Download Progress report Dec.2010

Identification and heterologous expression of enzymes
degrading xyloglucan in biomass
State Research Institute of Genetics and Selections of Industrial
Microorganisms (GosNIIgenetika)
Project leader Dr. Sergei Yarotsky
Technische Universität München, Dept. Microbiology
Project leader Prof. Wolfgang Liebl
Project is funded by the Ministry of education and Science of Russia
and the German Federal Ministry of Education and Research
Dr. Oksana Berezina
GosNII Genetika
Xyloglucan is one of the major structural polysaccharides
in primary plant cell walls
Primary plant cell wall, type I
pectin
crosslinks of
polygalacturonic acids
extensin
xyloglucan
cellulose microfibril
Xyloglucan connects cellulose microfibrils
by hydrogen bonding
Dr. Oksana Berezina
GosNII Genetika
German Jurgens, 29 April 2011
In some plants xyloglucan is a storage polysaccharide
Tamarindus indica is a tropical Asian evergreen tree
«Köhler’s Medizinal-Pflanzen», 1887
Dr. Oksana Berezina
GosNII Genetika
German Jurgens, 29 April 2011
Xyloglucan structure
Tamarind seed xyloglucan consist of cellotetraose units that have
three branched backbone residues and a fourth unbranched
backbone residue
Gal
Gal
2
2
α↓
Xyl
1
Xyl
1
6 β
α↓
Xyl
1
6 β
α↓
6
β
β
14
1 4
α↓
Xyl
1
6 β
2
Xyl
Xyl
1
α↓
1
α↓
α↓
α↓
Xyl
Gal
1
1
1
6 β
α↓
6
β
β
14
1 4
α↓
Xyl
1
Xyl
1
6 β
α↓
1
6 β
α↓
6
β
β
14
1 4
Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc →
14
14
14
14
14
14
Xylose may be derivatized with arabinose, and galactose with fucose
depending on the plant type
Dr. Oksana Berezina
GosNII Genetika
German Jurgens, 29 April 2011
Xyloglucan-degrading enzymes
GH74, GH5, GH12, GH31, GH44, GH7, GH16
GH74: endo-glucanases XEGs and exo-gluconases OXG-RCBH
Gal
Gal
α↓
α↓
Xyl
1
Xyl
1
6 β
α↓
Xyl
1
6 β
α↓
6
β
β
14
1 4
α↓
Xyl
1
6 β
2
Xyl
Xyl
1
α↓
1
α↓
2
2
Xyl
Gal
1
α↓
1
1
6 β
α↓
6
β
β
14
1 4
α↓
Xyl
1
Xyl
1
6 β
α↓
1
6 β
α↓
6
β
β
14
1 4
Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc → Glc →
14
14
14
14
XEGs
randomly hydrolyze
glycosidic bonds
of unbranched glucose
residues
Dr. Oksana Berezina
GosNII Genetika
German Jurgens, 29 April 2011
14
14
OXG-RCBHs
release two glucose residues
from reducing end of the main
chain
The first Glu must be
unbranched.
Project goals
Identification, isolation and heterologous production in
microbial hosts of new efficient enzymes degrading
xyloglucan in plant biomass
Xyloglucan utilization is part of the complete utilization of biomass, especially
of byproducts from agriculture, food and feed production, and forestry, as part
of a biorefinery process
The novel enzymes may be also utilized:
• for the partial digestion of xyloglucan to defined oligosaccharides; those may
be assayed for their effect on human, animal or plant cells e.g. as
nutraceuticals
• for the definition of the structure of such oligosaccharides; concomitantly the
mechanism of xyloglucan degradation will be elucidated
Dr. Oksana Berezina
GosNII Genetika
1
Project tasks
• New genes encoding xyloglucan degrading enzymes will be isolated
from bacteria and fungi
• These genes will be expressed in appropriate hosts
• Novel recombinant xyloglucanases will be purified and characterized
• Best strains will be optimized the for enhancing xyloglucanase
production
• Structure and properties of the oligosaccharides produced from
different types of xyloglucan by enzymatic hydrolysis will be
investigated
• Efficiency of xyloglucan hydrolysis with new recombinant enzymes in
combination with commercial enzymes will be evaluated
Dr. Oksana Berezina
GosNII Genetika
1
Tasks of the Russian side for the first stage of
the project
• Preparation of xyloglucanes of different structure
(galacto-, arabino- and fuco-xyloglucan) from various plant
sources
• Screening VKPM collection for xyloglucan degrading
fungi strains
Dr. Oksana Berezina
GosNII Genetika
1
Preparation of xyloglucan from tamarind seeds
Fruits and
seeds of
Tamarindus
indica
Tamarind seeds
contain 70 % of
xyloglucan
Suspension of
homogenized
seeds
Tamarind seed xyloglucan is used as a model substrate in study of
enzymatic properties of xyloglucanases
Dr. Oksana Berezina
GosNII Genetika
1
Preparation of xyloglucan from tamarind seeds
1
2
3
4
5
1. C (-)
2. C(+) XG Megazyme
3. Suspension of homogenized
seeds
4. deproteinized and defatted XG
5. Purified tamarind seed XG
1
2
3
1. C (-)
2. Corn starch
3. Purified tamarind
seed XG
Dr. Oksana Berezina
GosNII Genetika
2
VKPM - Russian National Collection of Industrial Microorganisms
VKPM contains 607
fungi strains
25 strains utilizing plant
biomass were selected
7 of them produced
thermostable enzymes
hydrolyzing xyloglucan at
50-60°С
Dr. Oksana Berezina
GosNII Genetika
3
Effect of temperature on the activity of xyloglucan degrading
enzymes
Strain
Reaction temperature
50 °С
Activity
U/ml
60 °С
Activity
U/ml
70 °С
Activity
U/ml
1,4
1,68
1,36
0,99
1,17
0,93
0,8
0,87
0,66
0,84
0,93
0,65
1
2
3
4
Dr. Oksana Berezina
GosNII Genetika
1
Thermostability of the fungi xyloglucan degrading enzymes
Strain
Incubation
temperature,
оС
Incubation time, min
20
40
60
Residual activity, %
1, 2, 3
50
100
100
100
1
60
100
88
80
2
3
1, 2, 3
60
60
70
100
100
0
86
80
0
78
70
0
Dr. Oksana Berezina
GosNII Genetika
Conclusions
• Xyloglucanes of different structure were isolated from
tamarind and nasturtium seeds and apple fruits
- Tamarind and nasturtium seeds contain storage galactoxyloglucane.
- Apple fruits contain structural arabinofucogalactoxyloglucan
• Fungi strains producing thermostable xyloglucan
degrading enzymes were selected from VKPM collection
Dr. Oksana Berezina
GosNII Genetika
1
Future plans
• New genes encoding fungi xyloglucan degrading
enzymes will be isolated and expressed in Pichia pastoris
• Novel recombinant xyloglucanases will be purified and
characterized
• Different types of xyloglucans will be used as a substrate
for study of the enzyme properties
Dr. Oksana Berezina
GosNII Genetika
1