My master thesis presentation.

1. Xylanases and xylanase
inhibitors in barley
Exploring the potential for improved grain quality
by Mik Marstrand
Enzyme and Protein Chemistry, DTU
Supervisors: Christine Finnie
Abida Sultan
13-09-2011

2. 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

3. 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

4. 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

5. 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

6. Testing for xylanase activity
Picture from Dornez et al. [2006] - Wheat-Kernel-Associated
Endoxylanases Consist of a Majority of Microbial and a Minority of Wheat
Endogenous Endoxylanases.
13-09-2011

7. 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

8. Previous studies
 Example of a xylanase assay
◦ Total absorbance is needed to calculate
activity
Xylanase measurementtwotwo barley
Xylanase activity of of barley
cultivars
cultivars
0.14
14
0.12
12
0.1
10
0.08
Activity 8
EU / grams
OD590 Absorbance for extracts
0.06
6
Total absorbance with control
40.04 enzyme
20.02
0 0
Cabaret Barke
Cabaret Barke Cabaret
Cabaret Barke Barke
Rinsing Rinsing
Rinsing Rinsing Washing
Washing Washing
Washing
Liquid Liquid
Liquid Liquid Liquid
Liquid Liquid
Liquid
13-09-2011

9. Analyzing differences between
cultivars
 Big difference in xylanase activity in
new ”Cabaret” cultivar
Activity of two cabaret cultivars from
different years of harvest
0.08
0.07
0.06
0.05
Activitiy
0.04
EU/grams
0.03
0.02
0.01
0.00
Cabaret 2009 Cabaret 2010 #1 Cabaret 2010 #2
Weather during June July August June July August
growth 09/10 2009 2009 2009 2010 2010 2010
Average 13,9 17,2 17,4 13,9 18,7 16,2
temperature ⁰C
Rainfall (mm) 64 86 68 52 69 124
Hours of sun 280 220 200 248
13-09-2011 247 151

10. Analyzing differences between
cultivars
 Inhibitory assay containing all of the
available barley cultivars
Inhibition in percentage (%) between different cultivars
%
100
90
80
70
60
50
40
30
20
10
0
13-09-2011

11. Analyzing differences between
cultivars
 New xylanase assay using Cabaret 2010
◦ Popov to determine same concentration of
proteins
◦ A noteable variance between from cultivars
Xylanase assay with inhibition in % the various
barley cultivars
100
90
80
70
60 Frozen and thawed
% OD590 after added extract
50
inhibitors
40 Fresh extract
30
20
10
0
13-09-2011

12. Analyzing differences between
cultivars
 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
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13. Analyzing differences between
cultivars
13-09-2011

14. Analyzing differences between
cultivars Spot nr
(cultivar)
Accession # Protein identified Organism #peptides
(MS/MS
Score Theor /
exp MW
Theor /
exp pI
Sequence cov
(%)
 Mass peptides)
2 gi|326497617 predicted protein Hordeum vulgare 5(1) 100 35992 6,74 16
Spectrometry (Cab) / 37600 / 7,21
16 gi|157093712 PR17c precursor Hordeum vulgare 4(2) 121 24431 9,32 22
(Cab) subsp. vulgare / 20100 / 8,85
85 gi|31615809 Chain A, Crystal Wheat 3(2) 126 30494 8,27 10
(Scar-S) Structure Of / 32600 / 7,74
 26 spots from Xylanase Inhibitor
Protein (Xip-I)
each gel excised 85
(Scar-S)
gi|326497365 predicted protein Hordeum vulgare
subsp. vulgare
3(2) 137 33664
/ 32600
7,68
/ 7,74
13
for MS 113
(Scar-S)
gi|326488467 predicted protein Hordeum vulgare
subsp. vulgare
9(1) 157 41632
/ 39600
8,84
/ 8,31
23
113 TC200929 Ferredoxin-NADP Zea mays (Maize) 128 26049 7
 14 significant hits, (Scar-S) reductase precursor / 39600 / 8,31
9 from barley 124 TC206610 Translated in frame 71 44743 6,3
Scar-S) 5 / 18000 / 8,87
 1 desired hit on 204
(Scar-E)
gi|157830301 Chain A, Crystal
Structure Of Barley
Hordeum Vulgare 9(1) 88 34260
/ 39600
6,51
/ 7,60
24
Grain Peroxidase 1
spot 85, XIP
216 gi|475602 BiP isoform A glycine max 50 73633 5,11 9
(Scar-E) (soybean) / 19200 / 7,58
237 gi|326519983 predicted protein Hordeum vulgare 8(2) 139 26569 6,97 36
(Scar-E) subsp. vulgare / 22400 / 8,24
238 gi|168052170 predicted protein Physcomitrella 7 83 48213 5,11 26
(Scar-E) patens subsp. / 23900 / 7,96
patens
247 gi|21693553 beta-1,3-glucanase II Hordeum vulgare 6 87 35227 9,01 26
(Scar-E) subsp. vulgare / 36100 / 8,87
255 gi|326533906 predicted protein Hordeum vulgare 74 12
(Scar-E) subsp. vulgare
257 gi|75262903 Basic endochitinase Rye Seed Chitinase 4(1) 76 28683 8,82 16
(Scar-E) C / 40100 / 9,16
292 gi|157093712 PR17c precursor Hordeum vulgare 3(1) 104 24431 9,32 17
(Scar-E) subsp. vulgare / 20400 / 9,20
295 gi|255079504 predicted protein Micromonas sp. 12 80 107418 8,93 30
(Scar-E) 13-09-2011 RCC299 / 25500 / 9,05

15. Analyzing differences between
cultivars
 One xylanase inhibition protein identified on
Scarlett Sejet
◦ Able to identify the the same protein on different
cultivars
100
90
80
70
% OD590 60
after added 50
inhibitors 40
30
20
10
0
13-09-2011

16. 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
13-09-2011

17. 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 engineering
13-09-2011

18. 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

19. Thank you for listening!
Questions?
13-09-2011