Z. Wu et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 32–38
33
as carrier. Secondly, lipase was immobilized on MCP through a
simple process. Then, the optimal conditions were investigated to
obtain the MCP-lipase with the highest activity. At the same time,
the catalytic property and stability of MCP-lipase were measured.
Furthermore, FT-IR spectra, SEM and BET-N2 method were used
for characterization. At last, to measure the practical application of
MCP-lipase, synthesis of cinnamyl acetate, a kind of important spice
widely used in daily detergents [10], was selected as model reac-
tion to evaluate the efficiency of enzyme. Considering the improved
properties, the MCP-lipase would have wide prospects for hydrol-
ysis, degradation and synthesis applications.
The activity and activity yield of lipase were calculated based on
the following equations:
(
Vsample − Vblank) × 50
Lipase activity (U) =
Activity yield (%) =
(1)
(2)
Me × 30
Eimmobilized
Efree
× 100
where Vsample (mL) is the volume of NaOH (0.05 mol/L) solution
used to neutralize the acetic acid liberated by lipase hydrolysis;
Vblank (mL) is the volume of NaOH (0.05 mol/L) solution consumed
by glyceryl triacetate solution, while in this situation, the lipase
◦
was heated at 90 C for 24 h. Me is the lipase mass added into the
2
. Materials and methods
glyceryl triacetate solution. 50 is the conversion factor of NaOH to
acetic acid. 30 (min) is the reaction time. Eimmobilized is the activity
of all MCP-lipase obtained from the original lipase solution after
immobilization, and Efree is the activity of all free lipase before
immobilization.
2.1. Material
Lipase from procine pancreas, Type II (PPL) (CAS 9001-62-1) was
purchased from Sigma Chemical Company (USA); cinnamyl alcohol
E1226034) was purchased from Aladdin Company; glyceryl triac-
(
2.5. Catalytic properties of lipase or MCP-lipase
etate, ethyl acetate, silica, calcium oxide, magnesium oxide were
purchased from Guangfu Company (Tianjin, China). Other chemi-
cals were analytical grade and obtained from common commercial
sources without further purification.
Reaction temperature: The activities of free lipase and Ni-MCP-
lipase were determined by adding the enzyme samples (30 mg of
free lipase or 1 g of Ni-MCP-lipase) into 30.0 mL substrate solution
(
(
saturated glyceryl triacetate solution) at different temperatures
25–55 C) for 30 min, and pH value was maintained 6.3.
Reaction pH: The activities of free lipase and Ni-MCP-lipase were
◦
2.2. Carrier preparation
determined by adding the enzyme samples (30 mg of free lipase
or 1 g of Ni-MCP-lipase) into 30.0 mL substrate solution (saturated
glyceryl triacetate solution) under different pH (5–10) for 30 min,
The carrier preparation process had been mentioned in our
previous work [11], briefly, metal hydroxide precipitation was pre-
pared by dissolution of metal chloride in deionized water and
precipitated by NaOH solution. The precipitation was filtered and
washed for 3 times. Then, the obtained metal hydroxide precipita-
◦
and the temperature was maintained 35 C.
◦
tion was dried at 70 C for 24 h.
2.6. Stabilities of lipase or MCP-lipase
Subsequently, metal ceramic powder (MCP) was prepared by
mixing of 22 g matrix (containing 10 g SiO , 5 g Al O , 5 g Na SiO ,
2
2
3
2
3
The thermal stabilities of free lipase (1 mg/mL) and Ni-MCP-
lipase (50 mg/mL) were determined by measuring the residual
activities of enzyme samples incubated in phosphate buffer at pH
1
g MgO, 1 g CaO), 5 g metal hydroxide precipitation and 10 g deion-
◦
ized water. Then the MCP was obtained by drying in muffle at 150 C
for 2 h and calcining through 1 C/min, 3 C/min, 5 C/min, 7 C/min
to 850 C, respectively, and the temperature was preserved for 4 h.
◦
◦
◦
◦
◦
6.3 and 70 C. The incubating time was from 1 h to 5 h and the time
◦
interval was 1 h. The storage stabilities of free lipase (1 mg/mL)
and Ni-MCP-lipase (50 mg/mL) were determined by measuring the
residual activities of enzyme samples in phosphate buffer at pH 6.3
Meanwhile, the ceramic powder (CP) was also prepared with the
same process as the control without mixing metal hydroxide pre-
cipitation.
◦
and 4 C. The storage time was set from 1 to 7 days.
2.7. Characterizations
2.3. Lipase immobilization
The XRD (X’Pert Pro, 3.0 kV, Cobalt bomb), SEM (S4800, 5 kV,
Surface activation: MCP surface was activated by 5% (w/w) sul-
high power mode), BET-N2 adsorption (F-sorp 2400, liquid nitro-
gen temperature), EDS (S-4800, 15 kV, 15,000× magnification) and
IR spectrum (Bio-Rad FTS 6000, FTIR, KBr disk method) were
performed to characterize the properties of Ni-MCP and Ni-MCP-
lipase.
furic acid for 1 h under vigorous stirring at room temperature. Then,
the activated MCP was washed with deionized water for 6 times.
Immobilization process: 5.0 g of activated MCP was added into
0.0 mL lipase solution with certain concentration and stirred at
C for different time, then the immobilized lipase (MCP-lipase)
1
4
◦
was washed by deionized water for 3 times to remove the un-
immobilized lipase.
2.8. Enzymatic synthesis of cinnamyl acetate
Synthesis reactions were carried out in 50 mL shaking flask at
5 C under 150 rpm for 10 h. The reaction mixture contained 0.12 g
2.4. Enzyme activity assay
◦
3
acetic acid, 0.268 g cinnamyl alcohol, 15.0 mL n-hexane and certain
amount of free lipase or Ni-MCP-lipase.
The cinnamyl acetate (CA) yield was determined by measuring
the acetic acid content in the reaction mixture through titration
with NaOH solution (0.1 mol/L) and calculated based on the fol-
lowing equation:
The activities of free lipase and MCP-lipase were determined
by hydrolysis of glyceryl triacetate [12]. The enzyme samples were
added into 30.0 mL saturated glyceryl triacetate solution (pH 6.3)
and the NaOH solution (0.05 mol/L) was added into the mixture to
maintain the solution with pH 6.3. Meanwhile, the reaction time
was set as 30 min. One unit activity (U) of free lipase or MCP-lipase
was defined as the amount of enzyme needed to liberate 1.0 mol
C − Cs
0
CA yield (%) =
× 100
(3)
◦
of acetic acid in 1 min at 35 C and pH 6.3.
Cc