xylanase presentation for thesis
DESCRIPTION
My master thesis presentation.TRANSCRIPT
Xylanases and xylanase
inhibitors in barley
Exploring the potential for improved grain quality
13-09-2011
by Mik Marstrand
Enzyme and Protein Chemistry, DTU
Supervisors: Christine Finnie
Abida Sultan
Overview
◦ Introduction
◦ Theory
◦ Aim of project
◦ Characterizing various barley cultivars Differences in xylanase activity and inhibition
◦ Analyzing differences between cultivars 1D and 2D immunoblotting
Mass spectrometry
◦ Conclusion
◦ Future prospects
◦ Acknowledgements
◦ Questions
13-09-2011
Introduction
Barley is a cereal grain, which has many
uses
◦ Animal fodder, human
consumption, malting
Widely adaptable crop
◦ Can be grown as a summer or winter
crop, depending on environment
◦ Naked or hulled
◦ Continually exposed to pathogens
Major industry
13-09-2011
Theory
Xylanase◦ A group of enzymes that break down
components of the cell wall matrix of plants
◦ Both from kernel and microorganisms on outer layers of kernel
◦ Easier degradation of cell wall leads to easier uptake of nutrition in animal feed
Xylanase inhibitor◦ Inhibits bacterial and/or fungal xylanases
◦ Functions as plant defense
◦ 3 different inhibitors – TAXI, XIP and TLXI
13-09-2011
Aim of project
To characterize xylanase activity and
inhibition amongst different barley
cultivars in order to identify the
differences, and potentially combining
these traits into an ideal cultivar
13-09-2011
13-09-2011
Picture from Dornez et al. [2006] - Wheat-Kernel-Associated
Endoxylanases Consist of a Majority of Microbial and a Minority of Wheat
Endogenous Endoxylanases.
Testing for xylanase activity
Characterization of barley
cultivars Major variance in xylanase activity
between cultivars
◦ Up to tenfold difference in measured
activity between 26 cultivars
Cultivar ”Cabaret” showed highest
amount of xylanase activity
Inhibitors from all cultivars obtained
and measured against each other
13-09-2011
Previous studies
Example of a xylanase assay
◦ Total absorbance is needed to calculate
activity
13-09-2011
0
2
4
6
8
10
12
14
Cabaret Rinsing Liquid
Barke Rinsing Liquid
Cabaret Washing
Liquid
Barke Washing
Liquid
OD590
Xylanase measurement of two barley cultivars
Absorbance for extracts
Total absorbance with control enzyme
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Cabaret Rinsing Liquid
Barke Rinsing Liquid
Cabaret Washing
Liquid
Barke Washing
Liquid
ActivityEU / grams
Xylanase activity of two barley cultivars
Analyzing differences between
cultivars
Big difference in xylanase activity in
new ”Cabaret” cultivar
13-09-2011
Weather during
growth 09/10June
2009
July
2009
August
2009
June
2010
July
2010
August
2010
Average
temperature ⁰C
13,9 17,2 17,4 13,9 18,7 16,2
Rainfall (mm) 64 86 68 52 69 124
Hours of sun 280 220 200 248 247 151
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Cabaret 2009 Cabaret 2010 #1 Cabaret 2010 #2
ActivitiyEU/grams
Activity of two cabaret cultivars from different years of harvest
Analyzing differences between
cultivars
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Inhibitory assay containing all of the
available barley cultivars
0
10
20
30
40
50
60
70
80
90
100
%
Inhibition in percentage (%) between different cultivars
Analyzing differences between
cultivars
New xylanase assay using Cabaret 2010
◦ Popov to determine same concentration of
proteins
◦ A noteable variance between the cultivars
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0
10
20
30
40
50
60
70
80
90
100
% OD590 after added inhibitors
Xylanase assay with inhibition in % from various barley cultivars
Frozen and thawed extract
Fresh extract
Analyzing differences between
cultivars
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1D immunoblot of 9 different cultivars using 3 different xylanase inhibitors – TAXI, XIP, TLXI◦ Visible difference in amount of inhibitor between cultivars
◦ 3 cultivars chosen for further examination by 2D immunoblots
Analyzing differences between
cultivars
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Analyzing differences between
cultivars
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Spot nr
(cultivar)
Accession # Protein identified Organism #peptides
(MS/MS
peptides)
Score Theor /
exp MW
Theor /
exp pI
Sequence cov
(%)
2
(Cab)
gi|326497617 predicted protein Hordeum vulgare 5(1) 100 35992
/ 37600
6,74
/ 7,21
16
16
(Cab)
gi|157093712 PR17c precursor Hordeum vulgare
subsp. vulgare
4(2) 121 24431
/ 20100
9,32
/ 8,85
22
85
(Scar-S)
gi|31615809 Chain A, Crystal
Structure Of
Xylanase Inhibitor
Protein (Xip-I)
Wheat 3(2) 126 30494
/ 32600
8,27
/ 7,74
10
85
(Scar-S)
gi|326497365 predicted protein Hordeum vulgare
subsp. vulgare
3(2) 137 33664
/ 32600
7,68
/ 7,74
13
113
(Scar-S)
gi|326488467 predicted protein Hordeum vulgare
subsp. vulgare
9(1) 157 41632
/ 39600
8,84
/ 8,31
23
113
(Scar-S)
TC200929 Ferredoxin-NADP
reductase precursor
Zea mays (Maize) 128 26049
/ 39600
7
/ 8,31
124
Scar-S)
TC206610 Translated in frame
5
71 44743
/ 18000
6,3
/ 8,87
204
(Scar-E)
gi|157830301 Chain A, Crystal
Structure Of Barley
Grain Peroxidase 1
Hordeum Vulgare 9(1) 88 34260
/ 39600
6,51
/ 7,60
24
216
(Scar-E)
gi|475602 BiP isoform A glycine max
(soybean)
50 73633
/ 19200
5,11
/ 7,58
9
237
(Scar-E)
gi|326519983 predicted protein Hordeum vulgare
subsp. vulgare
8(2) 139 26569
/ 22400
6,97
/ 8,24
36
238
(Scar-E)
gi|168052170 predicted protein Physcomitrella
patens subsp.
patens
7 83 48213
/ 23900
5,11
/ 7,96
26
247
(Scar-E)
gi|21693553 beta-1,3-glucanase II Hordeum vulgare
subsp. vulgare
6 87 35227
/ 36100
9,01
/ 8,87
26
255
(Scar-E)
gi|326533906 predicted protein Hordeum vulgare
subsp. vulgare
74 12
257
(Scar-E)
gi|75262903 Basic endochitinase
C
Rye Seed Chitinase 4(1) 76 28683
/ 40100
8,82
/ 9,16
16
292
(Scar-E)
gi|157093712 PR17c precursor Hordeum vulgare
subsp. vulgare
3(1) 104 24431
/ 20400
9,32
/ 9,20
17
295
(Scar-E)
gi|255079504 predicted protein Micromonas sp.
RCC299
12 80 107418
/ 25500
8,93
/ 9,05
30
Mass
Spectrometry
26 spots from
each gel excised
for MS
14 significant hits,
9 from barley
1 desired hit on
spot 85, XIP
Analyzing differences between
cultivars
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One xylanase inhibition protein identified on Scarlett Sejet◦ Able to identify the the same protein on different
cultivars
0102030405060708090
100
% OD590 after added inhibitors
Conclusion
Variation in xylanase activity and inhibition
could cause anomalies in product qualities
Many factors influencing xylanase, such as
genotype, growing site, year-to-year
differences etc.
One inhibitor protein identified in this
project, which confirms the method used
By identifying more of these proteins, a
more reliable comparison of inhibitors can
be made in between different cultivars
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Future prospects
Further characterizing cultivars as shown in
this project
Exploring the microbial growth on the
kernels – identifying
microorganisms, optimizing growth
conditions for them
Controlled field experiments to determine
optimal environment for desired xylanase
activity and inhibition
Identifying genes for xylanase inhibitors for
genetic engineering13-09-2011
Acknowledgements
Abida Sultan
Christine Finnie
Birgit Andersen
Sejet Plant Breeding
Antibodies obtained from◦ Kurt Gebruers -Laboratory of Food
Chemistry, Katholieke Universiteit
Leuven, Belgium.
13-09-2011
Thank you for listening!
Questions?
13-09-2011