Projects per year
Abstract
Enzymatic treatment of biomass is an environmentally friendly method to obtain a range of value-
added products, such as biofuels, animal feed or food ingredients. The objective of this PhD study
was to biocatalytically produce biofunctional food ingredients – human milk oligosaccharides
decorated with sialic acid from casein glycomacropeptide obtained from dairy side streams. In
addition, the biocatalysts employed in this study,
i.e.
, a sialyltransferase and a sialidase, were
subjected to protein engineering to alter the enzyme’s regioselectivity and to improve hydrolase
activity, respectively.
A recombinant
Pasteurella multocida
sialyltransferase (EC 2.4.99.-), namely PmST, exhibiting
promiscuous trans-sialidase activities was examined. The enzyme catalysed α-2,3- and α-2,6-
sialylation of lactose using either 2-
O
-(p-nitrophenyl)-α-
D
-
N
-acetylneuraminic acid or casein
glycomacropeptide as a sialyl donor. This is the first study reporting α-2,6-trans-sialidase activity of
this enzyme. Using response surface design allowed identification of two differently optimised
conditions for PmST-catalysed production of 3'-sialyllactose and 6'-sialyllactose, giving maximum
yields of 2.8 mM and 3.3 mM from casein glycomacropeptide (9 mM bound sialic acid),
respectively. The
k
cat
/
K
m
value for PmST catalysing 6'-sialyllactose synthesis using 3'-sialyllactose
as donor was 23.2±0.7 M
-1
s
-1
. Further, the enzyme was capable of catalysing synthesis of both 3'-
and 6'-sialylated galactooligosaccharides with use of galactooligosaccharides as acceptors.
Secondly, we examined the regioselectivity of five designed mutants of PmST catalysing synthesis
of 3'- and 6'-sialyllactoses using casein glycomacropeptide and lactose as substrates. The mutants
PmST
E271F
, PmST
R313Y
and PmST
E271F/R313Y
preferentially catalysed synthesis of 3'-sialyllactose
over 6'-sialyllactose. The best mutant PmST
E271F/R313Y
for α-2,3-trans-sialylation gave a maximum
3'-sialyllactose yield of 4.5 mM from casein glycomacropeptide (9 mM bound sialic acid). Another
mutant PmST
P34H
displayed a distinct preference for 6'-sialyllactose synthesis throughout the
reaction, though the total sialyllactose yield was consistently and significantly lower than that using
the wild type enzyme. PmST
P34H
had a 980-fold increase in α-2,6-sialyltransferase activity
compared to the wild type enzyme, while its α-2,3-sialyltransferase activity was almost abolished.
The
k
cat
/
K
m
value for PmST
P34H
catalysing 6'-sialyllactose synthesis using 3'-sialyllactose as donor
was 31.2 M
-1
s
-1
. Moreover, both the wild type enzyme and PmST
P34H
were capable of catalysing
the hydrolysis and transfer of α-2,6 bound sialic acid.
Original language | English |
---|
Publisher | DTU Chemical Engineering |
---|---|
Number of pages | 154 |
ISBN (Print) | 978-87-93054-43-1 |
Publication status | Published - 2014 |
Fingerprint
Dive into the research topics of 'Enzymatic production of human milk oligosaccharides'. Together they form a unique fingerprint.Projects
- 1 Finished
-
Enzymatic Production of Human Milk Oligosaccharides
Guo, Y. (PhD Student), Mikkelsen, J. D. (Main Supervisor), Willer, M. (Supervisor), Pinelo, M. (Examiner), Schols, H. (Examiner) & Rasmussen, S. K. (Examiner)
15/08/2010 → 24/09/2014
Project: PhD