Angewandte
Chemie
XD-D1 system (Shimadzu Ltd.). The synthetic cellulose was first
treated by thorough washing with a nitric acid solution. Cotton
cellulose powder (CF1, Whatman Ltd.) was completely mercerized by
[
17]
immersion into 16% NaOH aqueous solution.
The conversion rates were calculated by dividing the amount of
water-insoluble synthetic cellulose by the amount of cellobiose
supplied to the reaction system. The DP values were obtained by
viscometric analysis. Briefly, the intrinsic viscosity [h] of the cellulose
molecules dissolved in 0.5m cupriethylenediamine solution was
measured by using an Ostwald-type viscometer at 278C, and the
DP values were calculated according to the Mark–Houwink–Sakur-
À3
ada equation: [h] = 5.7 10 DP. The standard deviations were
within 5% for the conversion rates and 3% for the DP values.
Acetylation of synthetic cellulose was carried out according to the
[
18]
previous report. Pyridine (0.5 mL) was added to a solution (5 mL)
of synthetic cellulose (25 mg), LiCl (0.4 g), and DMAc (5 mL), and
then acetic anhydride (0.7 mL) was added dropwise at 708C for 8 h.
The molecular weight of acetylated cellulose was analyzed in
tetrahydrofuran by GPC with a refractive index detector in accord-
Figure 3. Conversion rates (empty bars) and DP values (filled bars) of
the synthetic cellulose that is insoluble in water.
[
7]
À1
ance with the previous report. The flow rate was 1.0 mLmin
Calibration curves were obtained by using polystyrene standards.
.
same way, when pure cellobiohydrolase from Trichoderma
viride was applied to this system, the conversion rate reached
up to approximately 20% but the DP values were less than 20.
The pH value of the water phase of the W/O emulsion
during preparation of the CS complex greatly affected both
the conversion rates and the DP values, even though cellulose
synthesis was carried out in a nonaqueous system. Specifically,
a higher pH value brought about higher conversion and
longer cellulose chains. Such pH-dependent enzymatic activ-
ity has been reported in relation to the cryo-preservation of
higher-order protein structure and the state of the active
Received: September 28, 2006
Revised: December 28, 2006
Published online: February 9, 2007
Keywords: cellulose · enzyme catalysis · glycosynthesis ·
.
polymerization · surfactants
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center.
In the present study, phosphate buffer solution
proved effective, even in pH value regions where there is no
buffering effect. Similar phenomena have been reported with
[
15,16]
regard to glycosylation with chitinase,
but the details
[2] T. S. Raju, J. B. Briggs, S. M. Chamow, M. E. Winkler, A. J. S.
remain to be solved and are currently under investigation.
In summary, by using our unique system, effective
elongation of cellulose chains during synthesis in vitro
became possible for the first time by using a CS complex as
a catalyst and nonaqueous LiCl/DMAc as a reaction medium.
This novel method of utilizing the substrate specificity of
glycohydrolases in organic solvents allows us to precisely
control the regio-and stereoselectivity and to design struc-
tural polysaccharides that are often difficult to prepare owing
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tages are summarized as follows: 1) it is possible to make
structural sugar chains that are insoluble in water and
common organic solvents, 2) aprotic polar solvents, which
frequently destroy enzymatic activity, are applicable, 3) cheap
enzymes can be utilized and enzyme/surfactant complexes are
easy to form by using a simple preparation process, 4) the
synthetic products are easily obtained in a one-pot reaction
process, and 5) tedious preparation of precursor sugar deriv-
atives as starting materials by complicated chemical synthesis
is unnecessary. Therefore, biocatalysts acting in organic
solvents would have potentially wide applications in the
glycoengineering field.
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Experimental Section
XRD profiles were acquired by using Ni-filtered CuKa radiation (l =
1
.5418 ) at a voltage of 30 kVand a current of 40 mA in the scanning
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range from 5–408 at a diffraction angle of 2q, as determined by an
352.
Angew. Chem. Int. Ed. 2007, 46, 2063 –2065
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