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

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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

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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

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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

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Previous studies

Example of a xylanase assay

◦ Total absorbance is needed to calculate

activity

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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

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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.

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Thank you for listening!

Questions?

13-09-2011