kasetsart journal natural science

138
Determination of enzyme activities
Carboxymethyl cellulase activity (CMcellulase)
was detemined by the modified method
of Okeke and Obi (1995) by performing the
reaction mixture of 0.1 ml of sample and 0.1 ml
of 1 % (w/v) CMC (Sigma) in 50 mM citrate
phosphate buffer pH 5.5 at 50°C for 20 min. The
amount of reducing sugar released was determined
by Dinitrosalicylic acid (DNS) method (Miller,
1959). One unit of enzyme was defined as the
amount of enzyme that released glucose equivalent
to 1 µmol of glucose per min.
β-1,3-1,4-glucanase activity was
measured using the same procedure as
carboxymethyl cellulase activity. However, 1 %
(w/v) of barley β-glucan (Sigma) was used as
substrate. One unit of enzyme was defined as
mentioned above
Xylanase activity was measured by the
same method described by the modified method
of Okeke and Obi (1995), but 1% (w/v) of oat spelt
xylan (Sigma) was used as substrate. One unit of
enzyme was defined as mentioned above.
Pectin methylesterase activitiy was
measured using the modification method of Huang
and Mahomey (1999) by titration of carboxyl
groups released from pectin (Sigma) at 50°C for
20 min. Approximately 0.5 ml of enzyme sample
was mixed with 4.5 ml of 0.5% pectin in 50 mM
cirtrate phosphate buffer pH 5.5. The acid released
was titrated with 0.01 N NaOH. One unit of pectin
methylesterase was defined as the amount of
enzyme which released 1 mmole of carboxyl
groups per min under the assay condition.
Pectin lyase activitiy was determined
according to the modification method of Huang
and Mahomey (1999). 0.1 ml enzyme sample was
mixed with 0.4 ml of 0.5 % pectin in 50mM cirtrate
phosphate buffer pH 5.5, incubated at 50°C for 20
min. 4 ml of 0.01 M HCl was added and
absorbance was measured at 235 nm. One unit of
pectin lyase was defined as the amount of enzyme
which released 4-5 unsaturated trans-elimination
Kasetsart J. (Nat. Sci.) 40(1)
products showing absorbance of 0.2 at 235 nm.
Pectate lyase activitiy was determined by
the same method of pectin lyase activity
determination, except that 0.5% polygalacturonic
acid (PGA) (Sigma) was used as substrate. One
unit of pectate lyase was defined as the amount of
enzyme which released unsaturated transelimination
products showing absorbance of 0.2
at 235 nm.
Polygalacturonase activity was measured
by the method of pectate lyase activity assay as
mensioned above, except that 1% pectin was used
as a substrate. One unit of enzyme was defined as
the amount of enzyme that released galactose
equivalent to 1 µmol of galacturonic acid per min.
Polymethylgalacturonase activity was
measured by the method of carboxymethyl
cellulase activity assay mensioned elsewhere,
except that 1% PGA was used as substrate. One
unit of enzyme was defined as the amount of
enzyme that released reducing sugar equivalent
to 1 µmol of galacturonic acid per min.
Protein assay
Protein concentration was determined by
the method of Lowry et al. (1951) using bovine
serum albumin as a standard.
Determination of grass degradation activity
Cell-free culture fluid (CFC) containing
enzyme components was filtrated with 0.2 micron
filter membrane. One ml of CFC was added to the
tube containing 1 g of grass powder prepared by
milling and sieving (mesh number 120). The
mixture was incubated for 24 h at experimental
temperatures. Then the extract solution was
prepared using the method of Ohmomo et al.
(1995). In brief, 10 ml of distilled water was added
into the reaction mixture, shaked at 100 rpm for 2
h and then filtrated through Whatman No.4 paper
filter. To remove all residual soluble carbohydrates,
the retentate was washed with 10 ml distilled water
twice. All filtrates were pooled and adjusted to the