產品貨號：ZZK-K-CELLG3

產品品名：內切纖維素酶檢測試劑盒

英文品名：endo-Cellulase Assay Kit

規格型號：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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