High stability in organic solvent of heme proteins immobilized in the interlayers of magadiite nanoparticles

Article abstract of DOI:10.1246/cl.2004.1210

Enzymatic activities of heme proteins, myoglobin (Mb) and hemoglobin (Hb), immobilized in the interlayers of magadiite nanoparticles under mild condition, toward the oxidation of o-phenylenediamine (OPD) in an organic solvent were firstly reported. The immobilized heme proteins showed higher stability than that of free Mb and Hb in organic toluene solution.

Full text of DOI:10.1246/cl.2004.1210

1210
Chemistry Letters Vol.33, No.9 (2004)
High Stability in Organic Solvent of Heme Proteins Immobilized in the Interlayers
of Magadiite Nanoparticles
Shuge Peng, Qiuming Gao,ꢀ and Jianlin Shi
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics,
Chinese Academy of Sciences, Shanghai 200050, P. R. China
(Received May 11, 2004; CL-040535)
Enzymatic activities of heme proteins, myoglobin (Mb) and
vantages of layered materials with nanomaterials, firstly report
the catalytic activities of two kinds of water-soluble heme pro-
teins, Mb and Hb, immobilized in the interlayers of magadiite
nanoparticles toward the oxidation of o-phenylenediamine
(OPD) in organic solvents. We have found that the stability or
durability of immobilized heme proteins were higher than that
of free Mb and Hb.
hemoglobin (Hb), immobilized in the interlayers of magadiite
nanoparticles under mild condition, toward the oxidation of
o-phenylenediamine (OPD) in an organic solvent were firstly
reported. The immobilized heme proteins showed higher stabil-
ity than that of free Mb and Hb in organic toluene solution.
Stable layered silicate sol consisting of 10–100-nm diameter
particles of tetrabutylammonium hydroxide (TBAOH) interca-
lated magadiite (TBA-magadiite) was successfully obtained by
intercalation method.8a Mb or Hb-intercalated magadiite (Mb-
or Hb-magadiite) nanocomposites were achieved by dripping
TBA-magadiite suspensions (2.0 mM) into Mb (60 mM) or Hb
(15 mM) (pH was controlled at 7 by K2HPO4) solutions, respec-
tively, and stirring for 1 h at room temperature. Drying of Mb- or
Hb-magadiite was achieved by lyophilizing the samples.
Catalytic activities and kinetic constants of Mb and Hb were
measured by spectrophotometric handles. The addition of tert-
butyl hydroperoxide (10 mM) to the reaction system containing
1.0 mg free or immobilized heme proteins and o-phenylenedia-
mine (2.5 mM) in toluene solvent resulted in the formation of
2,3-diaminophenazine, as observed in the absorption spectra. It
has found that the intensity of the absorbance at 496 nm increas-
es along with the increased reaction time. The kinetics of enzy-
matic reaction was studied by the initial rate method and the
steady-state assumption. The Michaelis–Menten constants Km
and the maximum rate Vmax were obtained from Lineweaver–
Burk plots and the transformation constant Kcat was obtained
from Vmax ¼ Kcat ꢁ ½Eꢂ0, where ½Eꢂ0 is the initial concentration
of enzyme. The Kcat values represent the catalytic activities.
The characterization of novel heme proteins, Mb and Hb,
immobilized in the interlayers of magadiite was described in an-
other paper.8b SEM and HRTEM images showed that the com-
posites of about 30-nm diameters with a certain aggregation
were formed. UV–vis and FTIR spectra showed that no visible
denaturation occurred. Immobilized Mb and Hb retained their
activity and could be readily accessible for substrates in aqueous
solvent. The effect of organic solvent on the activity of Mb and
Hb immobilized in the interlayers of magadiite nanoparticles
was studied here. Catalytic oxidation of o-phenylenediamine
by the free and immobilized proteins with the absorbance at
496 nm in toluene solvent was shown in Figure 1. The immobi-
lized Mb and Hb in the galleries of layered magadiite nanopar-
ticles exhibited higher stability than that of the free Mb and Hb.
The formation amount of 2,3-diaminophenazine for immobi-
lized Mb and Hb increased 60.0 and 113.0% compared to that
of free Mb and Hb after 30 min, respectively. When the immo-
bilized Mb and Hb were stored in toluene solvent for 30 days un-
der stirring condition, the formation amount of product for im-
Enzymes have great potential for the use in organic synthet-
ic chemistry due to their high efficiencies and specific catalytic
characters. Since the pioneering work of Zarks and Klibanov,1
organic solvents have been used extensively in enzymatic syn-
theses and considerable progresses have been achieved in this
field. The advantages for the use of organic solvents as media
for enzymatic reactions are obvious, such as increased thermal
stability2 and lower inhibition effects3 due to a reduced product
concentration at the enzyme resulting from the normally better
solubility of the product in the surrounding organic phase than
in the microenvironment of the hydrophilic enzymatic surface.
Owing to these obvious merits, an increasing interest on enzy-
matic catalysis in nonaqueous media has undergone rapid expan-
sion, particularly over the past decades. However, drastic reduc-
tions of catalytic efficiency for enzymes in organic media can oc-
cur, which is attributed to the phenomena such as aggregation or
irreversible conformational changes. It confined the applications
of enzyme in industrial chemistry in a large scale.
To effectively utilize enzymatic catalysis, different ap-
proaches to the construction of biocatalytic systems based on or-
ganic solvents have been successfully developed,4 such as sur-
face modification of enzyme with surfactant and polymer, re-
versed micelles and immobilization of enzyme. While enzyme
immobilization seems to be the most promising approach to pre-
vent enzyme denaturation not only in aqueous solvents but also
in organic media,5 immobilization may greatly increase the sta-
bility of enzymes, be separated from products and reused easily.
In order to improve the enzymatic catalytic efficiency in organic
solvents, it is the key to increase the contact probability between
enzyme and substrate. Nanomaterials provide larger specific sur-
face areas, which are good candidates of support matrixes for en-
zyme immobilization. Enzymes immobilized on ordered sur-
faces, such as layered inorganic materials6 and phosphate bilayer
membranes,7 have been reported. Hamachi realized the function-
al conversion of Mb bound to synthetic bilayer membranes from
an oxygen storage protein to a redox enzyme. The advantages for
the layered inorganic materials as support matrixes are obvious
in aqueous solvent to preserve biological activity of enzyme. So
far, it is unknown for enzymes immobilized in the interlayer re-
gion of layered materials whether the enzymatic activity is still
preserved in organic media. In this paper, we combined the ad-
Copyright ꢀ 2004 The Chemical Society of Japan

Chemistry Letters Vol.33, No.9 (2004)
Table 1. Kinetic constants of free and bound heme proteins in toluene solvent
1211
Heme Proteins
Km/mM
Vmax/mM minꢃ1 mgꢃ1
Kcat ꢀ 103/minꢃ1
ðKcat=KmÞ ꢀ 103/minꢃ1 mMꢃ1
Free Mb
Bound Mb
Free Hb
0.54
0.28
3.23
1.75
0.78
0.42
0.72
0.48
6.61
3.56
12.24
12.71
7.58
24.49
16.33
Bound Hb
9.33
mobilized Mb and Hb indicated that the catalytic activities were
lower than that of the free Mb and Hb. Although the catalytic ac-
tivity for the immobilized Mb and Hb was low, a larger amount
of products were obtained after 30 min, which indicated that it
was still an efficient method to protect enzyme from harming
by organic solvents.
In conclusion, Mb and Hb immobilized in the interlayers of
magadiite nanoparticles showed relatively lower initial or intrin-
sic activity and higher stability or durability than that of free Mb
and Hb, which demonstrated that the layered magadiite nanopar-
ticle matrix could efficiently prevent the conformational changes
of Mb and Hb in the organic solvent. The encouraging results
would be useful for and applicable to industrial processes and
other applications, especially some environmentally benign
enzymatic reactions.
free Hb
0.8
0.6
0.4
0.2
immobilized Hb
TBA-magadiite
free Mb
immobilized Mb
0.0
0
5
10
15
20
25
30
Time,minute
Figure 1. tert-Butyl hydroperoxide activated peroxidase activ-
ity for oxidation of o-phenylenediamine to 2,3-diaminophena-
zine with the absorbance at 496 nm at different interval times
for free and immobilized Mb and Hb. ([tert-butyl hydroperox-
ide] = 10 mM, [o-phenylenediamine] = 2.5 mM, [Mb] = [Hb]
= 1 mg).
This work was financially supported by Chinese National
Science Funding (No. 20201013) and ‘‘Plan of Outstanding
Talents’’ of Chinese Academy of Science.
References
mobilized heme proteins decreased 71.1% (Mb) and 68.9%
(Hb), respectively, while the enzymatic activities for the free
heme proteins were almost totally lost under the same condi-
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of the reactants through the layered support and facile access to
the active sites of the immobilized proteins in the organic media.
The higher stability of the immobilized Mb and Hb composites
demonstrates that the layered magadiite nanoparticle matrix
could efficiently prevent the conformational changes of Mb
and Hb in the organic solvent.
Comparison of the values of the kinetic constants based on
the Michaelis–Menten equation for the free and the bound Mb
and Hb is represented in Table 1. It is evident from the table that
the free Mb and Hb have larger values of Km and Vmax than that
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and Vmax values might be due to the existence of product inhib-
ition. This could be due to the restricted environments imposed
by the rigid matrix. The reductions in Vmax and Kcat for the im-
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Published on the web (Advance View) August 21, 2004; DOI 10.1246/cl.2004.1210