K. Sangseethong, M.H. Penner / Carbohydrate Research 314 (1998) 245–250
249
NMR spectra of synthesized compound 3
matched that of commercially available com-
pound 3 (Sigma Chemical Co., St. Louis,
MO).
Glucose is included in the specified mobile
phases to enhance the association of CBH II
with the ligand [2,21]. Chromatography was
performed at 4 °C with the mobile phase flow
rate at 0.5 mL min . Adsorbed CBHs were
eluted by the addition of cellobiose, 0.01 M,
to the mobile phase. Protein-containing frac-
tions were detected by monitoring absorbance
at 280 nm.
Endo- and exo-type cellulase activities were
measured as described previously [5] using
hydroxyethylcellulose (HEC; medium viscos-
ity, Fluka Chemical Co., Ronkonkoma, NY)
and microcrystalline cellulose (MCC; Avicel
PH 101, FMC, Philadelphia, PA) as sub-
strates. The amount of reducing sugar liber-
ated was determined by a colorimetric method
−
1
p-Aminophenyl
i-cellobioside
(PAPC)
(
4).—Compound 4 was obtained by catalytic
hydrogenation of 3 using 10% Pd on activated
charcoal as a catalyst. TLC (solvent B) indi-
cated the complete conversion of the nitro
compound 3 into its amino derivative 4.
Preparation of PAPC-deri6atized agarose.
—
PAPC was coupled to Affigel 10 (BioRad
Laboratories, Hercules, CA), under anhy-
drous condition to minimize non-specific
protein–matrix interactions [2], as recom-
mended by the manufacturer. The gel (10
mL), after being washed with cold 2-propanol
(
p-hydroxybenzoic acid hydrazide, PAHBAH)
(
(
50 mL), was added to a solution of PAPC
200 mmol) in anhyd MeOH (15 mL). The
[
22]. b-Glucosidase activities were measured
using p-nitrophenyl b-D-glucopyranoside as
substrate [23]. Enzyme activities were mea-
sured at 50 °C in 50 mM NaOAc buffer, pH 5.
slurry was rotated end-over-end for 3 h at
room temperature. Ten milliliters of 1 M
ethanolamine, pH 8, was then added to the
slurry and allowed to react for 1 h in order to
block active ester groups remaining on the gel.
The resulting gel was washed with 0.1 M
NaOAc, pH 5, containing 1 mM gluconolac-
tone (100 mL) and loaded into 1×8 cm glass
columns.
Acknowledgements
This study was supported in part by funds
award number 98-35103-6558) from the NRI
(
Competitive Grants Program/United States
Department of Agriculture. This paper is Ore-
gon State University Agricultural Experiment
Station Technical Publication Number 11,427.
Protein-binding capacities of the PAPC gels
were determined in partition equilibrium ex-
periments under chromatographic conditions.
Control experiments, using non-PAPC-cou-
pled/ethanolamine-blocked gels, demonstrated
negligible binding of T. reesei proteins to the
underivatized matrix.
References
Affinity chromatography of cellobiohydro-
lases.—Crude cellulase produced by T. reesei
[
1] P. Tomme, S. McCrae, T.M. Wood, M. Claeyssens,
Methods Enzymol., 160 (1988) 187–193.
(
Spezyme™-CP, Environmental BioTechnolo-
[2] K. Piyachomkwan, M.H. Penner, Anal. Biochem., 255
1998) 223–235.
(
gies, Menlo Park, CA) was initially fraction-
ated by anion-exchange chromatography
using DEAE-Sepharose [20]. The resulting
partially purified CBH I and CBH II prepara-
tions (10–25 mg) were further fractionated
using PAPC-derivatized agarose as the affinity
matrix. CBH I was applied to the column in
[
[
[
[
[
[
[
3] H. van Tilbeurgh, R. Bhikhabhai, L.G. Pettersson, M.
Claeyssens, FEBS Lett., 169 (1984) 215–218.
4] C. Orgeret, E. Seillier, C. Gautier, J. Defaye, H. Driguez,
Carbohydr. Res., 224 (1992) 29–40.
5] K. Piyachomkwan, K.P. Gable, M.H. Penner, Carbo-
hydr. Res., 303 (1997) 255–259.
6] B. Capon, J.W. Thomson, Bioorg. Chem., 8 (1979) 147–
173.
7] M.V. Deshpande, K.E. Eriksson, L.G. Pettersson, Anal.
Biochem., 138 (1984) 481–487.
8] M. Claeyssens, H. van Tilbeurgh, P. Tomme, T.M.
Wood, S.I. McRae, Biochem. J., 261 (1989) 819–825.
9] D.C. Irwin, M. Spezio, L.P. Walker, D.B. Wilson, Bio-
technol. Bioeng., 42 (1993) 1002–1013.
0
.1 M NaOAc, pH 5, containing 1 mM glu-
conolactone. CBH II was applied to the
column in 0.1 M NaOAc, pH 5, containing 1
mM gluconolactone and 0.2 M glucose. Glu-
conolactone is included in the mobile phase
for suppression of b-glucosidase activity [3].
[
10] M. Vrsanska, P. Biely, Carbohydr. Res., 227 (1992) 19–
27.