Biomedical Engineering Reference
In-Depth Information
speed in increasing production after the mutagenic treatment. Prokaryotic gene
recombination includes transformation, transduction, conjugation, and protoplast
fusion. Eukaryotic microorganism gene recombination includes sexual hybridiza-
tion, quasi-hybridization, protoplast fusion, and genetic transformation.
Improving cellulase activity by protoplast fusion technology began with the
work of Tayama et al., who researched
Trichoderma reesi
genetics using protoplast
fusion technology. The stable CMCase activity was one time higher than that of
the parent. In China, cellulose microbial protoplasm preparation, regeneration, and
fusion were all explored. Qiu et al. [
38
] researched the progeny of protoplast fusion
from
Trichoderma viride
N6 and
Aspergillus niger
856. By passage, fermentation,
and screening, three strains—AT23, AT16, and AT34—with high cellulase activity
and stability were obtained from 11 screening fusants. Their CMCase activities were
2.2 times, 1.4 times, and 1.2 times higher than the parental
Trichoderma viride
N6,
respectively. It was proved that AT23, AT16, and AT34 were gene recombination
fusions and had the advantages of crossbreeding by nystatin resistance testing and
soluble protein gel electrophoresis analysis. Zhang et al. carried out protoplast
fusion with
Aspergillus kawachii
genetically engineered brain TR12 and
Aspergillus
niger
34309 as the original strain. Then, with UV radiation mutation breeding, a
new strain with high enzyme activity was obtained. The acid resistance
'
-amylase
mL
1
, respectively.
Trichoderma reesei
is able to synthesize a large amount of cellobiohydrolase (CBH)
and endoglucanase (ED). However, the vitality of cellobiase (CB) from
T. reesei
was low, and that from
Aspergillus niger
was high. To fully exploit the interspecific
complementary strength traits between two distant sources,
Trichoderma reesei
and
A. niger
, protoplast fusion could be researched for
T. reesei
and
A. niger
to obtain
fusants with two genera advantages.
But, in general, it is seldom reported that cellulase activity could be significantly
improved through integration technology. Crossbreeding methods are complex. In
the breeding of industrial microorganisms, it is rare to cultivate high-yield strains
used in production practice by transformation, transduction, or bonding recombinant
technology.
mL
1
and glucoamylase activity reach 91.2 U
and 3216.2 U
11.2.3.3
Protein-Directed Evolution Technology
Under the condition that it is not necessary to knowledge spatial structure and
catalytic mechanisms of a biocatalyst, mutations could be introduced into genes
encoding biocatalyst through various mutagenesis methods. A mutated gene could
be reassembled to improve gene evolution efficiency by a recombinant method such
as DNA shuffling or the like. These transformed genes can produce a biocatalyst
with better performance. Then, a mutant strain fulfilling the requirements can be
obtained in a larger library using a filter or a selection method.
Wu et al. [
39
] carried out directed mutation for ED
Cel1
B from extremely heat-
resistant
Thermotoga maritime
by an error-prone PCR method. For the recombinant
plasmid pET-20b-Cel12B carrying the
Cel1
2B gene, error-prone PCR conditions
