Enzymology- Lecturer: Prof.

Zhang Wenbin
Fulfilled by student: Do Thi Van Thanh ( 杜氏去清 )
ID number: 6160102907
Homework 2
Novel Co-immobilization Technique And Its Application
The development of coimmobilized multi-enzymatic systems is increasingly driven by economic and
environmental constraints that provide an impetus to develop alternatives to conventional multistep
synthetic methods. as in nature, enzyme-based systems work cooperatively to direct the formation of
desired products within the defined compartmentalization of a cell. There are three primary reasons for
the utilization of coimmobilized enzymes: to enhance the effciency of one of the enzymes by the in-situ
generation of its substrate, to simplify a process that is conventionally carried out in several steps and/or
to eliminate undesired by-products of an enzymatic reaction.as such, coimmobilization provides benefits
that span numerous biotechnological applications, from biosensing of molecules to cofactor recycling and
to combination of multiple biocatalysts for the synthesis of valuable products(Betancor and Luckarift
2010).

Co-immobilization Of Enzymes With The Help Of A Dendronized Polymer And

Mesoporous Silica Nanoparticles
Fundaments Of Technique
The two enzymes Aspergillus sp. glucose oxidase (GOD) and horseradish peroxidase (HRP) were
co-immobilized on solid silica supports in a spatially controlled way by using mesoporous silica
nanoparticles (Hiroshima Mesoporous Materials, HMM) and a polycationic dendronized polymer
(denpol).The silica support was first coated with the denpol, followed by the deposition of the mesoporous
silica nanoparticles into which – in a next step – GOD was adsorbed. Finally, the GOD-loaded silica
nanoparticles were coated with a denpol–HRP conjugate constituting of several HRP molecules which
were covalently bound to the denpol via bis-aryl hydrazone (BAH) bonds. The entire immobilization
process was followed in real time with quartz crystal microbalance with dissipation monitoring (QCM-
D)(Gustafsson, Küchler et al. 2015).

Principle
First, coating of a silica surface with the denpol allowed efficient adsorption of the mesoporous silica
nanoparticles, in which the first type of enzyme (GOD) was immobilized. Second, the second type of
enzyme was added in the form of a denpol–BAH–enzyme conjugate, where the denpol served as a
macromolecular scaffold bringing several copies of the second enzyme (HRP) in close proximity to each
other and mediating the formation of a stable covering layer on the mesoporous silica nanoparticles Fig.1.
As a side effect, the presence of the denpol–BAH–HRP layer increased the stability of GOD loaded in the
particles Fig. 2. Probably due to a prevention of enzyme leaching from the particles. QCM-D was used as
a simple and robust measuring technique for the real time study of the stepwise co-immobilization of the
two enzymes, and their catalytic activity was measured with suitable enzymatic activity assays.

Fig.1 An illustration of the co-immobilized system prepared in a way resembling the layer-by-layer methodology. The first
layer is formed by the denpol de-PG2, the second by GOD-loaded mesoporous silica nanoparticles, and the third by the de-
PG2–BAH–HRP conjugate. In this way, a spatially controlled localization of the two enzymes on a solid silica support can be
achieved. The two enzymes catalyze a sequential cascade reaction: GODoxidizes D-glucose with dissolved dioxygen, yielding
glucono-d-lactone and hydrogen peroxide, which serves as oxidizing agent for the HRP catalyzed oxidation of OPD (o-
phenylenediamine) yielding DAP (2,3-diaminophenazine). GOD: glucose oxidase, PDB code 3QVP; HRP: horseradish
peroxidase (isoenzyme C), PDB code 1H5A; BAH: bis-aryl hydrazone. Please note that the carbohydrates on the surface of the
two glycosylated enzymes are not shown(Gustafsson, Küchler et al. 2015).

The proper choice of the mesoporous particle type and pore size is expected to depend on the enzyme of
interest. Therefore, experiments for finding the optimal particle system have always to be carried out for
achieving optimal performance. Once the optimal mesoporous silica particles are chosen and the denpol–
BAH–enzyme conjugate is synthesized, the immobilization procedure for obtaining a spatially controlled
enzyme co-immobilization is rather simple, like it is the case for the conventional layer-bylayer deposition
methodology(Sakr and Borchard 2013).

Its application
As a general result, the combination of the two different enzyme immobilization approaches – using HMM
nanoparticles as well as denpol–BAH–enzyme conjugates–allows a spatially controlled coimmobilization
of different types of enzymes on solid supports. (Gustafsson, Küchler et al. 2015) indicated that this type
of enzyme co-immobilization may find applications in the field of bioelectrode fabrications where a
defined localization of redox enzymes is essential for the efficient electron transfer between the
immobilized enzymes and the electrode(Roberto A. S. Luz 2014, Yang, Wei et al. 2014).

Fig.2 Apparent activity and stability of GOD immobilized in HMM nanoparticles attached on de-PG2-coated glass coverslips
without any additional coating with a denpol layer (a), and with an additional layer of de-PG2–BAH– HRP (b). Since the
activity of GOD in the latter case was measured without additional HRP in the assay mixture, the activity can be assigned
entirely to the surface localized enzymatic cascade reaction. Storage temperature: 40C; PBS pH = 7. The reaction scheme of the
enzymatic cascade reaction used for the activity measurements is given in (c). See Materials and methods for details(Gustafsson,
Küchler et al. 2015)
Glucose Oxidase And Catalase Co-Immobilization On Biosynthesized Nanoporous
SiO2
Fundaments of Technique
Nanoporous SiO2 is synthesized by sol–gel method using dry yeast cells as a template. Sol–gel method
(Stober method) is selected for nanoporous silica synthesis in the present of dry yeast cells as a structure
directing agent. So as to obtain high surface area to achieve high load of enzymes on it. Bio-synthesized
nanoporous SiO2 has the specific surface area of 460 m2/g and total pore volume of 0.36 cm3/g. Co-
immobilization of glucose oxidase and catalase by adsorption method was done on biosynthesized
nanoporous SiO2.

Performance For Co-Immobilization Of GOx And Catalase

Process conducted by (Mahdizadeh and Eskandarian 2014) 1.0 g of the nanoporous SiO2 was dispersed
in 10 ml of 150 U/ml GOx and 200 U/ml solution in acetate buffer at pH 5.50 with stirring by a magnetic
stirrer for 3 h at 200C. The experiments showed that use of higher dosages of GOx and catalase had not
more effect on the amount of enzymes immobilization in the support. The obtained hybrid catalyst was
separated from solution by filtration and was stored at 4 ±0.1 0C until use.

The morphology of the SiO2 sample was studied by the scanning electron microscopy (SEM). Particles
sizes were clearly identified by SEM method Fig. 1a. Average size of particles was 300– 400 nm. A SEM
of SiO2 after GOx and catalase immobilization has been shown in Fig. 1b that illustrates enzyme sticking
on surface of SiO2 spherical partials and the distance between them.

Fig.1 SEM images of the biosynthesized mesoprous SiO2 (A) before immobilization and (B) after immobilization of catalase
and GOx (Mahdizadeh and Eskandarian 2014)

Its Application
Co-immobilization of glucose oxidase and catalase on biosynthesized SiO2 was performed for application
in water dissolved oxygen removal for avoiding pitting corrosion in boilers. Central composite design
method based on response surface methodology was used to achieve optimum conditions in an enzymatic
water deoxygenation process. Optimum conditions obtained were found as (Mahdizadeh and Eskandarian
2014): pH = 5.3, T = 420C, substrate initial concentration [S] = 74.3 mmol/L and glucose oxidase activity
[E] = 34.8 mg/L. The removal of oxygen dissolved in water, in optimal conditions was predicted to be
98.8% during 1 min and was obtained 97.5% in experiment.

References
Betancor, L. and H. R. Luckarift (2010). "Co-immobilized coupled enzyme systems in biotechnology."
Biotechnology and Genetic Engineering Reviews 27(1): 95-114.
Gustafsson, H., et al. (2015). "Co-immobilization of enzymes with the help of a dendronized polymer and
mesoporous silica nanoparticles." J. Mater. Chem. B 3(30): 6174-6184.
Mahdizadeh, F. and M. Eskandarian (2014). "Glucose oxidase and catalase co-immobilization on biosynthesized
nanoporous SiO2 for removal of dissolved oxygen in water: Corrosion controlling of boilers." Journal of Industrial
and Engineering Chemistry 20(4): 2378-2383.
Roberto A. S. Luz, A. R. P., Jo¼o C. P. de Souza, Fernanda C. P. F. Sales, and Frank N. Crespilho (2014). "Enzyme
Biofuel Cells: Thermodynamics, Kinetics and Challenges in Applicability." ChemElectroChem 1: 1751-1777.
Sakr, O. S. and G. Borchard (2013). "Encapsulation of enzymes in Layer-by-Layer (LbL) structures: latest advances
and applications." Biomacromolecules 14(7): 2117-2135.

Yang, H., et al. (2014). "Monodispersed silica nanoparticles as carrier for co-immobilization of bi-enzyme and its
application for glucose biosensing." Spectrochim Acta A Mol Biomol Spectrosc 125: 183-188