Successes in engineering glucansucrases to enhance glycodiversification - Carbohydrate Chemistry: Chemical and Biological Approaches - page 639

As described earlier in this manuscript, GS are known to glucosylate
more or less eciently a wide range of hydroxylated molecules. The ef-
ficiency of this non-natural reaction will strongly depend on how well the
exogenous acceptor is able to compete with the natural products re-
sulting from sucrose utilization and present in the reaction media. The
development of biocatalysts with enhanced or even new glucosylation
capabilities is thus challenging and has only scarcely been considered.
Mutagenesis experiments applied to the amylosucrase from Neisseria
polysaccharea have been recently used to improve the acceptor glucosy-
lation rate using sucrose as donor. In this report, seven residues were
targeted for saturation mutagenesis and 133 mono-variants were
constructed. The eciency of the glucosylation of 2-acetamido-2-deoxy-a-
D -glucopyranoside was remarkably enhanced by some single mutants to
reach conversion degrees over 90% which were accompanied by up to
130-fold enhanced catalytic eciency. This library was also assayed
toward another molecule, the methyl-a- L -rhamnopyranoside, non-recog-
nized by the wild-type enzyme. Fifteen mutants harbouring mutations at
either positions 228 or 290 displayed a remarkable novel specificity
toward the exogenous acceptor and they were able to glucosylate it with
remarkable conversion degrees going up to 44% after protein purifi-
cation. 113,114 The pairwise recombination of these mutations was further
applied and led to the isolation of several double mutants displaying a
spectacular 400-fold improvement of their catalytic eciency toward
2-acetamido-2-deoxy-a- D -glucopyranoside. 115 A structure-based engin-
eering of this amylosucrase using stability change predictions was recently
reported. 22 The reshaping of subsite 1 was investigated leading to the
evaluation of 57 single mutants. Some variants were found more stable
than the wild-type enzyme or able to synthesize a series of oligosaccharides
with original distribution profile. Protein engineering appears to be
noteworthy for developing novel glucansucrases with unprecedented
properties that need to be further investigated in this purpose.
4 Screening methods applied to detect novel or
improved glucansucrases
Enzyme engineering strategies often require generating an important
number of variants and positive selection pressure usually have to be
applied for isolating original mutants from large libraries. High-
throughput screening methods have been developed to assay glucansu-
crase libraries. Automated protocols have been proposed for the isolation
of amylosucrase variants with improved biochemical properties such as
thermostability or organic solvent tolerance or activity. These methods
enabled already the identification of mutants generated by random
mutagenesis approach using a human mutagenic DNA polymerase dis-
playing up to 25-fold increased activity at 50 1C as compared to the par-
ental NpAS. 116,117 Variants with increased activity (up to 5-fold) were
identified using automated reducing sugar assay. 83 Methods for the
isolation of effective transformants displaying desired properties have
also been developed. A pH sensitive high-throughput screening has been