内切纤维素酶检测试剂盒 endo-Cellulase Ass

产品货号：K-CELLG3

品牌：Megazyme

产品品名：内切纤维素酶检测试剂盒

英文品名：endo-Cellulase Assay Kit

规格型号：180/360

A soluble substrate for the specific measurement of cellulase (endo-1,4-β-glucanase) in enzyme preparations and fermentation products.

INTRODUCTION:

Cellulase (endo-1,4-β-glucanase) plays a key role in the hydrolysis
of cellulosic biomass to fermentable sugars. This enzyme also finds
widespread industrial applications in the modification of cellulosic
materials and in the degradation of mixed linkage 1,3;1,4-β-glucans.
Numerous methods are available for the measurement of cellulase,
including those based on increase in reducing sugar levels on
hydrolysis of CM-cellulose or 1,3:1,4-β-glucan1. endo-Cellulase
can be specifically assayed using viscometric methods with soluble
CM-cellulose 7M as substrate, or by employing soluble or insoluble
(crosslinked) dyed cellulose or mixed-linkage β-glucan. In general,
assays based on the use of dyed polysaccharides are standardised
against a reducing sugar method that employs either CM-cellulose or
β-glucan as substrate. The problem here is that these polysaccharide
substrates are not completely defined and thus lead to some variation
in the values obtained.
The Megazyme CellG3, cellulase test reagent, employs high purity
β-glucosidase and benzylidene blocked, 2-chloro-4-nitrophenyl-β-Dcellotrioside
(BClPNPβ-G3). The level of β-glucosidase used ensures
maximum sensitivity of the assay. On hydrolysis of BClPNPβ-G3 to
benzylidene blocked cellobiose and 2-Cl-4-nitrophenyl-β-D-glucose
by cellulase, the 2-Cl-4-nitrophenyl-β-D-glucose is immediately
cleaved to D-glucose and free 2-Cl-4-nitrophenol (ClPNP) by the
β-glucosidase present in the substrate mixture (Scheme 1). Thus, the
rate of release of ClPNP relates directly to the rate of hydrolysis of
BClPNPβ-G3 by cellulase. The reaction is stopped, and the phenolate
colour is developed, on addition of Trizma base solution (pH 9).
Standard curves relating enzyme activity to increase in absorbance
at 400 nm on hydrolysis of BClPNPβ-G3 by Trichoderma and A. niger
cellulases are shown in Figures 1 and 2. A standard curve relating
enzyme activity on CM-cellulose 4M (Nelson/Somogyi reducing
sugar assay2) to absorbance increase at 400 nm on incubation
of Trichoderma cellulase with BClPNPβ-G3 is shown in Figure 3.
Cellulase enzymes from different sources vary in their ability to
hydrolyse BClPNPβ-G3, so it is necessary to establish a specific
standard curve for each cellulase to allow accurate quantitation. The
assay can be used at temperatures up to 80°C and in the pH range of
4.5 to 8.0. With cellulase enzymes with activity at high pH values, it
is necessary to terminate the reaction with tri-sodium phosphate
(pH 11.0).
Of the cellulases evaluated in the current study, that with the lowest
rate of hydrolysis of BClPNPβ-G3 was the cellulase from A. niger
(E-CELAN, Megazyme). BClPNPβ-G3 substrate solution as supplied
in dimethyl sulphoxide (DMSO) is very stable when stored at 4°C or
1
-20°C. The substrate, CellG3 is stable for at least 7 days at 4°C and
for > 2 years at -20°C.

KITS:

Kits suitable for performing 180 / 360 assays are available from
Megazyme. The kits contain the full assay method plus:
Bottle 1: (x2) Benzylidene blocked, 2-chloro-4-nitrophenyl-β-Dcellotrioside
(BClPNPβ-G3; 3 mL) in DMSO.
Stable for > 2 years at -20°C.
Bottle 2: KCl solution (15 mL, 100 mM) plus
sodium azide (0.02% w/v).
Stable for approx. 4 years at 4°C.
Bottle 3: Thermostable β-glucosidase (0.40 mL, 400 U/mL) in
50% w/v ammonium sulphate solution plus sodium
azide (0.02% w/v).
Stable for approx. 4 years at 4°C.
Bottle 4: Trichoderma cellulase standard solution (5 mL,
~ 1.6 U/mL; actual value stated on the vial label) in
50% aqueous glycerol plus sodium azide (0.02% w/v).
Stable for approx. 4 years at -20°C.

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