides and oligopeptides, and amino acids that are present during the proteolysis. Eq. (1) [19] shows the Michaelis–Menten model that was fitted to the data of whey proteins hydrolysis, catalyzed by immobilized Alcalase® . VN =

kEN KM (1 + (I/KI )) + N

(1)

In this equation, VN is the hydrolysis rate of peptide bonds (mmol/min/gbiocat ). N is the molar concentration of substrate peptide bonds that can be hydrolyzed by Alcalase® (mM). I is the molar concentration of inhibitor (mM). E is the concentration of enzyme, expressed in UBAEE /gbiocat , and k (mmol/min/UBAEE ), KM (mM) and KI (mM) are the kinetic parameters. In this work the same model (Eq. (1)) was fitted to experimental data of hydrolysis of polypeptides (the product of the action of trypsin, chymotrypsin and CPA on cheese whey proteins), catalyzed by Alcalase® immobilized on 10% glyoxylagarose. However, it was observed that the kinetic parameters (k, KM and KI ) changed, according to the degree of hydrolysis of the pre-hydrolyzed substrate (in steps 2–4, Fig. 1). To use Eq. (1), it is necessary to know the concentration of peptide bonds that might be cleaved by Alcalase® . In this work, the substrate was the product of the sequential action of trypsin, chymotrypsin and CPA. Hence, the concentration of peptide bonds that might be hydrolyzed by Alcalase® was equal to the number of peptide bonds in the whey proteins, minus the sum of the degrees of hydrolyses obtained with the two other endoproteases (trypsin and chymotrypsin). The peptide bonds cleaved by CPA (essentially an exoprotease) did not significantly reduce the concentration of peptide bonds to be cleaved by Alcalase® (an endoprotease). Previous long-term batch assays (6–8 h) showed that it was possible to obtain a maximum degree of hydrolysis (DHtotal ) of ca. 30% after the action of the three endoproteases. Above that value, a darkening of the reaction medium was observed,

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Fig. 2. Hydrolysis (A) and transpeptidation (B) reactions of a polypeptide chain, showing the competition between water and free amino groups for the nucleophilic attack of the peptide bond [3].

probably due to Maillard reactions. Therefore, 30% will be the maximum degree of hydrolysis of the substrate used in this work. Thus, the initial concentration of peptide bonds to be cleaved by Alcalase® (N0 , in mM), after the sequential action of trypsin and chymotrypsin on cheese whey proteins, was given by Eq. (2). N0 =

CH htot (DHtotal − DHinitial ) 100

(2)

where CH is the whey protein concentration (g/l), htot is the total concentration of peptide bonds in the whey proteins (8.8 mmol/g, according to Adler-Nissen ([3], p. 146–147), DHtotal is the maximum degree of hydrolysis of the cheese whey proteins after the hydrolysis with the three endoproteases (in this work, 30%) and DHinitial is the “initial” (for the Alcalase® ) degree of hydrolysis of the cheese whey proteins, i.e. the degree of hydrolyses after the proteolyses with trypsin and chymotrypsin (in this work, from 2 to 20%). Making use of a pH-stat, it is possible to observe the progress of the reaction by monitoring the consumption of a titrant, necessary to maintain the pH of the system during batch hydrolysis assays carried out in a laboratory-scale reactor. Initial reaction-rate assays in a pH-stat, with different substrate concentrations and different initial degrees of hydrolysis made it possible to estimate Michaelis–Menten kinetic parameters (k and KM ) as a function of the initial degree of hydrolysis of the substrate. Data from long-term batch assays provided estimates of the third parameter (KI ), taking into account the inhibitory effect of the products. That parameter was estimated for different initial degrees of hydrolysis (i.e. after steps 2 and 3, Fig. 1) using the Marquardt algorithm [20]. The quality of fit was always verified using visual comparisons between experimental and evaluated rates, and checking the standard deviations of the parameters.

2. Materials and methods 2.1. Materials “Minas Frescal” cheese whey (in natura) was donated by Cooperativa de Lactic´ınios S˜ao Carlos (Brazil). Trypsin (EC

3.4.21.1) from bovine pancreas and Alcalase® 2,4 L (EC 3.4.21.62) from Bacillus licheniformis were donated by Novo Nordisk A/S (Denmark). Chymotrypsin (EC 3.4.21.4) and carboxypeptidase A (EC 3.4.17.1), both from bovine pancreas, N-benzoyl-l-arginine etyl ester (BAEE), hyppuryl-lPhe (H-PHE), o-phthaldehyde (OPA) and mercaptoethanol were purchased from Sigma (USA). Sepharose CL-6B (6% agarose) was purchased from Amersham Pharmacia Biotech AB (Sweden) and 10% agarose was donated by Hispanagar (Spain). Bry-35 (polyethylene glycol dodecyl ether) was purchased from Fluka (Switzerland). All other reagents were of analytical grade. 2.2. Substrate preparation Cheese whey (pH between 4.5 and 5.0 and protein concentration between 6.0 and 7.0 g/l) was sterilized by microfiltration using 0.45 ␮m membrane (A/G Technology Corp., USA), concentrated to 45–50 g/l by ultra-filtration using Amicon® 10 kDa cutoff membrane (Amicon Inc., USA) and frozen to be used afterwards. Concentrations of proteins were measured by Kjeldahl method [21]. 2.3. Biocatalysts preparation Trypsin, chymotrypsin and CPA were immobilized on 6% agarose beads (weight basis, particles with average diameter of 100 ␮m) activated with 75 ␮eq. of aldehyde groups/ml of support (6% glyoxyl-agarose) according to protocols described by Blanco and Guis´an [14], Guis´an et al. [15] and Tardioli et al. [2]. Alcalase® was immobilized on 10% agarose beads (weight basis, particles with average diameter of 100 ␮m) activated with 210 ␮eq. of aldehyde groups/ml of support (10% glyoxyl-agarose), according to Tardioli et al. [10]. The trypsin and chymotrypsin derivatives were loaded with 14 mg of protein/g of support and all initial activities were measured using the immobilized enzyme. The CPA derivative was loaded with 1.6 mg of protein/g of support and had a recovered activity of 34.3 UH-PHE /g. The Alcalase® derivatives were prepared with three different enzymatic loads: the low-load derivatives had 0.2 and 1.4 mg of pro-

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tein/g of support, and the high-load one, 21 mg/g of support. The derivatives had recovered activities of 0.4, 2.7 and 45.7 UBAEE /g of support, respectively for the low-load and the high-load biocatalyst. 2.4. Enzymatic activity assays Enzymatic activities were measured spectrophotometrically by recording the increase in absorbance that accompanies the hydrolysis of the substrate. The assays were performed in a 1 cm light path quartz cuvette, thermostatically controlled at 25 ◦ C, using a Ultrospec 2000 spectrophotometer (Pharmacia Biotech, USA), adapted with magnetic stirring and water cell holder (circulation bath required). 2.4.1. BAEE activity It was measured according to Blanco and Guis´an [14]. Typically, 250 ␮l of enzymatic solution or immobilized enzyme suspension were added to the assay solution (2.5 ml of N-benzoyl-l-arginine etyl ester, BAEE, 0.5 mM, prepared in 50 mM phosphate buffer, pH 7.6) and the increase in absorbance was measured during 5–10 min, at 253 nm. Within the region of constant slope, the activity was calculated using a molar coefficient of 980 l/mol/cm. One BAEE unit (UBAEE ) was defined as the amount of enzyme that hydrolyses 1 ␮mol of BAEE/min under the conditions described. 2.4.2. H-PHE activity It was measured according to protocol reported by Folk and Schirmer [22] with slight modifications, as described by Tardioli et al. [2]. Typically, 100 ␮l of enzymatic solution or immobilized enzyme suspension were added to the assay solution (2.90 ml of hyppuryl-l-Phe, H-PHE, 1 mM, prepared in 25 mM Tris–HCl buffer, containing 0.5 M NaCl) and the increase in absorbance was measured during 5–10 min, at 254 nm. Within the region of constant slope, the amount of hippuric acid released by the action of CPA was calculated from the molar extinction coefficient (360 l/mol/cm). One H-PHE unit (UH-PHE ) was defined as the amount of enzyme that hydrolyses 1 ␮mol of H-PHE/min under the conditions described. 2.5. Hydrolysis reactions Hydrolyses of cheese whey protein concentrates were performed in pH-stat Titrino model (Metrohm, Switzerland), with magnetic stirring and controlled pH and temperature. The protons released during the hydrolysis were titrated with base solution, in an appropriated concentration for each assay. The initial rates of hydrolyses (VH ) were calculated by Eq. (3). BNb

1 = VH t α

(3)

where B is the volume of base consumed during the hydrolysis (ml), Nb is the normality of the base (meq./ml), α is the degree of dissociation of the ␣-amino group (Eq. (4)). VH is the initial rate of hydrolysis, i.e. the slope of the linear region of the curve of peptide bonds hydrolyzed (BNb (1/α)) as a function of the time (mmol/min), and t is the reaction time (min). α=

10(pH−pK) 1 + 10(pH−pK)

(4)

where pK is the average value of the ␣-amino groups liberated during the hydrolysis, pK values varies significantly with temperature, but is fairly independent of the substrates as such. pK values are supplied by Adler-Nissen ([3], p. 123). For substrates hydrolyzed by trypsin, chymotrypsin and Alcalase® , the degree of hydrolysis was calculated by Eq. (5) ([3], p. 122). DH = BNb

1 1 1 × 100% α MP htot

(5)

where DH is the degree of hydrolysis, defined as the percentage of peptide bonds cleaved during the proteolytic reaction, and MP is the mass of protein (g). Using Eq. (6) the degree of hydrolysis for substrates hydrolyzed by CPA was determined. DH =

MRA × 100% MTA

(6)

where MRA is the mass (g) of amino acids released by CPA/100 g of proteins and MTA is the total mass (g) of amino acids/100 g of whey proteins (115.54 g of amino acids/100 g of proteins, according to Adler-Nissen [3]). Hydrolyses catalyzed by trypsin and chymotrypsin were performed at 55 ◦ C, pH 8.0 [23]; hydrolyses catalyzed by CPA were performed at 45 ◦ C, pH 7.0 [2] and hydrolyses catalyzed by Alcalase® were performed at 50 ◦ C, pH 9.5 [19]. 2.6. Amino acids analysis Amino acids were analyzed by high-performance ionexchange chromatography using a Shim-pack ISC-30 aminoNa column (Shimadzu, Japan) and mobile phases A (0.2N sodium citrate buffer, pH 3.2), B (0.6N sodium citrate-boric acid buffer, pH 10) and C (0.2N sodium hydroxide) in a gradient elution at 0.50 ml/min and run for 72 min. The aftercolumn reagents were RA: carbonic acid–boric acid solution, pH 10, containing 0.04% (v/v) sodium hypochlorite and RB: 0.08% (w/v) o-phthalaldehyde solution, containing 0.04% (w/v) Bry-35 and 0.2% (v/v) 2-mercaptoethanol, at 0.20 ml/min for each solution. The temperature was 60 ◦ C and the amino acids were detected by fluorescence at 350 (excitation λ) and 450 nm (emission λ). The amino acid concentration (CAA ) was determined using standard curves (CAA versus chromatographic area, AC ) for each of the 20 standard amino acids (purchased from Sigma, USA).

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Fig. 3. Initial rates of hydrolyses of polypeptides, catalyzed by Alcalase® glyoxyl loaded with 0.4 UBAEE /g of support (low-load), at 50 ◦ C, pH 9.5. Substrate: cheese whey proteins hydrolyzed by trypsin-glyoxyl (E/S of 1/140, w/w, DH of 2%), chymotrypsin-glyoxyl (E/S of 1/190, w/w, DH of 5%) and CPA-glyoxyl (E/S of 25 UH-PHE /l of reactor, DH of 2.9%).

559

Fig. 4. Initial rates of hydrolyses of polypeptides (cheese whey proteins previously hydrolyzed, with DH from 2 to 20%), 50 ◦ C and pH 9.5, catalyzed by Alcalase® -glyoxyl (low-load derivative: 1.4 mg/g of support). Reaction conditions: 20 ml of substrate (2.5–40 g/l) were hydrolyzed with 20 UBAEE of Alcalase® /l of reactor for 10 min, using 40 mM NaOH for pH control.

3. Results and discussion 3.1. Selection of the enzyme concentration Twenty milliliters of a solution (ca. 19 g/l) of polypeptides and free amino acids (cheese whey proteins hydrolyzed by trypsin-, chymotrypsin- and CPA-glyoxyl, DH of 9.9%) were hydrolyzed by Alcalase® -glyoxyl (low-load derivative: 0.20 mg/g of support) for 10 min in a pH-stat Titrino model, with a 40 mM NaOH solution for pH control. Using the volume of NaOH consumed during the reaction, the initial rates of hydrolyses (VH ) were assessed by Eq. (3), for each enzyme concentration within the reactor. The linearity of the enzyme concentration (UBAEE /l of reactor) versus VH (meq./min) curve, Fig. 3, shows that mass transfer effects were not masking the kinetic results, for enzyme concentrations within the range studied here. This result also show that in the studied range the reaction kinetics follow a Michaelis–Menten like model (at standard conditions of pH, temperature and substrate concentration the initial rates of hydrolysis is a linear function of the enzyme concentration). Hence, 20 UBAEE /l were chosen to be used hereafter. This value was sufficiently high to provide products within a relatively short reaction time, with measurable concentrations, within high experimental accuracy. 3.2. Estimation of k and KM Preliminary assays showed that a Michaelis–Menten-like model of the hydrolysis of polypeptides would not be accurate if the upstream proteolyses were not taken into account. This is a direct consequence of the keystone hypothesis of this kind of proteolytic model, namely: that the complex mixture of peptides and amino acids that constitute the enzyme substrate is lumped into a single variable, a “concentration of

hydrolysable peptide bonds” (N, in Eq. (1)). This is a rather drastic, but necessary simplification, since considering each possible individual molecule that might appear after a partial hydrolysis would clearly result in an intractable system. However, a substrate containing free amino groups that inhibit the hydrolysis will certainly present a different kinetic behavior than a pool of proteins, and this effect must be considered somehow. Inhibitory effects may be lumped in an apparent constant, ap KM . Adler-Nissen [3] verified an approximately five-fold deap crease of KM (from 0.5 to 0.11%, in terms of degree of hydrolysis) when 9.5% previously hydrolyzed casein was the substrate of Alcalase® , instead of intact casein. Vmax also had a four-fold decrease, reflecting the inhibitory (products, amino acids) and competitive (transpeptidation) effects. Table 1 shows the kinetic parameters (Vmax and KM ) for different initial degrees of hydrolyses (i.e. for different degree of advance of the proteolyses by trypsin and chymotrypsin, that occurred before the reaction catalyzed by Alcalase® ). These parameters were estimated by the Levenberg–Marquardt method (using Origin® 6.0). The 95% confidence level intervals of the estimates are also shown. Initial values of the parameters were obtained using the Lineweaver–Burk method. Different initializations were also tested, but the method always converged to the same pairs (Vmax , KM ). As it is usual for Michaelis–Menten models, the two parameters were correlated, with an average correlation coefficient of 0.77. Standard deviations of the parameters were low (less than 12% for KM and 3% for Vmax ) and the model (Michaelis–Menten without inhibition) was very accurate (see Fig. 4). Table 2 shows that the values of KM was within the magnitudes reported by the literature. Fig. 4 shows that the rates of hydrolysis decrease when the degrees of pre-hydrolysis increase, for the same initial con-

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Table 1 Inherent kinetic parametersa of the Michaelis–Menten model (without inhibition), with 95% confidence intervals Previous DH (%)

Vmax (mmol/min/gbiocat )

E (UBAEE /gbiocat )

k = Vmax /E (mmol/min/UBAEE )

KM (mM)

2b 5b 7c 10c 15d,e 20e

0.057 ± 1.6 × 10−3 0.068 ± 1.9 × 10−3 0.046 ± 6.0 × 10−4 0.043 ± 1.1 × 10−3 0.021 ± 4.0 × 10−4 0.010 ± 5.0 × 10−4

1.97 2.68 1.97 2.68 2.39 2.15

0.029 0.025 0.023 0.016 0.009 0.005

13.7 9.1 8.0 5.5 4.9 4.0

± ± ± ± ± ±

1.5 0.9 0.6 1.0 0.4 0.8

Hydrolysis of polypeptides catalyzed by Alcalase® -glyoxyl (low-load derivative: 1.4 mg/g of support), 50 ◦ C, pH 9.5, E/S 20 UBAEE /l. a Observable Thiele modulus, Φ ([24], p. 133 ff), was less than 0.1 for all assays, providing an effectiveness factor, η, essentially equal to 1.0. b Cheese whey proteins previously hydrolyzed by trypsin-glyoxyl (E/S = 1/50, w/w). c Cheese whey proteins previously hydrolyzed sequentially by trypsin-glyoxyl and chymotrypsin-glyoxyl (E/S = 1/50, w/w). d Cheese whey proteins previously hydrolyzed simultaneously by a mixture (1:1) of trypsin-glyoxyl/chymotrypsin-glyoxyl (E/S = 1/50, w/w). e Cheese whey proteins previously hydrolyzed sequentially by mixture (1:1) of trypsin-glyoxyl/chymotrypsin-glyoxyl and Alcalase® -glyoxyl (E/S = 4 UBAEE /g of protein).

centration of substrate, N0 . As previously mentioned, this behavior can be caused by the greater availability of free amino acids, released by trypsin and/or chymotrypsin (these enzymes contain exoproteases as impurities). Either competition of transpeptidation reactions or product-inhibition effects would then reduce the hydrolytic activity of Alcalase® . The previous action of chymotrypsin, an endo-enzyme that attacks preferably hydrophobic amino acids, may also play a role here. Hydrophobic amino acids are preferential cleavage points for subtilisins, as well, and therefore the overall activity of the Alcalase® -derivative would be further decreased by the pre-hydrolysis with chymotrypsin. Plotting KM and k as function of the degree of prehydrolysis (see Fig. 5), an exponential (first-order) and a linear decay with the previous DH can be observed, respectively to KM and k. These parameters can be correlated as functions

of DH:




KM = 3.9 + 14.5 exp

−DH 5.1

 (7)

k = 3.2 × 10−2 − 1.4 × 10−3 DH

(8)

3.3. Estimation of KI The Michaelis–Menten model with product inhibition (Eq. (1)) was fitted to long-term batch results. Constant values of Vmax and KM (reported in the Section 3.1) were adopted, and parameter KI could be estimated. The initial concentration of inhibitor (I0 ) was zero, because any inhibitory effect was already lumped in KM and Vmax . Table 3 lists the obtained KI values for different degrees of pre-hydrolysis. The standard deviations of the estimated

Table 2 Kinetic parameters of hydrolyses catalyzed by Alcalase® Biocatalysts

Substrate

KM (g/l)

k (Vmax /E) (per second)

Experimental conditions

Source Ferreira et al. [25]

Soluble Alcalase® -silicaglutaraldehyde Alcalase® -glyoxyl 6% agarose Soluble Alcalase®

Casein Casein

0.78 0.63

0.0036 0.0048

50 ◦ C,

Whey proteins

13–26

0.022–0.069a

50 ◦ C, pH 6–11

Casein

3.6

0.86

50 ◦ C , pH 8

Soluble Alcalase®

Soybean protein hydrolysate Casein Soybean proteins Soybean proteins Soybean proteins Gelatin Casein Whey protein hydrolysate

6.0

n.a.

50 ◦ C, pH 8

Sousa Jr. et al. [19] Mannhein and Cheryan [26] Adler-Nissen [3]

5.0 9.0 15.0 12.0 5.0 1–2 3.1–5.6b

n.a. n.a. n.a. n.a. n.a. n.a. 0.005–0.029a

60 ◦ C, pH 8 50 ◦ C, pH 10 30 ◦ C, pH 8 30 ◦ C, pH 8 50 ◦ C, pH 9.5

This work

Alcalase®

Alcalase® -glyoxyl 10% agarose

n.a.: not available. a Units expressed in mmol/min/U BAEE . b Conversion from mmol/l to g/l using Eq. (2), making C = K in g/l and N = K in mM. H M 0 M

pH 8

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561

Fig. 5. Dependency of (A) KM and (B) k with respect to the degree of prehydrolysis of the substrate (cheese whey proteins previously hydrolyzed by trypsin and chymotrypsin).

KI were in average less than 7%, and the model results (concentration of cleaved peptide bonds as function of time) were very accurate (Fig. 6). Higher deviations between simulated values and experimental data were observed only for the highest degree of pre-hydrolysis (20%), probably due to experTable 3 KI values (with 95% confidence intervals), estimated using the Michaelis–Menten model with product inhibition (Eq. (1)), fitted from long-term batch hydrolyses of polypeptides at 50 ◦ C, pH 9.5, catalyzed by Alcalase® -glyoxyl (low-load derivative: 1.4 mg/g of support) Previous DH (%)

N0 (mM)

KI (mM)

2 5 5 7 10 10 15 15 20 20

106.0 19.6 78.2 76.9 16.3 65.2 45.6 11.4 32.3 8.1

1.56 ± 4 × 10−2 1.15 ± 6 × 10−2 0.95 ± 4 × 10−2 0.83 ± 2 × 10−2 0.58 ± 4 × 10−2 0.46 ± 1 × 10−2 0.46 ± 3 × 10−2 0.31 ± 4 × 10−2 0.17 ± 2 × 10−2 0.21 ± 4 × 10−2

Fig. 6. KI estimate: long-term batch hydrolyses of polypeptides (cheese whey proteins pre-hydrolyzed by trypsin and/or chymotrypsin with DH from 2 to 20%) at 50 ◦ C, pH 9.5, catalyzed by Alcalase® -glyoxyl (lowload derivative: 1.4 mg/g of support). Reaction conditions: 20 ml of substrate (34.6–43.0 g/l for (A) or 8.6–9.3 g/l for (B)), 4 h, 200 mM NaOH for pH control and E/S of 4 or 2 UBAEE /g of protein. Symbols are experimental data (molar concentration of peptide bonds, determined by Eq. (2)) and lines are values predicted by the kinetic model (Eqs. (1), (7)–(9)).

imental errors: the measured rates were very low. Yet, the standard deviation of the parameter was less than 18%. Fig. 7 shows that KI decays exponentially (first-order) with the degree of pre-hydrolysis, according to:
 −DH −1 KI = 1.1 × 10 + 1.9 exp (9) 7.6 3.4. Validation of the model The kinetic model, which predicts the inherent rate of hydrolysis of polypeptides catalyzed by Alcalase® -glyoxyl, was validated against long-term batch runs using cheese whey proteins sequentially pre-hydrolyzed by trypsin (DH of 2

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Fig. 7. Dependence of KI with respect to the degree of pre-hydrolysis of the substrate (cheese whey proteins previously hydrolyzed by trypsin and chymotrypsin).

Fig. 9. Amino acids released from cheese whey proteins hydrolyzed (overall DH of 29%) by trypsin, chymotrypsin, CPA and Alcalase® immobilized on glyoxyl-agarose. The theoretical concentration of each amino acid present in the whey proteins [3] was considered as 100%.

An excellent agreement can be observed in Fig. 8, suggesting that the inherent kinetics (without diffusive effects) of the hydrolysis of polypeptides (at 50 ◦ C and pH 9.5), catalyzed by Alcalase® immobilized on 10% glyoxyl-agarose (low-load derivative: 1.4 mg/g of support), can be accurately described by a semi-empiric Michaelis–Menten equation with product inhibition, provided its kinetic parameters take into account the degree of pre-hydrolysis of the substrate. 3.5. Cheese whey protein hydrolysate composition Table 4 shows the molecular mass distribution of the final product of the hydrolysis of cheese whey proteins, for a total degree of hydrolysis of 29%, which was obtained using the four proteases. The final product was composed by small peptides (ca. 70%, w/w, with molecular mass less than 1.7 kDa) Fig. 8. Kinetic model validation: long-term batch hydrolyses of polypeptides at 50 ◦ C, pH 9.5, E/S = 2 UBAEE /g of proteins, catalyzed by Alcalase® glyoxyl (low-load derivative: 1.4 mg/g of support). Kinetic parameters: (䊉) and (
) KM = 7.58 mM and KI = 0.41 mM; ( ) KM = 7.58 mM and KI = 0.63 mM, and ( ) KM = 6.86 mM and KI = 0.29 mM. For all assays, k = 0.02 mmol/min/UBAEE .

and 0.81%), chymotrypsin (DH of 5 and 7.3%) and CPA (DH of 2.9, 7.08 and 9.98%) as substrate. It should be noticed that these are actual validation testes, since none of these substrates were used for the fitting of the kinetic model. Fig. 8 shows the results (Eq. (1), using the parameters assessed from Eqs. (7)–(9)). To estimate k and KM , the degree of pre-hydrolysis obtained with endoproteases (trypsin and chymotrypsin) was used. In the case of KI , the total degree of pre-hydrolysis (DH obtained with trypsin, chymotrypsin and CPA) was substituted in Eq. (9), so the inhibitory effect of the free amino acids released by CPA would be taken into account.

Table 4 Molecular mass distribution of the final product of the hydrolysis of cheese whey proteins (5%, w/w), sequentially catalyzed by trypsin-glyoxyla , chymotrypsin-glyoxylb , CPA-glyoxylc and Alcalase® -glyoxyld , with a total degree of hydrolysis of 29% Molecular mass distributione (kDa)

Mass composition (%)

MM > 5.0 1.7 < MM < 5.0 1.0 < MM < 1.7 0.6 < MM < 1.0 MM < 0.6

10.60 21.94 49.22 6.11 12.14

55 ◦ C, pH 8.0, E/S = 1/180, 2% DH. 55 ◦ C, pH 8.0, E/S = 1/200, 5% DH. c 45 ◦ C, pH 7.0, 8 h of reaction, E/S = 19 U H-PHE /g, 7% DH. d 50 ◦ C, pH 9.5, E/S = 2 U BAEE /g, 6 h 20 min of reaction, 15% DH. e Peptides were analyzed by high performance size exclusion chromatography, using a Superdex Peptide HR 10/30 column (Pharmacia Biotech, Sweden) and insulin (5 kDa), neurotensin (1672 Da), angiotensin II (1002.2 Da) and leucine enkephalin (555.6 Da) as standards. The molecular mass distribution of the resulting peptides was assessed following the methodology of Sousa et al. [27]. a

b

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563

Fig. 11. Hydrolyses of polypeptides at 50 ◦ C, pH 9.5, E/S of 2 UBAEE /g, catalyzed by Alcalase® -glyoxyl (high-load derivative: 21 mg/g of support). The inherent-kinetic parameters were: KM = 7.58 mM (DH of 7%), k = 0.02 mmol/min/UBAEE (DH of 7%) and KI = 0.49 mM (DH of 12.3%). Assumed effectiveness factor η = 0.49.

3.6. Effectiveness factor of hydrolyses catalyzed by a highly loaded catalyst

Fig. 10. (A) Release of aromatic amino acids at 45 ◦ C, pH 7.0, by action of CPA-glyoxyl. (B) Amino acids released by action of CPA-glyoxyl after 9 h of reaction at 45 ◦ C, pH 7.0 and DH of 10.4%. Substrate: cheese whey proteins hydrolyzed by trypsin (DH of 2%) and by chymotrypsin (DH of 5%).

and free amino acids (ca. 9.5%, w/w). Fig. 9 shows that from the theoretical total number of aromatic amino acids, ca. 35% were released, i.e. ca. 47% of Tyr, ca. 29% of Phe and ca. 27% of Trp. If a final product completely free of aromatic amino acids were pursuit, the proteolyses by trypsin and by chymotrypsin might proceed up to a higher degree of hydrolysis. All aromatic residues could then be placed in the C-terminal ends of the polypeptide chains. Besides, in the proteolysis catalyzed by CPA, the biocatalyst could be added by steps, to avoid or minimize enzyme inhibition by the released amino acids, especially phenylalanine and tyrosine, which are competitive inhibitors of CPA [2]. Fig. 10 shows that there were released ca. 71, 47 and 45%, respectively of Tyr, Phe and Trp, after 9 h of reaction at 45 ◦ C, pH 7.0, using this strategy.

Cheese whey proteins were sequentially hydrolyzed by trypsin-glyoxyl and by chymotrypsin-glyoxyl (E/S = 1/50, w/w, DHtotal of 7%), by CPA-glyoxyl (200 UH-PHE /l of reactor, DH of 5.3%) using a highly-loaded (21 mg of protein/g of support) Alcalase® -glyoxyl (E/S = 2 UBAEE /g of protein, 4 h of reaction). Data of N0 − N versus time of one assay (N0 of 60.7 mM) were used to estimate the effectiveness factor of the reaction with highly-loaded biocatalyst, η (i.e. ratio between the measured rates and the ones assessed by the kinetic model), using the inherent kinetics of the hydrolysis of polypeptides catalyzed by Alcalase® immobilized on 10% glyoxyl-agarose (lowly-loaded). Although this is a transient process, η was practically constant during the course of the reactions. Its standard deviation was less than 2%. Hence, a constant value was adopted in further simulations (η = 0.49 ± 0.01). It is clear that the high-load system has important diffusion effects. Independent assays were used to validate the estimated effectiveness factor. Fig. 11 shows that concentration time profiles, predicted by the model, and experimental results are very close.

4. Conclusions The inherent kinetics of the hydrolysis of polypeptides catalyzed by lowly-loaded Alcalase® -glyoxyl (1.4 mg of protein/g of support) could be represented by a Michaelis–Menten equation with product inhibition. How-

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ever, the kinetic parameters (k, KM and KI ) must vary with the degree of pre-hydrolysis of the substrate (with trypsin, chymotrypsin and carboxipeptidase A). KM ranged from 4.0 to 13.7 mM, k from 0.005 to 0.029 mmol of peptide bonds/min/UBAEE , and KI from 0.19 to 1.56 mM. The order of magnitude of KM agreed with the literature, for different Alcalase® -substrate systems. Apparent reaction rates of the hydrolysis of polypeptides catalyzed by highly-loaded Alcalase® -glyoxyl (21 mg of protein/g of support) was different from the inherent ones. An effectiveness factor of ca. 0.5 was obtained. Thus, the diffusion of the polypeptides inside the 10% glyoxyl-agarose support pores puts an important resistance to this process. The final product of the sequential hydrolysis of cheese whey proteins catalyzed by four proteases (total DH of 29%), using the conditions for high thermal stability (55 ◦ C and pH 8.0 for trypsin and chymotrypsin; 45 ◦ C and pH 7.0 for CPA and 50 ◦ C and pH 9.5 for Alcalase® ), was composed primarily by small peptides (ca. 70%, w/w, with molecular mass below of 1.7 kDa) and free amino acids (ca. 9.5%, w/w). In those conditions, and for a degree of hydrolysis of 17% (obtained after hydrolyzing cheese whey proteins with trypsin-, chymotrypsin-, CPA- and Alcalase® -glyoxyl), ca. 71, 47 and 45%, respectively of tyrosine, phenylalanine and tryptophan, were released. Higher withdraws of Phe may be achieved using other process operational conditions. Acknowledgments The authors thank the Brazilian research funding agencies FAPESP and PADCT/CNPq for support, Cooperativa de Lactic´ınios S˜ao Carlos (Brazil) for the gift of the cheese whey and Viviane P.S. Folly from Novozymes Latin America Ltd. for the generous donation of the enzyme Alcalase® (Novo Nordisk A/S, Denmark). References [1] UNILIVRE Bulletin. Impacts of commercial liberalization on the environmental quality of developing nations (Portuguese): available in http://www.unilivre.org.br/boletimunilivre/folhatec02.htm. Access on June 28th, 2003. p. 1–7. [2] Tardioli PW, Fern´andez-Lafuente R, Guis´an JM, Giordano RLC. Design of new immobilized-stabilized carboxypeptidase A derivative for production of aromatic free hydrolysates of proteins. Biotechnol Prog 2003;19:565–74. [3] Adler-Nissen J. Enzymic hydrolysis of food proteins. London: Elsevier Applied Science Publishers; 1986. [4] Kukman IL, Zelenik-Blatnik M, Abran V. Isolation of low-molecularmass hydrophobic bitter peptides in soybean protein hydrolysates by reversed-phase high-performance liquid chromatography. J Chromatogr A 1995;704:113–20. [5] Hossain MMD, Stanley RA. Selective removal of hydrophobic peptides from protein hydrolysates in a continuous supported liquid membrane process. Sep Sci Technol 1996;31(10):1443–62.

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