Enzyme and Microbial Technology 31 (2002) 300309

A new kinetic model proposed for enzymatic hydrolysis of lactose by a -galactosidase from Kluyveromyces fragilis
E. Jurado , F. Camacho, G. Luzn, J.M. Vicaria
Departmento Ingenier a Qu mica, Facultad Ciencias, Universidad de Granada, Granada 18 071, Spain Received 3 January 2002; accepted 21 March 2002

Abstract We study the enzymatic hydrolysis of lactose by a commercial enzyme from a selected strain of Kluyveromyces fragilis. The variables analyzed were: temperature (2540 C), enzyme concentration (0.13.0 g l1 ), lactose concentration (0.02780.208 M), and initial galactose concentration (0.0347 M). On the basis of the data analyzed, both published and in the present work, we propose a MichaelisMenten kinetic model with inhibition by the product (galactose), which reveals that the substrate (lactose) and the product (galactose) present similar afnity for the active site of the enzyme. 2002 Elsevier Science Inc. All rights reserved.
Keywords: Lactose hydrolysis; Kluyveromyces fragilis; Kinetic model; -Galactosidase

1. Introduction Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry because of the potentially benecial effects on the assimilation of foods containing lactose, as well as the possible technological and environmental advantages of industrial application, including: 1. Elimination of lactose intolerance (370% depending on the populational group [1]), encouraging the utilization of lactose as an energy source, as well as calcium and magnesium assimilation from milk. 2. Formation of galacto-oligosaccharides during lactose hydrolysis to favor the growth of intestinal bacterial microora. The presence of these compounds is considered desirable in foods [2,3]. 3. Improvement in the technological and sensorial characteristics of foods containing hydrolyzed lactose from milk or whey [47] such as: increased solubility (avoidance of lactose crystallization and the grainy aspect of ice creams and condensed or powdered products); greater sweetening power and thus lower caloric content of the products (glucose and galactose monosaccharides have greater sweetening power than does lactose); formation of monosaccharides, which are easier to ferment in cer

tain products such as yogurt [8]; lower freezing point of ice creams (increasing softness and creaminess); and reduction of the Maillard reaction. 4. Greater biodegradability of whey in which the lactose has been hydrolyzed [9]. The commercial enzymes used for lactose hydrolysis are -galactosidases of diverse origins [5,6,10]. Yeast and fungal enzymes have the greatest commercial interest. Many studies have been made with -galactosidases obtained from Escherichia coli, although their use is not viable for products intended for human consumption [1115]. The optimal operating conditions are described in Table 1. Fungal enzymes are usually used to hydrolyze lactose from products with acidic pH values, such as whey. Yeast enzymes are habitually used for products with neutral pH values [16] such as milk and sweet whey. The mechanism of enzymatic hydrolysis of lactose by -galactosidase applied to different substrates (lactose solutions, whey and skim-milk) under different experimental conditions has been studied by several authors. Table 2 presents the kinetic models proposed by different researchers, showing that most propose a MichaelisMenten kinetic model, with competitive inhibition by galactose. However, there is great dispersion of the values of the kinetic constants proposed (see Table 3). The present work provides a survey of the models proposed by different authors, presents an experimental study of enzymatic hydrolysis in a stirred tank with -galactosidase

Corresponding author. E-mail address: ejurado@ugr.es (E. Jurado).

0141-0229/02/$ see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 1 0 7 - 2

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Nomenclature E L Ga Gl EL EGa k KM KI r e x R Ea ( Hf )a T concentration of free enzyme present in the reaction medium (g enzyme preparation l1 ) concentration of monohydrate lactose (M) concentration of galactose (M) concentration of glucose (M) concentration of the enzymelactose complex concentration of the enzymegalactose complex rate constant of Eq. (2) (mol g1 h1 ) MichaelisMenten constant (M) equilibrium constant of Eq. (3) (M) reaction rate (mol g1 h1 ) concentration of total active enzymatic complex (g l1 ) conversion (adimensional) constant of the ideal gases (cal K1 mol1 ) activation energy (kcal mol1 ) enthalpy of formation (kcal mol1 ) temperature (K)

Subscripts 0 initial concentrations

from Kluyveromyces fragilis, and proposes a simplied kinetic model for the action of the enzyme.

2. Materials and methods The chemical products used (PRS quality) are glucose, citric acid, K2 HPO4 , KCl, trichloroacetic acid (supplied

by Panreac), MgCl2 6H2 O (Prolabo), monohydrate lactose (Scharlau), and galactose (Across). The enzyme used was a commercial -galactosidase, lactozym 3000L HP-G [EC.3.2.1.23], which has a protein content of 35 g l1 , supplied by Novo Nordisk, derived from a selected strain of the yeast K. fragilis, = 1.2 g ml1 , with a declared activity of 3000 LAU ml1 (1 LAU = commercial enzyme which can obtain 1 mol glucose min1 in standard conditions: 4.7% lactose concentration, pH 6.5, 30 C, 30 min, standard milky buffer [4]). This enzyme satises the specications recommended for food enzymes. The glucose was analyzed applying the GOD-Perid method proposed by Werner et al. [36] using a commercial reagent (Behringer Mannheim Gmbh). The galactose and lactose present in the medium had no inuence on the glucose determination. As the reaction medium, lactose solutions were prepared on a buffer of 0.01 M K2 HPO4 , 0.015 M KCl, and 0.012 M MgCl2 6H2 O at pH 6.75 adjusted with citric acid. The enzymatic activity was measured in test tubes at 30 C in the following way: 1 ml of 50 g l1 monohydrate lactose solution prepared on the buffer indicated was added to 1 ml of 10 g l1 enzyme solution prepared on the same buffer. The test tube was incubated at 30 C for 10 min, after which 1 ml was extracted. The reaction was stopped by mixing with 1 ml of 0.1N trichloroacetic acid. Afterwards, the glucose concentration was measured by the GOD-Perid method. The enzymatic activity remained constant for the entire period of use. The enzymatic reaction took place in a 200 ml stirred tank reactor with pH and temperature controls. For a maximum period of 2 h, samples were extracted from the reactor. The enzyme was denatured with 0.1N trichloroacetic acid and the glucose concentration was measured by the GOD-Perid method. The variables tested are shown in Table 4.

Table 1 Experimental conditions of the most used enzymes Enzyme source Fungal (Aspergillus niger) Fungal (A. oryzae) Yeast (K. fragilis) Yeast (K. lactis) pH (optimal) 3.04.0 5.0 6.6 6.97.3 T ( C) (optimal) 5560 5055 37 35 Cofactors

Mn2+ , Mg2+ , K+ Mn2+ , Na+

Table 2 Kinetic models proposed by different authors for enzymatic hydrolysis of lactose Kinetic model proposed MichaelisMenten rst order MichaelisMenten without inhibition by product (galactose) (with enzymelactose complex formation) MichaelisMenten with competitive inhibition by product (total galactose) MichaelisMenten with competitive inhibition by product (- and - galactose) MichaelisMenten with competitive inhibition by product (glucose) Di-, tri- and tetra-saccharides formation References [17] [18] [9,11,12,1831] [32,33] [24] [34]

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Table 3 Kinetic constants proposed by different authors for the enzymatic hydrolysis of lactose References [19] Enzyme source A. A. A. A. oryzae oryzae oryzae oryzae (sol.) (imm.) (sol.) (imm.) pH 4.5 4.5 6.5 6.5 4.5 T ( C) 50 50 50 50 50 50 7.0 21 KM (M) 0.112 0.122 0.178 0.160 0.0357 0.0539 0.00298 0.00346 0.00434 0.00332 0.00373 0.0469 0.0490 0.0536 0.0519 0.0533 0.0436 0.1370 0.0797 0.0764 0.0733 0.0705 0.0679 0.0656 0.0635 0.0615 0.006 0.0286 0.052 0.0833 0.0809 0.0799 0.0795 0.079 0.0786 0.0782 0.1019 0.0995 0.0986 0.0982 0.0977 0.0973 0.0969 0.021 0.0278 0.0544 0.0365 0.0004 0.0046 0.023 KI (M) 0.0095 0.0105 0.0025 0.0045 0.0201 0.00092 (), 0.0118 (), 0.0109 (complex) 0.035 0.034 0.082 0.039 0.034 0.0200 0.0200 0.0321 0.0055 0.0946 0.0519 0.2340 0.0000613 0.0000620 0.0000627 0.0000633 0.0000640 0.0000646 0.0000652 0.0000658 0.012 0.0024 0.00022 (), 0.042() 0.000472 0.000528 0.000553 0.000565 0.000578 0.00059 0.000601 0.000527 0.000584 0.000609 0.000622 0.000634 0.000646 0.000658 0.0292 0.0316 0.0869 0.0114 0.00041 0.0036 0.0153

[35] [32] [11]

A. oryzae (sol.) A. niger (sol.) E. E. E. E. E. coli1 (imm.) coli2 (imm.) coli3 (imm.) coli4 (imm.) coli5 (imm.)

[20] [21]

A. oryzae (sol.) A. oryzae (imm.) A. niger (imm.)

4.5 4.5

50 50 30 40 50 43 25 30 35 40 45 50 55 60 23 37 30 8 30 40 45 50 55 60 8 30 40 45 50 55 60 28 35 28 35 5 25 40

[22] [23]

K. fragilis (sol.) K. fragilis (imm.) A. niger (imm.)

6.9 4.0

[26] [33] [27]

K. fragilis (imm.) A. niger (imm.) A. oryzae (imm.) A. niger (sol.) (Model 1)

5.2 4.5 4.0

A. niger (sol.) (Model 2)

4.0

[28]

K. marxianus (sol.) K. marxianus (imm.)

6.6 6.6 6.57.2

[9]

K. fragilis (sol.)

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Table 3 (Continued ) References [30] Enzyme source K. marxianus (imm.) pH 6.6 T ( C) 10 15 20 22.5 25 27.5 30 32.5 35 37.5 KM (M) 0.0154 0.0183 0.0219 0.0239 0.0248 0.0275 0.0317 0.0330 0.0379 0.0383 KI (M) 0.0247 0.0370 0.0561 0.0695 0.0801 0.0969 0.1097 0.1351 0.1620 0.2008

Table 4 Variables tested in the hydrolysis experiments performed T ( C) 25 L0 (102 M) 2.78 6.94 13.9 20.8 2.78 3.47 6.94 13.9 20.8 2.78 6.94 13.9 20.8 Ga0 (102 M) 0 0 0 0 0 3.47 0 0 0 0 0 0 0 e0 (g l1 ) 0.5 0.1, 0.5, 1.0, 3.0 0.1, 0.5, 1.0, 3.0 0.5 0.1, 0.5 0.1, 0.1, 0.1, 0.5, 1.0 0.5, 1.0 0.5, 1.0 0.5, 1.0

The variation in the glucose concentration over time can be dened as: L0 (k/KM )Le dx = dt 1 + (L/KM ) + (Ga/KI ) kL0 (1 x)e = KM + L0 (1 x) + (KM /KI )(Ga0 + L0 x)

(7)

30

Separating variables and integrating, we would arrive at an expression of the following type: KM 1 + =k
0 t

S0 KI e dt

[ln(1 x)] + 1

KM KI

L0 x (8)

40

0.5 0.1, 0.5, 1.0, 3.0 0.1, 0.5, 1.0, 3.0 3.0

where: S0 = L0 + Ga0 (9)

3. Results and discussion 3.1. Kinetic model of enzymatic hydrolysis with competitive inhibition of galactose The widely accepted kinetic model to explain enzymatic lactose hydrolysis is competitive inhibition by the product (galactose): E + L EL ELEGa + Gl EGaE + Ga
KI k

In the case that the enzymatic deactivation does not occur in the hydrolysis process, the active enzyme concentration would remain constant throughout the experimental period, giving e = e0 , and thus: KM 1 + S0 KI [ln(1 x)] + 1 KM KI L0 x = ke0 t (10) an expression that enables the t of the experimental data by a linear regression according to: e0 t KM S0 [ln(1 x)] (1 (KM /KI )) = 1+ + L0 x k KI x k [ln(1 x)] =A +B (11) x an expression previously proposed by several authors [27]. As an example, Fig. 1 shows the linearization of the experimental results at 30 C, applying Eq. (11). All the experiments performed at the same substrate concentration and different enzyme concentrations were aligned, indicating that, during the reaction period and under the experimental conditions, no enzymatic denaturing occurred (Fig. 1). Similarly, in the experiments in which the sum of the initial molar concentrations of galactose and lactose were the same,

(1) (2) (3)

The lactose, galactose, and glucose concentrations were dened as a function of the conversion: L = L0 (1 x) Ga = Ga0 + L0 x Gl = Gl0 + L0 x (4) (5) (6)

considering the concentrations of the enzymatic complexes EL and EGa to be negligible.

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Fig. 1. Experiments of lactose hydrolysis at T = 30 C; e0 t/x vs. [ln(1 x )]/x.

the data were tted to the same line (experiment made with 0.0347 M initial galactose concentration). Nevertheless, the kinetic constants resulting from the application of Eq. (11) did not lead to completely satisfactory results, prompting us to reconsider the kinetic model proposed. The result is due to the fact that the tting used, Eq. (11), is not appropriate to

calculate the KM /KI relationship, because it implies extrapolation distant from the experimental interval, and an experimental deviation can heavily inuence the KM /KI value (for conversions ranging between 0 and 0.95 the value of [ln(1 x )]/x uctuates between 1 and 3, far from the origin axis).

Fig. 2. Representation of the kinetic constants KM and KI proposed by different authors.

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Fig. 3. Fitting of the kinetic constant k to the Arrhenius equation by author.

The literature reveals the dispersion of the kinetic constants values KM and KI proposed by different authors who have applied a similar kinetic model (Table 3 and Fig. 2). According to our review, regardless of the enzyme origin, the value proposed for KM may be more or less than the KI value, contradicting reports [5,6] that galactose inhibition is stronger in fungal enzymes than in yeast or bacterial enzymes. The inuence of the t used, Eq. (11), disappears when the kinetic parameters are calculated by non-linear regression

from the kinetic model, as in the results of Santos et al. [9], where the values of KM and KI were similar. For kinetic constant k proposed by different authors, Fig. 3 shows the t to the Arrhenius equation. The value of the ordinate at the origin varies in different works, depending on the way in which the enzyme concentration is dened (protein mass per liter, mass of enzymatic solution per liter). The values of the resulting slopes were similar, indicating that the values of the activation energy for k were comparable.

Fig. 4. Fitting of the kinetic constant KM to the Vant Hoff equation by author.

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Fig. 5. Fitting of the kinetic constant KI to the Vant Hoff equation by author.

Figs. 4 and 5 show the temperature dependence on the kinetic parameters KM and KI , as the Vant Hoff equation suggests. However, in some cases, this dependence proves anomalous. For example, in Fig. 4, corresponding to KM , the slope changes sign according to the author, while in Fig. 5 the KI values do not t to the Vant Hoff equation in some cases. However, in these gures the results from the

non-linear regression [9] have very similar slopes for both constants. 3.2. Kinetic model under the assumption KM = KI The above explanations lead us to assume that KM = KI , implying that the enzyme has almost the same

Fig. 6. Experimental data of lactose hydrolysis at 40 C, Ga0 = 0 M; e0 t vs. [ln(1 x )].

E. Jurado et al. / Enzyme and Microbial Technology 31 (2002) 300309 Table 5 Kinetic constants of lactose hydrolysis calculated applying Eq. (12) T ( C) 25 30 40 k (103 mol g1 h1 ) 168 2 227 1 415 5 KM (104 M) 60 10 70 6 100 10

307

afnity for the substrate (lactose) as for the reaction product (galactose). The simplication proposed in Eq. (11) leads to Eq. (12): e0 t = KM 1 + S0 [ln(1 x)] k k (12)

The good t of all the experimental data according to Eq. (12), as well as the t of the kinetic constants k and KM to the Arrhenius and Vant Hoff equations, respectively, veries that the assumption made is correct. For example,

the results at 40 C are shown in Fig. 6, where, as indicated by Eq. (12), the ordinate at the origin is 0. Eq. (12) implies a rst-order kinetic model, as indicated by some authors, although the pseudo-rst-order kinetic constant would depend on S0 . This result suggests that the kinetic reaction is determined by the break down of the enzymesubstrate complex. That is, the limiting step is (2), implying that the Michaelis constant represents the dissociation constant of the enzymesubstrate complex and that the lactose is bonded to the enzyme by the galactosyl group [3,37,38]. Only in this way it can be explained in a simple way that KM and KI values are similar. Table 5 lists the values of the kinetic constants calculated by non-linear regression from the t of the experimental data to Eq. (12). The activation energy, enthalpy of formation and pre-exponential factor values in this and other works are shown in Table 6.

Table 6 Kinetic parameters calculated in this work and proposed by other authors for the enzymatic hydrolysis of lactose References k Ea [25] [9] [31] [29,30] This work (kcal mol1 ) KM Pre-exponential-factor values (M) 2.48 2.06 5.23 1.44 1012 107 102 102 ( H f )a 7.38 20.1 13.3 5.87 5.92 (kcal mol1 ) KI Pre-exponential-factor values (M) 4.73 9.87 2.66 1.44 1010 108 108 102 ( Hf )a (kcal mol1 ) 16.3 17.9 15.5 13.0 5.92

7.7 11.7 10.9 10.4 11.2

Fig. 7. Fitting of the experimental data to the proposed model at 25 C, Ga0 = 0 M.

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E. Jurado et al. / Enzyme and Microbial Technology 31 (2002) 300309 [7] Gonzlez-Siso MI. The biotechnological utilization of cheese whey: a review. Bioresour Technol 1996;57:111. [8] Shah NP, Spurgeon KR, Gilmore TM. Use of dry whey and lactose hydrolysis in yogurt bases. Milk Sci Int 1993;48(9):4948. [9] Santos A, Ladero M, Garc a-Ochoa F. Kinetic modeling of lactose hydrolysis by a -galactosidase from Kluyveromyces fragilis. Enzyme Microb Technol 1998;22:55867. [10] Finocchiaro T, Olson NF, Richardson T. Use of immobilized lactase in milk systems. Adv Biochem Eng 1980;15:7188. [11] Heng MH, Glatz CE. Ion exchange immobilization of charged -galactosidase fusions for lactose hydrolysis. Biotechnol Bioeng 1994;44:74552. [12] Kim IH, Chang HN. Variable-volume hollow-ber enzyme reactor with pulsatile ow. AIChE J 1983;29(6):9104. [13] Walsh MK, Swaisgood HE. Characterization of a chemically conjugated -galactosidase bioreactor. J Food Biochem 1993;17: 28392. [14] Sungur S, Yildirim O. Batch and continuous hydrolysis of lactose using -galactosidase immobilized on gelatin-CMC. Polym Plast Technol Eng 1999;38(4):8219. [15] Fujikawa H, Itoh T. Differences in the thermal inactivation kinetics of Escherichia coli -galactosidase in vitro and in vivo. Biocontrol Sci 1997;2(2):738. [16] Harju M. Lactose hydrolysis. B Int Dairy Fed 1987;212:504. [17] Santos A, Ladero M, Garc a-Ochoa F. Kinetic model for the hydrolysis of lactose by -galactosidase from Kluyveromyces fragilis. Proceedings of the Seventh Mediterranean Congress of Chemical Engineering. Barcelona, 1996. p. 184. [18] Berrueta-Jimnez J, Garc a-Valle T. Hidrlisis enzimtica de lactosa en reactores de lecho jo. Ing Qu mica 1988;246:1416. [19] Friend BA, Shahani KM. Characterization and evaluation of Aspergillus oryzae lactase coupled to a regenerable support. Biotechnol Bioeng 1982;24:32945. [20] Huffman LM, Harper WJ. Lactose hydrolysis in batch and hollow bre membrane reactors. NZ J Dairy Sci Technol 1985;20:5763. [21] Ma H, Monsan P, Mayer R, Rouleau D. Hydrolyse enzymatique du lactose dans un reacteur type reservoir agite. Can J Chem Eng 1983;61:739. [22] Carrara CR, Rubiolo AC. Determination of kinetics parameters for free and immobilized -galactosidase. Proc Biochem 1996;31(3):2438. [23] Yang S-T, Okos MR. Effects of temperature on lactose hydrolysis by immobilized -galactosidase in plug reactor. Biotechnol Bioeng 1989;33:87385. [24] Cavaille D, Combes D. Characterization of -galactosidase from Kluyveromyces lactis. Biotechnol Appl Biochem 1995;22:5564. [25] Papayannakos N, Markas G, Kekos D. Studies on modelling and simulation of lactose hydrolysis by free and immobilized galactosidase from Aspergillus niger. Chem Eng J 1993;52:B112. [26] Korus RA, Olson AC. Use of -galactosidase, -galactosidase, glucose isomerase and invertase in hollow ber reactors. In: Pye EK, Weetall HH, editors. Enzyme engineering, vol. 3. London, New York: Plenum Press, 1978. p. 5439. [27] Yang S-T, Okos MR. A new graphical method for determining parameters in MichaelisMenten-type kinetics for enzymatic lactose hydrolysis. Biotechnol Bioeng 1989;34:76373. [28] Illanes A, Altamirano C, Aillapan A, Tomasello G, Zuiga ME. Packed-bed reactor performance with immobilized lactase under thermal inactivation. Enzyme Microb Technol 1998;23:39. [29] Illanes A, Wilson L, Tomasello G. Temperature optimization for reactor operation with chitin-immobilized lactase under modulated inactivation. Enzyme Microb Technol 2000;27:2708. [30] Illanes A, Wilson L, Tomasello G. Effect of modulation of enzyme inactivation on temperature optimization for reactor operation with chitin-immobilized lactase. J Mol Catal B: Enzymatic 2001;11: 53140.

The activation energies proposed by almost all authors for the kinetic constant k is similar to the value calculated in the present work. The enthalpy of formation values for the constant KM and KI are very close for all the authors reviewed. In fact, one author shows the enthalpy of formation for KM to be higher than for KI in one work [9], but lower in another work [31], though this difference could derive from the immobilization of the enzyme. In Fig. 7, it is shown the results on applying the model and simplication proposed, revealing a close t. 4. Conclusions The conclusions of the present study can be summarized as follows: The kinetic mechanism of lactose hydrolysis by the enzyme -galactosidase responds to a model of inhibition by the product (galactose). It has been veried that the enzyme has similar afnity both for the substrate (lactose) and for the product (galactose) because both compounds are capable of occupying the active site of the enzyme with equal probability, so that the constants that govern the equilibrium semi-reactions have equal value. A simplied model has been proposed, in which the constants KM and KI are equal and the reaction rate can be calculated by the equation: r= kL0 (1 x) KM + L0 + Ga0

Kinetic constants have been formulated for the enzymatic hydrolysis of lactose according to temperature, which can be calculated from the following equations: k = 2.71 107 exp 11200 RT 5920 RT

KM = 1.44 102 exp

References
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E. Jurado et al. / Enzyme and Microbial Technology 31 (2002) 300309 [31] Ladero M, Santos A, Garc a-Ochoa F. Kinetic modeling of lactose hydrolysis with an immobilized -galactosidase from Kluyveromyces fragilis. Enzyme Microb Technol 2000;27:58392. [32] Flaschel E, Raetz E, Renken A. The kinetics of lactose hydrolysis for the -galactosidase from Aspergillus niger. Biotechnol Bioeng 1982;24:2499518. [33] Peterson RS, Hill CG, Amundson CH. Lactose hydrolysis by immobilized -galactosidase in capillary bed rector. Biotechnol Bioeng 1989;34:43846. [34] Iwasaki K-I, Nakajima M, Nakao S-I. Galacto-oligosaccharide production from lactose by an enzymic batch reaction using galactosidase. Proc Biochem 1996;31(1):6976.

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[35] Reichenbach LMH. Lactose hydrolysis with fungal -galactosidase in shaker ask reactors and hollow ber membrane reactors. M.S. Thesis, Ohio State University, Columbus, OH, 1979. [36] Werner W, Rey HG, Wielinger H. Properties of a new chromogen for the determination of glucose in blood according to the GOD/POD method. Anal Chem 1970;252:2248. [37] Klewicki R. Transglycosylation of a -galactosyl radical, in the course of enzymic hydrolysis of lactose, in the presence of selected polyhydroxyalchols. Biotechnol Lett 2000;22:10636. [38] In M-J, Jin J. Characterization of -galactosidase from a Bacillus sp. with high catalytic efciency for transgalactosylation. J Microbiol Biotechnol 1998;8(4):31824.