Role of Intestinal Microbiota in Ulcerative Colitis

Table 1
List of enzymes.
Enzyme Source pH optimum Temperature
optimum
J. Holck et al. / Process Biochemistry 46 (2011) 1039–1049 1041
EC number Family Reference
Pectin lyase Aspergillus nidulans 7.0 n.a. 4.2.2.10 PL 1 [29]
�-Galactosidase-1 Aspergillus niger 4.0–4.5 60 ◦ C 3.2.1.23 GH 35 Megazyme
�-Galactosidase-2 Kluyveromyces lactis n.a. n.a. 3.2.1.23 GH 2 n.a.
�-Galactanase Aspergillus niger 4.0–4.5 50 ◦ C 3.2.1.89 GH 53 Megazyme
�-Arabinofuranosidase Aspergillus aculeatus 4.0–4.5 30 ◦ C 3.2.1.55 – [50]
�-Arabinanase Aspergillus aculeatus 5.5 30 ◦ C 3.2.1.99 GH 43 [50]
RGI lyase Bacillus licheniformis 8.0–8.5 60–65 ◦ C 4.2.2.– – Unpublished data
phy to obtain the structural elements and avoids the use of organic
solvents. To assess the possible effect of different chain length, the
study includes evaluation of the biological activity of homogalacturonan
oligosaccharides DP4 and DP5 based on their capacity to
alter the ratio between Bacteroidetes and Firmicutes, which represent
the dominant bacterial phyla in the human intestine. This
is assessed by in vitro bacterial fermentation using human faecal
samples.
2. Materials and methods
2.1. Substrate
Sugar beet pectin was obtained from Danisco A/S (Nakskov, Denmark). The
pectin had been prepared by sequential acid extraction with nitric acid from sugar
beet pulp, involving removal of insoluble cellulose, ultrafiltration, and diafiltration
with a 50 kDa cutoff essentially as described by Buchholt et al. [1], except that precipitation
in isopropanol was replaced by spray drying. Degree of methoxylation
and acetylation were determined to be 59% and 20%, respectively.
2.2. Chemicals
Ammonium formate, Trizma-HCl, sodium acetate, acetic acid, hepes, manganese
chloride, d-glucuronic acid, d-galactose, d-arabinose, d-fucose, l-rhamnose monohydrate,
d-galacturonic acid monohydrate, and �-cyano-4-hydroxy-cinnamic acid
were purchased from Sigma–Aldrich (Steinheim, Germany) d-glucose and d-xylose
were purchased from Merck (Darmstadt, Germany). Trifluoroacetic acid was from
Riedel-de Haën (Seelze, Germany). High resolution Source 15Q anion exchange resin
and chromatography columns were obtained from GE Healthcare Biosciences (Uppsala,
Sweden). All chemicals used were analytical grade.
2.3. Enzymes
Pectin lyase was produced by fermentation essentially as described by Stratton
et al. [28]. The Pichia pastoris clone transformed with the pectin lyase gene
AN2569.2 was obtained from the Fungal Genetic Stock Center as described by
Bauer et al. [29]. Rhamnogalacturonase I from Bacillus licheniformis was cloned and
expressed in P. pastoris (unpublished results). Galactanase and �-galactosidase-1
were purchased from Megazyme (Bray, Co. Wicklow, Ireland), �-galactosidase-2
was purchased from Sigma–Aldrich (Steinheim, Germany). 1 and 2 denotes the origin
of gene, either Aspergillus niger (1) or Kluyveromyces lactis (2). Arabinanase and
�-l-arabinofuranosidase were obtained from Novozymes (Bagsvaerd, Denmark)
(Table 1).
2.4. Biorefining strategy
The sugar beet pectin was separated into homogalacturonan (HG) and rhamnogalacturonan
I (RGI) by sequentially applying monocomponent enzymes addressed
towards certain structures in the pectin molecule, followed by removal of the
released oligomers by membrane separation (Fig. 1B). Pectin lyase was employed
to catalyze the cleavage of methoxylated homogalacturonan by �-elimination. This
gave rise to a double bond in between carbon 4 and 5 of galacturonic acid in the nonreducing
end of one of the cleavage products. In turn, this allowed the assessment of
the molar level of released products in the HG permeate by UV spectroscopy. Next,
enzymes active towards arabinan and galactan side chains of the RGI were added
to catalyze the release of arabinose and galactose oligomers and monomers. These
products were separated into the side chain (SC) permeate (Fig. 1). Finally RGI lyase
was added to the retentate to catalyze the cleavage of the remaining RGI backbone
by �-elimination (Fig. 1) again introducing a detectable double bond. The released
RGI oligomers were separated into the RGI permeate. Any unprocessed material was
found in the final retentate.
2.5. Membrane reactions and separation
All reactions and separations were performed in a 200 mL stirred membrane
reactor model 8200 (Millipore, Billerica, MA) equipped with a 3 kDa regenerated
cellulose membrane (Millipore, Billerica, MA), connected to compressed nitrogen
for flux regulation. The reactor contents were mixed by magnetic stirring using a
RCT basic magnetic stirrer (IKA, Germany). Temperature control was maintained
using a Julabo ED5 water bath (Julabo, Germany).
2.6. Pectin lyase treatment
A 3% (w/w) SB pectin solution in 50 mM Tris buffer pH 8 was treated with 0.5%
E/S (enzyme/substrate) pectin lyase and incubated at 50 ◦ C for 15 h in a stirred and
thermostatically controlled reactor. The reaction mixture was transferred to the
membrane reactor and filtration was performed at 50 ◦ C and 2 bars. The retentate
was diafiltrated twice in 0.7 sample volumes of water. All permeate was pooled and
stored at 5 ◦ C until further use.
2.7. Side chain degrading treatment
A 1.4% (w/w) RGI retentate solution in 50 mM acetate buffer pH 5 was treated
with a mixture of 0.2% �-galactosidase-1, 4.7% �-galactosidase-2, 0.2% galactanase,
0.8% arabinofuranosidase, and 0.8% arabinanase (all percentages in % E/S) and incubated
in a membrane reactor at 45 ◦ C for 16 h. The enzymes were dosed according to
a tentative estimate of the available concentration of the specific structural substrate
element and the individual enzyme activity. Filtration was performed at 50 ◦ C and 2
bars. The retentate was diafiltrated once in 1 sample volume of water. All permeate
was pooled and stored at 5 ◦ C until further use.
2.8. RGI lyase treatment
Prior to the enzymatic treatment, RGI lyase was incubated in 100 mM MnCl2
for 0.5 h for activation [30]. A 0.75% (w/w) RGI backbone solution in 50 mM Hepes
buffer pH 7.8 was treated with 0.5% E/S RGI lyase and incubated in a membrane
reactor at 60 ◦ C for 2 h. Filtration was performed at 60 ◦ C and 2 bars. The retentate
was diafiltrated twice in 1 sample volume of water. All permeate was pooled and
stored at 5 ◦ C until further use.
2.9. Alkaline deesterification
Prior to ion exchange chromatography, methoxyl- and acetyl groups were
removed from pectic substances using cold alkaline conditions. The low temperature
was used in order to prevent spontaneous �-elimination [31]. Sample temperature
was lowered to approx. 5 ◦ C and cold 2 M NaOH was added under stirring until pH
13 and a final concentration of 40 mM NaOH was reached. Samples were incubated
for 24 h. After incubation pH was adjusted using 1 M acetate buffer pH 6.5 and 2 M
HCl. Samples were diluted to conductivity under or equal to the conductivity of the
ion exchange buffer.
2.10. Ion exchange chromatography
Anion separation was performed at room temperature with an ÄKTA purifier
100 work station equipped with a P-900 pump, UV-900 monitor, pH/C monitor,
and Frac-950 fraction collector, all controlled by UNICORN software. A HR16/10
column with a 23 mL packed bed of high resolution Source 15Q was used for separation.
The elution of the unsaturated oligosaccharides was monitored by the UV
absorption at 235 nm and subsequently quantified by use of the molar extinction
coefficient 4600 M −1 cm −1 [32]. Baseline correction was made with a blank run. Elution
was performed at a flow rate of 10 mL/min with water and ammonium formate
as eluents.
Before injection the column was equilibrated with 20 mM ammonium formate
for two column volumes (CV). After injection the column was washed with 20 mM
ammonium formate for three CV, followed by elution with a gradient specified
below. After elution the column was regenerated with 1000 mM ammonium formate
for two CV and with water for three CV. To obtain the oligomers in the HG
permeate, a sample was injected on the column and eluted using a linear gradient