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type polymer which is mainly composed of a-1,6 linkages. Mutagenesis at
position S268 was also applied what led to variants with modified
transglucosylation properties. Variants mainly synthesizing short-chain
oligosaccharides, among which mutants S268D and S268R, lost their
capacity to synthesize polymers probably by facilitating the release of
acceptor reaction products as well as the attack of GTFR-Glc intermediate
by water and acceptor substrates.
109
Amino acid residues located near the active site of DSRBCB4 dex-
transucrase from Leuconostoc mesenteroides B-1299CB4 were targeted by
site-directed mutagenesis. The triple mutant V532P/E643N/V644S was
constructed and showed to add a-1,3 and a-1,4 linkages onto the a-1,6
linked glucan mainly synthesized by the wild-type enzyme. The V535I/
S642N mutations were subsequently introduced by directed mutagenesis.
The resulting variant was shown to synthesize an increased amount of
a-1,4 linkages (up to 11%) compared to the triple-mutant.
110
The molecular basis of glucan production of amylosucrase from Neis-
seria polysaccharea was also investigated through the engineering of
subsites
þ
1,
þ
2,
þ
3 residues. An outstanding variant R446A was isol-
ated which synthesizes twice as much insoluble glucan as the parental
enzyme while generating lower amounts of by-products.
38
The use of glucansucrases might be impaired by uncontrolled levels of
sucrose hydrolysis which is a minor reaction occurring naturally. The
transglucosylation/hydrolysis ratio has thus to be considered when op-
timizing performances of GS in order to limit their side reactions. Hybrid
reuteransucrases have been constructed in this way. Some variants ex-
hibiting strongly increased transglucosylation activities were obtained by
targeting specific regions of the catalytic domains. The conversion of
sucrose into oligosaccharide and polysaccharide products was increased.
Two variants namely GTFO-A-dN and GTFO-dN-RS, derived from the
reuteransucrase GTFO from Lactobacillus reuteri ATCC55730, displayed
reduced hydrolysis activities as compared to their parental enzyme but
maintained the a-1,4 linkage specificity.
111
The transglucosylation/hy-
drolysis ratio appears thus to be controllable through rational protein
engineering.
Though mainly used for glucan synthesis, glucansucrases can also be
exploited for synthesizing glucoconjugates or short oligosaccharides,
polymerization being then undesired for this purpose. The polymer-
ization capacity of GS may be modulated to favour the production of
short glucosylated products. The gene encoding the amylosucrase from
Neisseria polysaccharea was submitted to high-rate segmental random
mutagenesis. A segment coding for amino acids involved in substrates
recognition was targeted. Two residues, D394 and G396 were identified as
playing a major role in the control of generated chain length. Indeed, by
substituting these residues with bulky amino acids, the synthesis of short
oligosaccharides (up to three units) was shown to be favoured. Steric
hindrance introduced at these sites was thought to interfere with the
elongation of amylose chains. The variants selected were specific of the
synthesis of mono and di-glucosylated products and could be considered
for the limited glucosylation of acceptors.
112
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