Biomedical Engineering Reference
In-Depth Information
buffer at pH 4.8. The mixture is reacted at 50
ı
C for 60 min. Then, 1 mL DNS
reagent is added to terminate the reaction. The mixture is put into a boiling water
bath for 5 min and immediately cooled. The amount of reducing sugar is measured at
550 nm. The concentration of the enzyme solution is adjusted repeatedly to obtain
2 mg reducing sugar generated in the reaction. It is really complicated to adjust
enzyme solutions from different sources to a similar reaction. In recent years, many
improved methods were provided, such as the improved method proposed by Gao
et al. But, conventional methods in the laboratory adjust the OD of the generated
sugar at about 0.4.
②
Cotton enzyme activity
For the cotton enzyme activity method, 50
˙
5 mg cotton, 2 mL acetic acid buffer at
L
1
, and 0.5 mL of appropriately diluted
enzyme solution are added together. The mixture is reacted at 50
ı
C for 24 h. The
reducing sugar is determined by the DNS method.
pH 4.8 and with a concentration of 0.2 mol
(3) Determination of cellulose component activity
CBH is a necessary component for the enzymatic hydrolysis of crystalline cellulose.
However, if it acts alone on cellulose, it is difficult to determine physical and
chemical changes. Reducing sugar would be generated only when it cooperates with
EG (endo-
“
-1,4-glucanase). It is obvious that the enzymatic activity is the activity of
different enzyme components while determining enzymatic hydrolysis with cotton
as substrate. Now, there are several methods to measure CBH activity.
①
-D-cellobiose (PNPC) method
For the PNPC method, 2 mL reaction solution contains 0.5 % PNPC, 0.1 % glucose
lactone, and 0.05 mol
P-Nitrophenol-
“
L
1
acetic acid buffer at pH 4.8, in which glucose lactone
keeps
-glucosidase (BG) from hydrolyzing cellobiose side chain. Then, 0.5 mL
appropriately diluted enzyme solution is added. The reaction solution is reacted at
50
ı
C for 15 min. Then, 0.5 mL 10 % Na
2
CO
3
is added to terminate the reaction.
The amount of generated p-nitrobenzoic acid is determined at 420 nm.
②
“
An improved method proposed by Gao et al.
Gao et al.'s improved method determines CBH according to the differences of
adsorption property of CBH-CBH, EG, and BG components in cotton fiber.
However, it is too complicated.
There are mainly two methods for EG: CMC method and Brix method.
①
CMC method
For the CMC method, 2 mL reaction solution containing 0.5 % CMC-Na, pH 4.8,
and 0.2 mol
L
1
acetate buffer. Then, 0.5 mL suitably diluted enzyme solution is
added. The mixture reacts at 50
ı
C for 30 min. Finally, the amount of reducing
sugar is measured by DNS.
②
Brix method
For the Brix method, 5 mL 0.5 % CMC-Na are added to an austenitic sugar meter,
then 20 mL of suitably diluted enzyme solution are added. The reaction is carried
out at 30
0.1
ı
C for 10 min. Finally, the Brix value change is measured.
˙
