A new kinetic model proposed for enzymatic hydrolysis of lactose by a -galactosidase from Kluyveromyces fragilis

Chia sẻ: Dao Manh Tuyen | Ngày: | Loại File: PDF | Số trang:10

0
15
lượt xem
0
download

A new kinetic model proposed for enzymatic hydrolysis of lactose by a -galactosidase from Kluyveromyces fragilis

Mô tả tài liệu
  Download Vui lòng tải xuống để xem tài liệu đầy đủ

We study the enzymatic hydrolysis of lactose by a commercial enzyme from a selected strain of Kluyveromyces fragilis. The variables analyzed were: temperature (25–40 ◦C), enzyme concentration (0.1–3.0 g l−1), lactose concentration (0.0278–0.208 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 Michaelis–Menten kinetic model with inhibition by the product (galactose), which reveals that the substrate (lactose) and the product (galactose) present similar affinity for the active site of the enzyme.

Chủ đề:
Lưu

Nội dung Text: A new kinetic model proposed for enzymatic hydrolysis of lactose by a -galactosidase from Kluyveromyces fragilis

Enzyme and Microbial Technology 31 (2002) 300–309<br /> <br /> A new kinetic model proposed for enzymatic hydrolysis of lactose<br /> by a ␤-galactosidase from Kluyveromyces fragilis<br /> E. Jurado∗ , F. Camacho, G. Luzón, J.M. Vicaria<br /> Departmento Ingenier´ıa Qu´ımica, Facultad Ciencias, Universidad de Granada, Granada 18 071, Spain<br /> Received 3 January 2002; accepted 21 March 2002<br /> <br /> Abstract<br /> We study the enzymatic hydrolysis of lactose by a commercial enzyme from a selected strain of Kluyveromyces fragilis. The variables<br /> analyzed were: temperature (25–40 ◦ C), enzyme concentration (0.1–3.0 g l−1 ), lactose concentration (0.0278–0.208 M), and initial galactose<br /> concentration (0.0347 M). On the basis of the data analyzed, both published and in the present work, we propose a Michaelis–Menten<br /> kinetic model with inhibition by the product (galactose), which reveals that the substrate (lactose) and the product (galactose) present<br /> similar affinity for the active site of the enzyme.<br /> © 2002 Elsevier Science Inc. All rights reserved.<br /> Keywords: Lactose hydrolysis; Kluyveromyces fragilis; Kinetic model; ␤-Galactosidase<br /> <br /> 1. Introduction<br /> Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry because<br /> of the potentially beneficial effects on the assimilation of<br /> foods containing lactose, as well as the possible technological and environmental advantages of industrial application,<br /> including:<br /> 1. Elimination of lactose intolerance (3–70% depending on<br /> the populational group [1]), encouraging the utilization<br /> of lactose as an energy source, as well as calcium and<br /> magnesium assimilation from milk.<br /> 2. Formation of galacto-oligosaccharides during lactose hydrolysis to favor the growth of intestinal bacterial microflora. The presence of these compounds is considered<br /> desirable in foods [2,3].<br /> 3. Improvement in the technological and sensorial characteristics of foods containing hydrolyzed lactose from<br /> milk or whey [4–7] such as: increased solubility (avoidance of lactose crystallization and the grainy aspect of<br /> ice creams and condensed or powdered products); greater<br /> sweetening power and thus lower caloric content of the<br /> products (glucose and galactose monosaccharides have<br /> greater sweetening power than does lactose); formation<br /> of monosaccharides, which are easier to ferment in cer∗<br /> <br /> Corresponding author.<br /> E-mail address: ejurado@ugr.es (E. Jurado).<br /> <br /> tain products such as yogurt [8]; lower freezing point of<br /> ice creams (increasing softness and creaminess); and reduction of the Maillard reaction.<br /> 4. Greater biodegradability of whey in which the lactose<br /> has been hydrolyzed [9].<br /> The commercial enzymes used for lactose hydrolysis are<br /> ␤-galactosidases of diverse origins [5,6,10]. Yeast and fungal enzymes have the greatest commercial interest. Many<br /> studies have been made with ␤-galactosidases obtained from<br /> Escherichia coli, although their use is not viable for products intended for human consumption [11–15].<br /> The optimal operating conditions are described in Table 1.<br /> Fungal enzymes are usually used to hydrolyze lactose from<br /> products with acidic pH values, such as whey. Yeast enzymes<br /> are habitually used for products with neutral pH values [16]<br /> such as milk and sweet whey.<br /> The mechanism of enzymatic hydrolysis of lactose by<br /> ␤-galactosidase applied to different substrates (lactose<br /> solutions, whey and skim-milk) under different experimental conditions has been studied by several authors.<br /> Table 2 presents the kinetic models proposed by different<br /> researchers, showing that most propose a Michaelis–Menten<br /> kinetic model, with competitive inhibition by galactose.<br /> However, there is great dispersion of the values of the<br /> kinetic constants proposed (see Table 3).<br /> The present work provides a survey of the models proposed by different authors, presents an experimental study of<br /> enzymatic hydrolysis in a stirred tank with ␤-galactosidase<br /> <br /> 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.<br /> PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 1 0 7 - 2<br /> <br /> E. Jurado et al. / Enzyme and Microbial Technology 31 (2002) 300–309<br /> <br /> Nomenclature<br /> E<br /> L<br /> Ga<br /> Gl<br /> EL<br /> EGa<br /> k<br /> KM<br /> KI<br /> r<br /> e<br /> x<br /> R<br /> Ea<br /> (−Hf )a<br /> T<br /> <br /> concentration of free enzyme present in the<br /> reaction medium (g enzyme preparation l−1 )<br /> concentration of monohydrate lactose (M)<br /> concentration of galactose (M)<br /> concentration of glucose (M)<br /> concentration of the enzyme–lactose<br /> complex<br /> concentration of the enzyme–galactose<br /> complex<br /> rate constant of Eq. (2) (mol g−1 h−1 )<br /> Michaelis–Menten constant (M)<br /> equilibrium constant of Eq. (3) (M)<br /> reaction rate (mol g−1 h−1 )<br /> concentration of total active<br /> enzymatic complex (g l−1 )<br /> conversion (adimensional)<br /> constant of the ideal gases (cal K−1 mol−1 )<br /> activation energy (kcal mol−1 )<br /> enthalpy of formation (kcal mol−1 )<br /> temperature (K)<br /> <br /> Subscripts<br /> 0<br /> initial concentrations<br /> <br /> from Kluyveromyces fragilis, and proposes a simplified<br /> kinetic model for the action of the enzyme.<br /> <br /> 2. Materials and methods<br /> The chemical products used (PRS quality) are glucose,<br /> citric acid, K2 HPO4 , KCl, trichloroacetic acid (supplied<br /> <br /> 301<br /> <br /> by Panreac), MgCl2 ·6H2 O (Prolabo), monohydrate lactose<br /> (Scharlau), and galactose (Across).<br /> The enzyme used was a commercial ␤-galactosidase, lactozym 3000L HP-G [EC.3.2.1.23], which has a protein content of 35 g l−1 , supplied by Novo Nordisk, derived from a<br /> selected strain of the yeast K. fragilis, ρ = 1.2 g ml−1 , with a<br /> declared activity of 3000 LAU ml−1 (1 LAU = commercial<br /> enzyme which can obtain 1 ␮mol glucose min−1 in standard conditions: 4.7% lactose concentration, pH 6.5, 30 ◦ C,<br /> 30 min, standard milky buffer [4]). This enzyme satisfies the<br /> specifications recommended for food enzymes.<br /> The glucose was analyzed applying the GOD-Perid<br /> method proposed by Werner et al. [36] using a commercial reagent (Böehringer Mannheim Gmbh). The galactose<br /> and lactose present in the medium had no influence on the<br /> glucose determination.<br /> As the reaction medium, lactose solutions were prepared<br /> on a buffer of 0.01 M K2 HPO4 , 0.015 M KCl, and 0.012 M<br /> MgCl2 ·6H2 O at pH 6.75 adjusted with citric acid.<br /> The enzymatic activity was measured in test tubes at 30 ◦ C<br /> in the following way: 1 ml of 50 g l−1 monohydrate lactose<br /> solution prepared on the buffer indicated was added to 1 ml<br /> of 10 g l−1 enzyme solution prepared on the same buffer.<br /> The test tube was incubated at 30 ◦ C for 10 min, after which<br /> 1 ml was extracted. The reaction was stopped by mixing with<br /> 1 ml of 0.1N trichloroacetic acid. Afterwards, the glucose<br /> concentration was measured by the GOD-Perid method. The<br /> enzymatic activity remained constant for the entire period<br /> of use.<br /> The enzymatic reaction took place in a 200 ml stirred<br /> 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<br /> acid and the glucose concentration was measured by the<br /> GOD-Perid method. The variables tested are shown in<br /> Table 4.<br /> <br /> Table 1<br /> Experimental conditions of the most used enzymes<br /> Enzyme source<br /> <br /> pH (optimal)<br /> <br /> T (◦ C) (optimal)<br /> <br /> Cofactors<br /> <br /> Fungal (Aspergillus niger)<br /> Fungal (A. oryzae)<br /> Yeast (K. fragilis)<br /> Yeast (K. lactis)<br /> <br /> 3.0–4.0<br /> 5.0<br /> 6.6<br /> 6.9–7.3<br /> <br /> 55–60<br /> 50–55<br /> 37<br /> 35<br /> <br /> Mn2+ , Mg2+ , K+<br /> Mn2+ , Na+<br /> <br /> Table 2<br /> Kinetic models proposed by different authors for enzymatic hydrolysis of lactose<br /> Kinetic model proposed<br /> <br /> References<br /> <br /> Michaelis–Menten first order<br /> Michaelis–Menten without inhibition by product (galactose) (with enzyme–lactose complex formation)<br /> Michaelis–Menten with competitive inhibition by product (total galactose)<br /> Michaelis–Menten with competitive inhibition by product (␣- and ␤- galactose)<br /> Michaelis–Menten with competitive inhibition by product (glucose)<br /> Di-, tri- and tetra-saccharides formation<br /> <br /> [17]<br /> [18]<br /> [9,11,12,18–31]<br /> [32,33]<br /> [24]<br /> [34]<br /> <br /> 302<br /> <br /> E. Jurado et al. / Enzyme and Microbial Technology 31 (2002) 300–309<br /> <br /> Table 3<br /> Kinetic constants proposed by different authors for the enzymatic hydrolysis of lactose<br /> References<br /> <br /> Enzyme source<br /> <br /> pH<br /> <br /> T ( ◦ C)<br /> <br /> KM (M)<br /> <br /> KI (M)<br /> <br /> [19]<br /> <br /> A.<br /> A.<br /> A.<br /> A.<br /> <br /> 4.5<br /> 4.5<br /> 6.5<br /> 6.5<br /> <br /> 50<br /> 50<br /> 50<br /> 50<br /> <br /> 0.112<br /> 0.122<br /> 0.178<br /> 0.160<br /> <br /> 0.0095<br /> 0.0105<br /> 0.0025<br /> 0.0045<br /> <br /> oryzae<br /> oryzae<br /> oryzae<br /> oryzae<br /> <br /> (sol.)<br /> (imm.)<br /> (sol.)<br /> (imm.)<br /> <br /> [35]<br /> <br /> A. oryzae (sol.)<br /> <br /> [32]<br /> <br /> A. niger (sol.)<br /> <br /> [11]<br /> <br /> E.<br /> E.<br /> E.<br /> E.<br /> E.<br /> <br /> coli1 (imm.)<br /> coli2 (imm.)<br /> coli3 (imm.)<br /> coli4 (imm.)<br /> coli5 (imm.)<br /> <br /> [20]<br /> <br /> 50<br /> <br /> 0.0357<br /> <br /> 0.0201<br /> <br /> 50<br /> <br /> 0.0539<br /> <br /> 0.00092 (␣), 0.0118 (␤),<br /> 0.0109 (complex)<br /> <br /> 7.0<br /> <br /> 21<br /> <br /> 0.00298<br /> 0.00346<br /> 0.00434<br /> 0.00332<br /> 0.00373<br /> <br /> 0.035<br /> 0.034<br /> 0.082<br /> 0.039<br /> 0.034<br /> <br /> A. oryzae (sol.)<br /> A. oryzae (imm.)<br /> <br /> 4.5<br /> <br /> 50<br /> 50<br /> <br /> 0.0469<br /> 0.0490<br /> <br /> 0.0200<br /> 0.0200<br /> <br /> [21]<br /> <br /> A. niger (imm.)<br /> <br /> 4.5<br /> <br /> 30<br /> 40<br /> 50<br /> <br /> 0.0536<br /> 0.0519<br /> 0.0533<br /> <br /> 0.0321<br /> 0.0055<br /> 0.0946<br /> <br /> [22]<br /> <br /> K. fragilis (sol.)<br /> K. fragilis (imm.)<br /> <br /> 6.9<br /> <br /> 43<br /> <br /> 0.0436<br /> 0.1370<br /> <br /> 0.0519<br /> 0.2340<br /> <br /> [23]<br /> <br /> A. niger (imm.)<br /> <br /> 4.0<br /> <br /> 25<br /> 30<br /> 35<br /> 40<br /> 45<br /> 50<br /> 55<br /> 60<br /> <br /> 0.0797<br /> 0.0764<br /> 0.0733<br /> 0.0705<br /> 0.0679<br /> 0.0656<br /> 0.0635<br /> 0.0615<br /> <br /> 0.0000613<br /> 0.0000620<br /> 0.0000627<br /> 0.0000633<br /> 0.0000640<br /> 0.0000646<br /> 0.0000652<br /> 0.0000658<br /> <br /> [26]<br /> <br /> K. fragilis (imm.)<br /> A. niger (imm.)<br /> <br /> 5.2<br /> <br /> 23<br /> 37<br /> <br /> 0.006<br /> 0.0286<br /> <br /> 0.012<br /> 0.0024<br /> <br /> [33]<br /> <br /> A. oryzae (imm.)<br /> <br /> 4.5<br /> <br /> 30<br /> <br /> 0.052<br /> <br /> 0.00022 (␣), 0.042(␤)<br /> <br /> [27]<br /> <br /> A. niger (sol.) (Model 1)<br /> <br /> 4.0<br /> <br /> 8<br /> 30<br /> 40<br /> 45<br /> 50<br /> 55<br /> 60<br /> <br /> 0.0833<br /> 0.0809<br /> 0.0799<br /> 0.0795<br /> 0.079<br /> 0.0786<br /> 0.0782<br /> <br /> 0.000472<br /> 0.000528<br /> 0.000553<br /> 0.000565<br /> 0.000578<br /> 0.00059<br /> 0.000601<br /> <br /> A. niger (sol.) (Model 2)<br /> <br /> 4.0<br /> <br /> 8<br /> 30<br /> 40<br /> 45<br /> 50<br /> 55<br /> 60<br /> <br /> 0.1019<br /> 0.0995<br /> 0.0986<br /> 0.0982<br /> 0.0977<br /> 0.0973<br /> 0.0969<br /> <br /> 0.000527<br /> 0.000584<br /> 0.000609<br /> 0.000622<br /> 0.000634<br /> 0.000646<br /> 0.000658<br /> <br /> K. marxianus (sol.)<br /> <br /> 6.6<br /> <br /> K. marxianus (imm.)<br /> <br /> 6.6<br /> <br /> 28<br /> 35<br /> 28<br /> 35<br /> <br /> 0.021<br /> 0.0278<br /> 0.0544<br /> 0.0365<br /> <br /> 0.0292<br /> 0.0316<br /> 0.0869<br /> 0.0114<br /> <br /> K. fragilis (sol.)<br /> <br /> 6.5–7.2<br /> <br /> 5<br /> 25<br /> 40<br /> <br /> 0.0004<br /> 0.0046<br /> 0.023<br /> <br /> 0.00041<br /> 0.0036<br /> 0.0153<br /> <br /> [28]<br /> <br /> [9]<br /> <br /> 4.5<br /> <br /> E. Jurado et al. / Enzyme and Microbial Technology 31 (2002) 300–309<br /> <br /> 303<br /> <br /> Table 3 (Continued )<br /> References<br /> <br /> Enzyme source<br /> <br /> pH<br /> <br /> T ( ◦ C)<br /> <br /> KM (M)<br /> <br /> KI (M)<br /> <br /> [30]<br /> <br /> K. marxianus (imm.)<br /> <br /> 6.6<br /> <br /> 10<br /> 15<br /> 20<br /> 22.5<br /> 25<br /> 27.5<br /> 30<br /> 32.5<br /> 35<br /> 37.5<br /> <br /> 0.0154<br /> 0.0183<br /> 0.0219<br /> 0.0239<br /> 0.0248<br /> 0.0275<br /> 0.0317<br /> 0.0330<br /> 0.0379<br /> 0.0383<br /> <br /> 0.0247<br /> 0.0370<br /> 0.0561<br /> 0.0695<br /> 0.0801<br /> 0.0969<br /> 0.1097<br /> 0.1351<br /> 0.1620<br /> 0.2008<br /> <br /> The variation in the glucose concentration over time can<br /> be defined as:<br /> <br /> Table 4<br /> Variables tested in the hydrolysis experiments performed<br /> T (◦ C)<br /> <br /> L0 (×102 M)<br /> <br /> Ga0 (×102 M)<br /> <br /> e0 (g l−1 )<br /> <br /> 25<br /> <br /> 2.78<br /> 6.94<br /> 13.9<br /> 20.8<br /> <br /> 0<br /> 0<br /> 0<br /> 0<br /> <br /> 0.5<br /> 0.1, 0.5, 1.0, 3.0<br /> 0.1, 0.5, 1.0, 3.0<br /> 0.5<br /> <br /> 2.78<br /> 3.47<br /> 6.94<br /> 13.9<br /> 20.8<br /> <br /> 0<br /> 3.47<br /> 0<br /> 0<br /> 0<br /> <br /> 0.1,<br /> 0.5<br /> 0.1,<br /> 0.1,<br /> 0.1,<br /> <br /> 2.78<br /> 6.94<br /> 13.9<br /> 20.8<br /> <br /> 0<br /> 0<br /> 0<br /> 0<br /> <br /> 0.5<br /> 0.1, 0.5, 1.0, 3.0<br /> 0.1, 0.5, 1.0, 3.0<br /> 3.0<br /> <br /> 30<br /> <br /> 40<br /> <br /> 0.5, 1.0<br /> 0.5, 1.0<br /> 0.5, 1.0<br /> 0.5, 1.0<br /> <br /> L0<br /> <br /> (k/KM )Le<br /> dx<br /> =<br /> dt<br /> 1 + (L/KM ) + (Ga/KI )<br /> kL0 (1 − x)e<br /> =<br /> KM + L0 (1 − x) + (KM /KI )(Ga0 + L0 x)<br /> <br /> Separating variables and integrating, we would arrive at<br /> an expression of the following type:<br /> <br /> <br /> <br /> <br /> S0<br /> KM<br /> KM 1 +<br /> [−ln(1 − x)] + 1 −<br /> L0 x<br /> KI<br /> KI<br />  t<br /> = k e dt<br /> (8)<br /> 0<br /> <br /> where:<br /> S0 = L0 + Ga0<br /> <br /> 3. Results and discussion<br /> 3.1. Kinetic model of enzymatic hydrolysis with<br /> competitive inhibition of galactose<br /> The widely accepted kinetic model to explain enzymatic<br /> lactose hydrolysis is competitive inhibition by the product<br /> (galactose):<br /> E + L ↔ EL<br /> k<br /> <br /> EL→EGa + Gl<br /> KI<br /> <br /> EGa↔E + Ga<br /> <br /> (1)<br /> (2)<br /> (3)<br /> <br /> The lactose, galactose, and glucose concentrations were<br /> defined as a function of the conversion:<br /> L = L0 (1 − x)<br /> <br /> (4)<br /> <br /> Ga = Ga0 + L0 x<br /> <br /> (5)<br /> <br /> Gl = Gl0 + L0 x<br /> <br /> (6)<br /> <br /> considering the concentrations of the enzymatic complexes<br /> EL and EGa to be negligible.<br /> <br /> (7)<br /> <br /> (9)<br /> <br /> In the case that the enzymatic deactivation does not occur<br /> in the hydrolysis process, the active enzyme concentration<br /> would remain constant throughout the experimental period,<br /> giving e = e0 , and thus:<br /> <br /> <br /> <br /> <br /> S0<br /> KM<br /> KM 1 +<br /> [−ln(1 − x)] + 1 −<br /> L0 x = ke0 t<br /> KI<br /> KI<br /> (10)<br /> an expression that enables the fit of the experimental data<br /> by a linear regression according to:<br /> <br /> <br /> e0 t<br /> KM<br /> S0 [−ln(1 − x)]<br /> (1 − (KM /KI ))<br /> =<br /> 1+<br /> + L0<br /> x<br /> k<br /> KI<br /> x<br /> k<br /> [−ln(1 − x)]<br /> =A<br /> +B<br /> (11)<br /> x<br /> an expression previously proposed by several authors [27].<br /> As an example, Fig. 1 shows the linearization of the<br /> experimental results at 30 ◦ C, applying Eq. (11). All the experiments performed at the same substrate concentration and<br /> different enzyme concentrations were aligned, indicating<br /> that, during the reaction period and under the experimental<br /> 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,<br /> <br /> 304<br /> <br /> E. Jurado et al. / Enzyme and Microbial Technology 31 (2002) 300–309<br /> <br /> Fig. 1. Experiments of lactose hydrolysis at T = 30 ◦ C; e0 t/x vs. [−ln(1 − x)]/x.<br /> <br /> the data were fitted to the same line (experiment made with<br /> 0.0347 M initial galactose concentration). Nevertheless, the<br /> kinetic constants resulting from the application of Eq. (11)<br /> did not lead to completely satisfactory results, prompting us<br /> to reconsider the kinetic model proposed. The result is due<br /> to the fact that the fitting used, Eq. (11), is not appropriate to<br /> <br /> calculate the KM /KI relationship, because it implies extrapolation distant from the experimental interval, and an experimental deviation can heavily influence the KM /KI value<br /> (for conversions ranging between 0 and 0.95 the value of<br /> [−ln(1−x)]/x fluctuates between 1 and 3, far from the origin<br /> axis).<br /> <br /> Fig. 2. Representation of the kinetic constants KM and KI proposed by different authors.<br /> <br />

CÓ THỂ BẠN MUỐN DOWNLOAD

Đồng bộ tài khoản