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2.3. Characterization of the immobilized preparations
2.5. Statistical analysis
2.3.1. Lipase activity assay
All experiments were performed in triplicate: the average values
were reported along with Standard deviation.
The hydrolytic activity of the free and the immobilized lipase
preparations was estimated titrimetrically using 0.05 M sodium hy-
droxide as titrant. All experiments were performed at constant tem-
perature under stirring for 30 min. The reaction mixture contained
0.1 mL tributyrin, 0.5 mg gum arabica, 5 mL sodium phosphate buffer
(50 mM, pH 7.0) and 5 mg immobilized enzyme (or 20 μL free lipase
with concentration of 8 mg/mL). For the control experiments equiva-
lent amount of buffer was added instead of enzyme solution. One li-
pase unit is defined as the amount of enzyme required to liberate
1 μmol butyric acid per min under the assay conditions.
3. Results and discussion
3.1. Support characteristics and immobilization
The textural properties of the tin dioxide and silica supports are
presented in Table 1 as well as in Figs. S1–S3 and Table S1 (see
Supporting Information). N2 physisorption isotherms show that
all studied samples exhibit mesoporosity and are characterized
with high specific surface area and narrow pore size distribution
in the mesopore range (Figs. S1 and S3). The more elaborate syn-
thetic procedure used in case of SnO2-B was chosen in order to
get material with larger pores similar to those obtained with
SBA-15. The X-ray diffraction patterns confirm that the obtained
SnO2 materials are crystalline with particle sizes below 10 nm
(Fig. S2), and SBA-15 possesses very well-organized pore system
of hexagonally packed cylindrical mesopores (Fig. S3). A lipase
from Rhizopus delemar was physically adsorbed on the three carriers.
The physical adsorption of lipases on hydrophobic surfaces is the most
frequently used method of immobilization. In comparison to covalent
attachment it is easily performed and does not involve hazardous
reagents therefore is milder for the enzyme and preserves its activ-
ity [11]. Many authors observed a hyper activation of lipases upon
contact with support [12]. It is due to interactions between the sur-
face of the carrier and the lid of lipase which covers the active site. A
conformational rearrangement of this lid occurs; it moves away
from the active site of the enzyme and facilitates the access of the
substrate to the catalytic center. RhDL is a small monomeric mole-
cule (32 kDa) and also shows interfacial activation [13]. We
achieved less protein loading yield with SBA-15, despite of its larger
surface area and similar to nanoSnO2-B pore size. In addition,
nanoSnO2-A-RhD and nanoSnO2-B-RhD exhibited higher specific
activity which we ascribe to the higher hydrophobicity of tin diox-
ide in respect to silica (Table 1). We supposed that due to stronger
hydrophobic interactions between tin dioxide and the lid region of
RhDL the enzyme molecule was attached in more appropriate con-
formation (closer to fully open). Similar activation by hydrophobic
polymeric carriers was reported in the literature for lipases from
Candida antarctica, Candida rugosa and Mucor miehei [4,12]. The
monolayer adsorption capacity of the two tin dioxide samples and
the reference material are comparable to the literature data for
RhDL immobilized on amphiphilic polymer particles and pure or
modified silica [14,15].
2.3.2. Stability and activity of the immobilized lipases
The pH activity and stability profiles of nanoSnO2-A(B)-RhD
and SBA-15-RhD were studied using variety of buffers at 50 mM
concentrations: citrate–phosphate buffer (pH 3.0–6.0), phosphate
buffer (pH 7.0–8.0), and carbonate–bicarbonate buffer (pH
9.0–10.0). The activity of the samples was determined by measur-
ing the release of butyric acid from tributyrin at different pH and
constant temperature (40 °C) [9].
For thermal-, pH- and solvent stability studies, 5 mg immobilized
lipase preparations were subjected to one-hour heating at various
temperatures or were soaked in 1 mL of various buffers (pH 3–10)
or solvents, respectively. The activity of the treated samples was de-
termined as described above. The residual activity was defined as
the ratio of the activity after and before the treatment of the
biocatalysts.
2.4. Synthesis of isoamyl acetate
In a screw-capped test vial, 4-nitrophenyl acetate (1 mM), isoamyl
alcohol (1–8 mM) and 50 mg immobilized RhDL were stirred
(200 rpm) for 12 h in presence of 1 mL organic solvent. The tempera-
ture and water activity (aw=0.33) were kept constant [10]. The
progress of the reaction was monitored on Shimadzu GC-17A instru-
ment equipped with a column HP-5 MS (0.25 μm×0.25 mm×30 m)
and fitted with a flame ionization detector. The column was maintained
at 50 °C for 3 min and then raised to 230 °C at 3 °C/min. The tempera-
ture of the injector and the detector was 250 °C. Nitrogen was used as
a carrier gas. The retention times of isoamyl alcohol and isoamyl acetate
were respectively, 2.4 min, and 4.8 min, respectively.
To test the stability of the immobilized preparations after each
batch reaction, the enzyme was recovered by filtration and added to
fresh substrate mixture without other treatment.
free RhDL
nanoSnO2-A-RhD
100
nanoSnO2-B-RhD
Table 1
Physical properties of the carriers and biochemical characteristics of Rhizopus delemar
SBA-15-RhD
80
lipase immobilized on tin dioxide and silica material.
Sample Characteristics of the pure
supports
Characteristics for immobilized
lipase
60
40
20
0
Specific
Total
pore
Main pore Total
Specific
activity
(U/mg
prot.)
Protein
surface
diametera activityb
loading
area (m2/ volume
(nm)
(U/g
carrier)
yieldc (%)
g)
(mL/g)
SBA-15 809
SnO2-A 170
SnO2-B 115
0.95
0.13
0.25
6.9
3.0
7.0
428 14.3 23.8 0.8
760 21.0 31.0 1.1
684 23.5 33.8 1.3
55.1 1.9
75.0 2.6
61.8 2.2
a
Main pore diameter was evaluated from NLDFT pore size distribution curves—half-
width at half maximum.
2
3
4
5
6
7
8
9
10
11
b
Tributyrin was used as a substrate (pH 7.0, 40 °C).
pH
c
The protein loading yield (%) was calculated from the ratio between the amount of
the immobilized protein and the amount of the protein in the initial loading solutions.
Fig. 1. Effect of the immobilization on pH stability of Rhizopus delemar lipase.