Identification of carbohydrate
phosphorylases from
Euglena gracilis and Ochromonas sp.
for β-1,3-glucan production
Sue Kuhaudomlarp
Email: [email protected]
Biological Chemistry, John Innes Centre, UK
β-1,3-glucan phosphorylase
Pi
Pi
Laminaribiose or
longer β(1,3) oligos
Pi
Laminaribiose phosphorylases
•  Synthesis of laminaribiose only
(DP = 2) from glucose as an
acceptor
•  Activity and sequences were
described. Belong to GH94
family
Laminaribiose α-Glucose-1-P
(acceptor)
(sugar donor)
β-1,3-oligoglucan
phosphorylases
•  Synthesis of β-1,3-glucan with
DP >= 3 from laminaribiose
•  Activity has been described but no
sequence candidates
β-1,3-glucan phosphorylase:
potential application
Β-1,3glucan
•  Potent immune stimulant
•  Anti tumour growth
•  Drug/vaccine adjuvant
Possible enzymatic synthesis
Laminaribiose phosphorylase
and β-1,3-oligoglucan
phosphorylase
Objective
β-1,3-oligoglucan phosphorylases
Sequence discovery
Ochromonas danica
Evolution and the presence of the
candidates in other species
Euglena gracilis
•  In vitro characterisation
Sequence discovery through
transcriptome mining
Subject 1:
20,000 seq
Queries
21 seq
BLAST 1
Ochromonas
transcripts
GH94
Subject 2:
30,000 seq
Queries
3 hits
BLAST 2
Euglena
transcripts
3 hits
4 hits
•  Localisation prediction (TargetP)
•  Postranslational modification such
as glycosylation
•  Transmembrane domain prediction
Phylogenetic analysis of the candidates
shows that they are not GH94 members
Internal strips
External strips
Acknowledgements
Prof. Rob Field
Dr Ellis O’Neill
Field group
IB carb organisers
Royal Thai
Government Scholarship