Biomedical Engineering Reference
In-Depth Information
substrate-Trp
170
-Leu
171
-heme; MnP crystal structure includes 17 % neutral sugars
and a large number of acidic amino acids. There is only one Mn
2C
binding site in
heme [
48
-
50
].
The copper number of different types of laccase is different. Generally, lacasse
contains four copper ions. Copper ions can be divided into three types according
to spectroscopic and magnetic characteristics. Type I has the characteristics of one
Cu
2C
, single-electron acceptor, paramagnetism, blue color, and special absorption
spectra at
614 nm. Type II has the characteristics of one Cu
2C
, single-electron
acceptor, paramagnetism, nonblue color, and no characteristic absorption spectra.
Type III has the characteristics of two Cu
4C
, dual-electron receptor, diamagnetism,
coupling ions (Cu
2C
-Cu
2C
), and broad absorption spectra at
œ
330 nm. The 3-D
structure of laccase is not yet clear, but it has been confirmed that copper ion is
located in an active site and plays a decisive role in the catalytic oxidation process
[
48
,
51
,
52
].
œ
11.2.4.2
Determination Method for Activity of Cellulose-Degrading
Enzymes
Enzyme activity is usually determined when the solubility of substrate is better and
the concentration of substrate is high. So, the reaction is zero stage for substrate.
Then, the specific activity of the enzyme can be obtained exactly by measuring
initial velocity. However, it is difficult to compare cellulase activity determined by
various methods and calculated with different methods because there is no common
standard. A major problem in current cellulose basic research is how to determine
cellulase activity accurately and rapidly. Problems in the determination of cellulase
activity and the current method used are discussed next.
1. Problems in the cellulase activity measuring process
(1) Substrate cellulose structure is heterogeneous, including an easily degradable
amorphous region and a difficultly degraded crystalline region. Most substrate is
water insoluble or sparingly soluble polymers. In terms of cellulase, substrate is
always unsaturated. The contact of cellulase enzyme with a solid substrate and
reaction product (such as CMC) is only partially soluble. The more the substituent
is, the greater the solubility is. In turn, the more the substituent is, the lower
the decomposability is as a cellulose enzyme substrate. Therefore, researchers
around the world determine cellulase activity in the state of unsaturated substrates.
The measured enzymatic activity is always low because of unsaturated substrate.
Moreover, the more the cellulose is used, the greater the unsaturation is. While
determining cellulase activity, different results are obtained for different enzyme
preparations or different dilutions.
(2) Because the substrate used also contains cellulose, hemicellulose, and lignin, the
saccharification effect of enzyme on substrate also includes hemicellulase activity
in general cellulase in addition to that of cellulose (approximately two times higher
than cellulase) because the sugar produced is reducing property sugar. Therefore,
impure substrate has a great impact on cellulase activity.
