THESIS
2012
xv, 172 p. : ill. ; 30 cm
Abstract
Background: The complete hydrolysis of cellulosic residues requires three types of
cellulases: endoglucanases [1], exoglucanases [2], and cellobiases (Cels) [3]. Before 2012,
only two cellobiase (cel) genes from Cellulomonas biazotea (C. biazotea), cba and cba2,
were sequenced and studied. The characterization and understanding of the functional roles of
C. biazotea Cels may enable their utilization in various industrial applications, such as
cellulosic waste management, and the production of biofuels and biomaterials. Consequently,
my research studies have focused on the cloning and characterization of novel cel genes from
C. biazotea, and the expression of recombinant Cels in Escherichia coli (E. coli).
Methodology/Principal Findings: To search for novel cels in C. biazotea,...[
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Background: The complete hydrolysis of cellulosic residues requires three types of
cellulases: endoglucanases [1], exoglucanases [2], and cellobiases (Cels) [3]. Before 2012,
only two cellobiase (cel) genes from Cellulomonas biazotea (C. biazotea), cba and cba2,
were sequenced and studied. The characterization and understanding of the functional roles of
C. biazotea Cels may enable their utilization in various industrial applications, such as
cellulosic waste management, and the production of biofuels and biomaterials. Consequently,
my research studies have focused on the cloning and characterization of novel cel genes from
C. biazotea, and the expression of recombinant Cels in Escherichia coli (E. coli).
Methodology/Principal Findings: To search for novel cels in C. biazotea, a shotgun
approach and PCR-based probing were employed. From the experiments, four cel candidates,
designated cba3, cba4, cba5 and Cbgh3d, were obtained. The cba3 [3], cba4 and cba5 genes
were fully characterized, and their encoded enzymes were demonstrated to be bona fide
secretory Cels, despite their absence of classical secretion signals. Biochemical studies on
Cba, Cba3, Cba4 and Cba5 support that Cba, Cba3 and Cba4 are multifunctional enzymes,
which exhibit not only Cel, but also hemicellulase activities. The results suggest that the four
Cels are unlikely to be functionally redundant in C. biazotea. The strategy of extracellular
production (excretion) of cellulases has many advantages over its intracellular counterpart in
biotechnological applications [4]. In my dissertation, I have also introduced and discussed an
innovative approach of employing histidine-tag sequences to greatly enhance the secretion of
recombinant Cba, Cba3 and Cba4 in E. coli.
Conclusions/Significance: A total of six cel genes have been cloned by our research group
from C. biazotea, of which cba3, cba4 and cba5 have been characterized in detail in my
studies. Moreover, an innovative approach is introduced for the efficient excretion of non-classical
secretory proteins in E. coli.
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