Chemistry Reference
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acceptors, the GBD-CD2 enzyme was able to catalyze transglucosylation
reactions and interestingly, it synthesized solely a-1,2 linkages.
92
The
mode of branching of this enzyme was investigated through the analysis
of a 1.5 kDa grafted dextran and revealed a stochastic branching
process.
93
In order to provide better insights on the sequence-structure-function
relationships of glucansucrases, attempts have been made to diversify
their three-dimensional organization by varying enzyme scaffolds. In vitro
constructions of chimeric glucansucrases have been attempted. Selected
sites of glucansucrases DSRS and DSRT5 from Leuconostoc mesenteroides
NRRL 512-F have been exchanged and six chimeric variants were con-
structed. Upon analysis, their products were found to differ from the
glucans synthesized by their parental enzyme in terms of solubility and
linkage specificity.
94
Fusion proteins DXSR harbouring dextransucrase
and dextranase activities were generated and successfully expressed in
E. coli and used for the production of linear isomalto-oligosaccharides
(IMO) further increased by the introduction of metal ions to reach a
30-fold increase in the production of IMO as compared to a mixture of the
two enzymes.
95
Another fusion protein namely DSXR has also been
constructed and the expression level was optimized using response sur-
face methodology to overcome the low productivity of DXSR but con-
serving similar properties.
96
Fusion protein involving glucansucrases
were also considered for transgenic investigations. Dextransucrase DSR-S
from Leuconostoc mesenteroides fused to the chloroplastic ferredoxin
signal peptide was used to transform two potato genotypes (cv. Kardal
and the amylose-free mutant (amf)). Dextrans were detected in potatoes
tuber juices from transformants of both species but with a two fold in-
creased concentration for Kardal. No dextran could be detected inside the
starch granule, however the morphology of this latter was altered prob-
ably due to an accumulation of dextran in the tuber juices.
97
A truncated
mutansucrase GtfICAT without starch-binding domain derived from GtfI
was expressed in Kardal. The production of mutan adhering to starch
granules was detected but not incorporated in the starch granules.
98
A
fusion protein comprising GtfICAT and a starch-binding domain (SBD) at
either N- or C- terminal end was introduced in two genetically different
potato backgrounds. The fusion protein was detected in starch granules.
Starches from the plant expressing GtfICAT contained less mutan than
GtfI expressing plant. However, the granule morphology was altered in
both genetic backgrounds. These results underline the fact that
expression of engineered glucansucrases can be used to interfere with
starch biosynthetic pathway in plants.
99
3.2 Semi-rational and rational engineering
Glucansucrases are mainly involved in the synthesis of a wide variety of
gluco-oligosaccharides and high molecular weight glucopolymers. De-
termination of the three-dimensional structures of glucansucrases have
enabled to investigate the role of the different subsites and glucan
binding domains of the enzymes. Intensive work has shown that prop-
erties of the glucan synthesized such as specificity of the osidic linkages
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