= ∑ d obs d pred d pred
3.5. Immobilization of β- D -galactosidase in organic-inorganic composites obtained by sol-gel entrapment combined with adsorption
3.6.4. Study of kinetic parameters of native and immobilized β- D -galactosidases on synthetic (o-nitrophenyl-β- D -galactopyranoside) and natural (lactose) substrates
The main aim of kinetic parameters determination for immobilized enzymes is to establish how is affected the transformation and the acces of the subtrate in the catalytic site by the immobilization method and parameters. As well as chemical reactions kinetics, initial reaction rates were determined especially for the natural substrates prefered by enzymes in vivo, but not only.
To establish the effect of covalent binding (on chitosan microspheres cross-linked with glutaraldehyde) and sol-gel entrapment of enzyme on the substrate access into the catalytic site and consequently of the product formation rate, the values of the kinetic constants Km and catalytic efficiencies kcat/ Km were determined towards ONPG and lactose hydrolysis, respectively. The two immobilized preparates, and native β-D-galactosidase as well, were incubated at 30ºC in the presence of various concentrations of ONPG and lactose. The ONPG concentration was varied in 21 levels for native enzyme, 12 levels for covalent
bonded and 18 levels for sol-gel entrapped β-D-galactosidase in the range of 0.1-30 mM.
Initial rates were determined as ONP release using discontinuous assay (section 2.4.1). The corresponding Michaelis-Menten plots are shown in Fig. 3.45-3.47, and in all cases a saturated dependence on substrates concentrations was observed. Thus, an individual determination of kcat and Km was possible by using Lineweaver-Burk plot (Lineweaver and Burk, 1934) with the help of SigmaPlot 11.0 version. The Km value for native β-D -galactosidase was in concordance with the value obtained by Gaur et al. (2006). They reported a Km value of 2.63 mM for native β-D-galactosidase when ONPG was used as substrate (Gaur et al., 2006). The Km values were higher after both immobilization methods than that of the free enzyme, which indicates that higher concentrations of substrates are needed for immobilized enzymes probably because the access of the substrate in the active site is hindered. The catalytic efficiency was more affected by sol-gel entrapment of the enzyme than by covalent binding.
ONPG [mM]
0 10 20 30
Vi [mM/min]
0.00 0.02 0.04 0.06 0.08 0.10
Fig. 3.45. Michaelis-Menten plot for determination of kinetic parameters in ONPG hydrolysis by native β-D-galactosidase. Initial rates were determined as function of ONP
release, using a discontinuous assay Km = 3.93 mM
kcat = 81.05 s-1
kcat/ Km = 20.62 L×mmol-1×s-1
ONPG [mM]
0 10 20 30
Vi [mM/min]
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16
Fig. 3.46. Michaelis-Menten plot for determination of kinetic parameters in ONPG hydrolysis by covalently bonded β-D-galactosidase. Initial rates were determined as
function of ONP release using a discontinuous assay
ONPG [mM]
0 10 20 30
Vi [mM/min]
0.0 0.5 1.0 1.5 2.0 2.5
Fig. 3.47. Michaelis-Menten plot for determination of kinetic parameters in ONPG hydrolysis by sol-gel entrapped β-D-galactosidase. Initial rates were determined as function
of ONP release using a discontinuous assay
The kinetic parameters for lactose hydrolysis were determined by varying the lactose concentration in 22 levels for native enzyme, 14 levels for covalently bonded and 20 levels for sol-gel entrapped β-D-galactosidase from 0.1-550 mM. Initial-rates were determined by
Km = 13.95 mM kcat = 12.72 s-1
kcat/ Km = 0.91 L×mmol-1×s-1
Km = 10.53 mM kcat = 23.68 s-1
kcat/ Km = 2.25 L×mmol-1×s-1
glucose release using a discontinuous assay (section 2.4.3). The corresponding Michaelis-Menten plots shown in Fig. 3.48-3.50 indicate a saturated dependence on substrates concentration in all three cases. The kcat and Km kinetic constants were calculated using the Lineweaver-Burk linearization with the help of SigmaPlot 11.0 version. Compared to the values obtained when ONPG was used as substrate for hydrolysis, the Km values obtained for lactose hydrolysis even for native enzyme were higher and in concordance values obtained by Carrara, who reported a Km value of 75 mM for native β-D-galactosidase (Carrara et al., 1994). The Km values for covalently bonded (Fig. 3.49) and sol-gel entrapped (Fig. 3.50) β-D-galactosidase were increased and the results indicate that the specificity of the enzyme towards lactose was affected after immobilization. The enzyme catalytic efficiency was more affected by sol-gel entrapment of the enzyme than by covalent binding.
Lactose [mM]
0 200 400 600
Vi [mM/min]
0.0 0.5 1.0 1.5 2.0 2.5
Fig. 3.48. Michaelis-Menten plot for determination of kinetic parameters in lactose hydrolysis by native β-D-galactosidase. Initial rates were determined as function of
D-glucose release using a discontinuous assay Km = 84.42 mM
kcat = 9.48×102 s-1
kcat/ Km = 11.23 L×mmol-1×s-1
Lactose [mM]
0 200 400 600
Vi [mM/min]
0 1 2 3 4 5 6
Fig. 3.49. Michaelis-Menten plot for determination of kinetic parameters in lactose hydrolysis by covalently bonded β-D-galactosidase. Initial rates were determined as
function of D-glucose release using a discontinuous assay
Lactose [mM]
0 200 400 600
Vi [mM/min]
0 1 2 3 4 5 6 7
Fig. 3.50. Michaelis-Menten plot for determination of kinetic parameters in lactose hydrolysis by sol-gel entrapped β-D-galactosidase. Initial rates were determined as function
of D-glucose release using a discontinuous assay Km = 123.65 mM
kcat = 62.89 s-1
kcat/ Km = 0.51 L×mmol-1×s-1 Km = 140.67 mM
kcat = 196.36 s-1
kcat/ Km = 1.40 L×mmol-1×s-1
3.6.5. Time course of lactose hydrolysis by native and immobilized β-D-galactosidases
To determine the hydrolytic activity of native and immobilized β-D-galactosidase towards lactose, the covalently bonded, sol-gel entrapped and native β-D-galactosidase were incubated with 30 mM ONPG for 8 h. High concentrations of lactose (550 mM) were used because the enzyme was supposed to be suitable for hydrolysis of higher concentrations from mammal milk (88-234 mM) and whey permeate (85% lactose), respectively, for 8 h (Elnashar and Yassin, 2008). Samples were taken hourly (as described in section 2.7). The hydrolytic activity corresponds to the concentration of D-glucose released. Thus, D-glucose was measured in each sample using the glucose assay method with GOD and POD.
Different amount of preparates were used in order to determine the hydrolytic constants kH
of each type of enzyme, which were calculated from the slope of linear increase of product concentration during 8 h (Fig. 3.51) divided by the concentration of enzyme used in the hydrolysis reactions. As it was expected, the hydrolytic constants of sol-gel entrapped enzyme kH = 46.05 min-1 and covalently bonded β-D-galactosidase kH = 220.09 min-1 were significantly affected compared to the native enzyme kH = 2981.75 min-1. However, substrates or product inhibition were not observed increasing the lactose concentration up to 550 mM.
Time [h]
0 2 4 6 8 10
Glucose [mM]
0 20 40 60
native ββββ-D-gal
sol-gel entrapped ββββ-D-gal covalently bonded ββββ-D-gal
Fig. 3.51. Time-dependent measurements of the hydrolysis of lactose catalyzed by native (■), sol-gel entrapped (●), covalently bonded (▼) β-D-galactosidases
3.6.6. Multiple utilization of immobilized β-D-galactosidases in batch hydrolysis of o-nitrophenyl-β-D-galactopyranoside and lactose
Reusability of the enzyme represents one of the main goals of enzyme immobilization, especially for cost-effective use of the enzyme in repeated batch or continuous systems.
The major advantage of the immobilization is reusability after an easy and elegant separation from the reaction mixture, thus the costs are reduced, particularly for continuous processes.
In this study the effect of reuse on the immobilized enzyme activity was studied for ONPG and lactose hydrolysis. After each reutilization, the immobilized enzyme was recovered, by filtration in case of covalent bind β-D-galactosidase, and by centrifugation in case of sol-gel entrapped β-D-galactosidase, respectively. The activity was determined based on product release in 10 min with ONPG substrate, and 30 min with lactose substrate. The results, shown in Fig. 3.52-3.53 indicate a decrease in the enzymatic activity of sol-gel entrapped β-D-galactosidase to 70% after first utilization by using ONPG as substrate and then no decrease in activity after five uses was observed (Fig. 3.52).
ONPG reutilization cycles
0 1 2 3 4 5 6
Relative activity [%]
0 20 40 60 80 100
120 sol-gel entrapped ββββ-D-gal
covalently bonded ββββ-D-gal
Fig. 3.52. Effect of reutilization on the activity of sol-gel entrapped (●) and covalently bonded β-D-galactosidase (▼), using ONPG substrate
Lactose reutilization cycles
0 1 2 3 4 5 6
Relative activity [%]
0 20 40 60 80 100
120 sol-gel entrapped ββββ-D-gal
covalently bonded ββββ-D-gal
Fig. 3.53. Effect of reutilization on the activity of sol-gel entrapped (●) and covalently bonded β-D-galactosidase (▼), using lactose substrate
Quite similar results were observed when lactose was used as substrate (Fig. 3.53), around 50% activity losses after first utilization and no decrease after 5 uses. When covalent bind β-D-galactosidase was tested a gradually decrease of the enzyme activity was observed, especially when lactose was used as substrate.