Biomedical Engineering Reference
In-Depth Information
(1) Cross-linking immobilization method for cellulase [
61
]
Wu took chitosan as a carrier to immobilize cellulase through a cross-linking
method. The effect of glutaraldehyde concentration and the amount of enzyme
on immobilized cellulase activity was investigated. The optimum temperature,
pH value, and
K
m
were also analyzed. Results showed that, compared with the
free enzyme, there were many advantages for immobilized cellulase obtained by
the cross-linking method. Dong prepared PEG-modified chitosan carrier (PEG-
CS, chitosan) by the cross-linked method to immobilize cellulase. Compared with
the free enzyme, the thermal stability of immobilized cellulase was significantly
improved, and the stability of the operation and storage was good.
(2) Immobilization of cellulase by the magnetic adsorption method [
62
]
For immobilization of cellulase by the magnetic adsorption method, 1.0 g magnetic
chitosan is weighted and added to 50 mL acetic acid-sodium acetate buffer (0.2 M)
to swell. Then, it is filtered. A certain amount of cellulase solution of 1.0 mg mL
1
is added. The mixture is shaken at 30
ı
C and 150 r
min
1
for a certain time on
the constant-temperature oscillator. Microspheres are precipitated under a magnetic
field. Then the supernatant is spilled out and washed with acetic acid-sodium
acetate buffer until no protein can be detected. Freeze-dried immobilized enzyme
is conserved in a 4
ı
C refrigerator for application.
(3) Superparamagnetic nanoparticle immobilization method for cellulase [
63
]
For the superparamagnetic nanoparticle immobilization method for cellulase, 50 mg
magnetic nanoparticles are added to 2 mL phosphate buffer (pH 6.0, containing
0.1 M NaCl), and 1 mL 1-ethyl-(3-dimethyl-aminopropyl) carbodiimide hydrochlo-
ride solution is added. Then, the mixture is treated with ultrasound for 10 min.
Enzyme solution (2 mL) is added. The new mixture is treated with ultrasound
again for 30 min. Then, it reacts at 25
ı
C for 24 h in a shaker at 150 r
min
1
.
Finally, separation is carried out with 3,800 Gs of surface magnetic strength. The
magnetic particles obtained are washed with deionized water. The hydroxyl groups
on the magnetic particles can cross-link the amino group on cellulase molecules
effectively. Magnetic particles of cellulase before and after immobilization both
have a superparamagnetic property.
Cellulase immobilized with superparamagnetic nanospheres can uniformly sus-
pend in a reactor and can achieve full contact with insoluble cellulose substrate.
Because the immobilized enzyme has a magnetic property, after hydrolysis, cel-
lulase can be separated and recycled rapidly in the reaction system by applying a
magnetic field. In addition, the movement manner and orientation of immobilized
magnetic cellulase can be controlled using an external magnetic field instead of
the traditional mechanical stirring. In this way, the catalytic efficiency of the
immobilized enzyme is enhanced. By the method of polymerization of monomer
or direct adsorption, cellulase can covalently couple with functional groups on the
surface of magnetic particle polymer.
