J. Li et al. / Carbohydrate Research 316 (1999) 133–137
137
like solid. (Buffer solutions with 50 mM and
four different pHs were used as follows:
sodium acetate buffer pH 4.85 for A. oryzae,
sodium acetate buffer pH 5.0 for B. circulans,
sodium phosphate buffer pH 6.0 for Gly001-
02 from CLONEZYME™, and sodium phos-
phate buffer pH 7.0 for E. coli.)
ity [p] was determined as the value of specific
viscosity psp, (pr −1)/c, at the limit of vanish-
ing c.
Acknowledgements
The authors thank Diversa, Inc (San Diego,
CA) for providing the CLONEZYME™ gly-
cosidase library. This work was generously
supported by Hercules, Inc, NSF (BES-
9728366), and the Herman Frasch Foundation
(449-HF97). J.L. is grateful for a Robert May-
tag Fellowship from the University of Miami.
Enzymatic assay of glycosidase acti6ities of
i-galactosidase from A. oryzae.—To a spec-
trophotometer cuvette at room temperature
was added 1 mL of enzyme solution in sodium
acetate buffer at pH 4.5 (2 mg/mL) and 10 mL
of 100 mM substrate solution using p-nitro-
phenyl b-
D
-glucopyranoside, p-nitrophenyl b-
glucopyranoside,
and o-nitrophenyl
b-galactopyranoside. The amount of p-nitro-
phenolate liberated was measured continu-
ously at 380 nm. One unit of specific activity
is defined as the amount of enzyme that cata-
lyzes the formation of 1 mmol p-nitropheno-
late per minute per mg protein at ambient
temperature.
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Peroxidase–chromogen test.—The mixed
enzyme–chromogen reagent was prepared as
follows: 0.5 mL of galactose oxidase (70
units), 0.5 mL of horseradish peroxidase (100
mg/L), 0.5 mL of o-tolidine (200 mg/L) and
0.5 mL of the substrate solution (the reaction
concentration was less than 1.39×10−4 M,
i.e., 278 nmol in 2 mL of reaction solution)
were mixed and placed in incubator at 30 °C
for 1 h. Maximum chromogenesis took place
within 60 min. The color developed was read
at 420 nm. Due to the complexity of the
Gal–HEC, galactose and lactose were both
used as standard curves in the range of 278
nmol for the detection of galactose. The Gal–
HEC reaction solution absorbance at 420 nm
was compared against the standard curve of
galactose and lactose.
Intrinsic 6iscosity.—Viscosity measurements
were conducted using an Ubbelohde viscome-
ter. The measurement was carried out using
H2O as the solvent in a constant temperature
bath at 25 °C with relative viscosity, pr =t/t0.
All flow times were sufficiently long to avoid a
kinetic energy correction. The intrinsic viscos-
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