Successes in engineering glucansucrases to enhance glycodiversification - Carbohydrate Chemistry: Chemical and Biological Approaches - page 637

or chain length are enzyme-dependent and may be modulated through
protein engineering.
Mutagenesis experiments of Q937 and D569 positions of glucansucrase
GTF-I from Streptococcus downei showed that single amino acid substi-
tutions can impact the glucan linkage. 100,101 Another enzyme, dex-
transucrase DSRS from Leuconostoc mesenteroides NRRL B-512F, has been
submitted to site-directed mutagenesis. Lysine residues were introduced
at the N-terminal end. Two single mutants, T350K and S455K, and the
corresponding double-mutant T350K/S455K were constructed. Their
products showed an enhanced amount of 1,6-linked Glcp going from 70
to 85% for the single mutants and the unusual presence of 2,6-linked
Glcp for the double-mutant. 102 Recent semi-combinatorial engineering of
this enzyme has also underscored the impact of amino acid mutations on
glucan structures and properties. Eight residues from the catalytic do-
main were targeted from sequence analysis and engineered using In-
corporating Synthetic Oligos via Gene Reassembling (ISOR) method. 103
Products obtained using the variants of a truncated DSRS (DSRS vardel
D4N) harbouring from one to four amino acid substitutions (F353T,
S512C, F353W, H463R/T464D/S512C, H463R/T464V/S512C, D460A/
H463S/T464L, D460M/H463Y/T464M/S512C) were analyzed and revealed
polymers differing in their a-1,3 linkage contents and their gel-like
properties in solution. This work provided a useful toolbox of glu-
cansucrases producing increasing amounts of a-1,3 linkages. 104
Reuteransucrase from Lactobacillus reuteri 121 GTF-A was also suc-
cessfully engineered. The role of N1134 located right next to the catalytic
residue D1133 was investigated and it was shown to be involved in both
product specificity and hydrolysis/transglucosylation ratio. Single mu-
tants at this position affected the total specific activity going from
45-75% loss for N1134Q, N1134G or N1134H up to two fold increase for
N1134A, N1134D and N1134S. 105 Another study on this enzyme converted
its reuteransucrase activity into a dextransucrase activity by increasing
the amount of a-1,6 linkage from

B B

35% up to 85% and decreasing the

amount of a-1,4 linkages from 5% for the quintuple

mutant P1026V/I1029V/N1134S/N1135E/S1136V. These results underline
the role of amino acid changes in enzyme mechanism and product
nature. 106
Bioengineering of glucansucrase GTF-180, an a-1,3/a-1,6 linkage syn-
thesizing enzyme, from Lactobacillus reuteri strain 180 was undertaken
leading to the creation of a triple mutant V1027P/S1137N/A1139S able to
synthesize a-1,4 linkages. Twelve other variants were also identified as
producing modified exopolysaccharides, some of them generating a-1,4
linkages. The products synthesized by these variants were analyzed and
showed discrepancies in their structural and physical properties such as
solubility, molecular weight or structure. 107,108
Specificity of glucansucrase GTF-R from Streptococcus oralis was also
modulated through random mutagenesis of a conserved motif sur-
rounding the transition state stabilizer. 109 A triple mutant R624G/V630I/
D717A was identified as producing a mutan type polymer harbouring
mainly a-1,3 linkages by opposition to the wild-type produced dextran

B 45% down to B