Biomedical Engineering Reference
In-Depth Information
targeting efficiency in P. decumbens, Li et al. [
94
] developed a highly efficient
gene targeting system by deleting pku70, which is involved in the nonhomologous
end-joining pathways, and succeeded in significantly improving gene targeting
efficiency. The development of gene manipulation platforms in species of cellu-
lase-producing fungi will efficiently improve the efficiency of engineering these
species for better lignocellulose degradation.
Aside from the genetic engineering of cellulase-producing fungal strains, pro-
tein engineering of cellulases is also a focus of research in China. Xiao et al. [
105
]
applied an error-prone PCR technique in random mutagenesis of EG III from
T. reesei, and obtained a psychrophilic enzyme called w-3. Catalytic analysis of this
enzyme suggested a 19% increase in specific activity and a 41% increase in K
cat
/K
m
at 30 C. The sequence analysis suggested a 25 amino acid residue deletion at the
C-terminus and a significant decrease in its hydrophobicity. The same technique was
used to investigate EG III from T. reesei in search of an alkaline EG. An N321T
mutant with an increased optimum pH was obtained. Further site-directed muta-
genesis suggests the important role of this amino acid residue for the activity of EG
III, and it resulted in an N321H mutant with a broader range of pH tolerance [
106
].
Another example of the genetic engineering of enzymes is the research on EG II from
T. reesei during the search for an alkaline EG. Qin et al. [
107
,
108
] subjected residue
342 to saturated mutagenesis and modified the enzyme through random mutagenesis
and two rounds of DNA shuffling. A series of modified enzymes were produced
during these investigations, including an N342 variant with an optimum pH of 5.8,
one unit higher than the wild-type enzyme and with a 1.5-fold higher K
cat
/K
m
at pH
6.5, an N39R/L218H/W276R/N342T variant that has a pH optimum of 6.2 and a
1.4-fold higher K
cat
/K
m
at pH 6.2, as well as three variants L218H, Q139R/N342T,
and Q139R/L218H/W276R/N342T that have more than 3.5-fold increased activity
at pH 7.0 [
107
,
108
].
Research on optimizing processes of cellulase production is another focus of
study in China, which aims to improve the productivity and efficiency of cellulase
production. Yu and Koo [
109
] used mixtures of Avicel and wheat bran as carbon
sources and obtained higher secreted cellulase activity [11.67 filter paperase units
(FPU) per milliliter] with T. reesei Rut C-30 in a 2.5-L fermentor. Duan et al.
[
110
] investigated the combined effects of cellulose powder CF11 and glucose on
EG production from T. pseudokoingii S38, and demonstrated that the use of both
substrates as carbon sources increases the volumetric product efficiency, as well as
the specific activity of EG production compared with the use of only one substrate.
Qu et al. [
76
] suggested that the high price of cellulases is partly due to the high
cost of cellulose powder and inorganic salts in media. A new cellulase production
process using industrial wastes was therefore developed with P. decumbens JU1.
In this process, spent ammonium sulfite liquor and cellulosic wastes (clarifier
sludge and digester fines) from a paper mill were used as the medium for fungal
growth, as well as cellulase production. Yang and Yu [
111
] used bagasse
pretreated with alkali and microwave radiation instead of cellulose powder,
thereby addressing the high prices of cellulose powders. In another study, solid
residues
from
the
evaporation
of
acidic
liquid
generated
during
industrial
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