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Enzyme tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry University of Toronto Supervisor: Professor Emma Master Committee members: Professor Bradley Saville and Professor Alexander Yakunin 1

Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

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Page 1: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

Enzyme tools and carbohydrate tools for

bioproducts development

Ruoyu Yan, PhD

Department of Chemical Engineering and Applied Chemistry

University of Toronto

Supervisor: Professor Emma Master

Committee members: Professor Bradley Saville and Professor

Alexander Yakunin

1

Page 2: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

Chem. Soc. Rev., 2014, 43, 7485-7500 Biomaterials

Enzymatic modification

Deconstruction or combustion

BioethanolLevulinic acids

Heat and power

Formic acids

Page 3: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

HXGum exudates

XSeaweed

GAXOat Spelt

AGXSpruce

GXBeechwood

AXWheat bran

Xylans represent abundant renewable resources for the development of

bioproducts.

3

Page 4: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

GH115 enzymes are the only known enzymes that target GlcA/MeGlcA from high

molecular-weight xylans.

Endo-(1→4)-β-xylanase

Acetylxylan esterase

α-L-Arabinofuranosidase

(1→2)-α-Glucuronidase

Ferulic acid esterase

Sidegroup chemistry Prebiotic activity, rheology, solubility, material attributes

4

Page 5: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

In nature, carbohydrate-active enzymes are produced in organisms from various habitats including harsh environmental

conditions

5

http://australianfungi.blogspot.ca

http://www.bugoutservice.com/termite-protection

https://en.wikipedia.org/wiki/Iceberg

http://wallpapersin4k.net

Page 6: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

6

Marine bacteriumSaccharophagus degradans

Alkaliphilic bacteriumAmphibacillus xylanus

Salt marsh cord grass in the Chesapeake Bay

Composts of manure with grass and rice straw in Japan

Tolerance in high salt condition?

Tolerance in alkaline condition?

Select bacteria from unique habitats

https://microbewiki.kenyon.edu https://microbewiki.kenyon.edu

Page 7: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

7

ATCGCTAGTACGGCATGCACTGTGCATAATTCCAGTACGTTTGGGATCG

ATCGCTAGTACGGCATGCACTGTGCATAATTCCAGTACGTTTGGGATCG

Source organism

Obtain gene of interest through genomic DNA or direct synthesis

Amplification

Insert into plasmid

Transform E. coli cells

Genetically modified E. coli cells

Bacterial culture

Induce protein expression

Enzyme purification

Produce enzymes using biotechnology

His

His

His

His

Page 8: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

Both enzymes demonstrated activity towards glucuronoxylans and oligomers

with preference towards internally substituted residues.

kcat=29.2 ± 1.6 s -1

Km=5.0 ± 1.0 mM kcat /Km=5.8 s-1mM-1

U4m2XX (mM)

0 10 20 30 40 0

500

1000

1500

2000

Activity (U

/µm

ol pro

tein

)

(C)

SdeA

gu11

5A

AxyA

gu115A

kcat(app)=535.8 ± 25.2 s -1

Km(app)=70.9 ± 5.2 mg/mL

K’m(app)=53.2 ± 3.9 mM

kcat(app) /K’m(app)=10.1 s-1mM-1

Activity (U

/µm

ol pro

tein

)

00 20 40 60 80

BEX concentration (mg/mL) 20000

15000

10000

5000

0 0 20 40 60

MeGlcA substituent concentration (mM)

(A)

Activity (U

/µm

ol pro

tein

)

kcat=72.7 ± 6.5 s -1

Km=2.4 ± 0.53 mM kcat /Km=30.3 s-1mM-1

0

1000

2000

3000

4000

Activity (U

/µm

ol pro

tein

)

XU4m2XX (mM) 0 2 4 6 8

(D)

Activity (U

/µm

ol pro

tein

)

Activity (U

/µm

ol pro

tein

)

Activity (U

/µm

ol pro

tein

)

)

BEX concentration (mg/mL) 20000 )

XU4m2XX, aldopentauronic acid U4m2XX, aldotetrauronic acid

BEX, beechwood glucuronoxylan

8

J. Biol. Chem. 291 (2016) 14120–

Enzyme Microb. Technol. 104 (2017) 22–2

Page 9: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

3 4 5 6 7 8 9 10 10.5 11-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

pH

(B)

3 4 5 6 7 8 9 10 10.5 11-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

pH

(A)

1 %

BE

X0

.5 %

BE

X1

% O

SX

0.5

% O

SX

Me

Glc

A c

on

ce

ntr

ati

on

(mg

/mL

)

1 %

BE

X0

.5 %

BE

X1

% O

SX

0.5

% O

SX

Me

Glc

A c

on

ce

ntr

ati

on

(mg

/mL

)AxyAgu115A demonstrated better performance in alkaline condition

AxyAgu115A performance was higher than SdeAgu115A, particularly at pH values above 9.0.

Increase in substrate solubility in alkaline condition increased substrate accessibility.

9

AxyAgu115A-24h

SdeAgu115A-24h

Enzyme Microb. Technol. 104 (2017) 22–2

Grey bars, beechwood glucuronoxy

White bars, Oat spelt xylan

Page 10: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

Consistent with the marine origin of SdeAgu115A,

salt activation of enzyme activity was observed for SdeAgu115A but not

AxyAgu115A

SdeAgu115A

Rela

tive a

cti

vit

y (

%)

120

100

80

60

40

20

0 0 300 600 900 1200 1500NaCl concentration (mM)

0

0

0

0

0

0

0

AxyAgu115A

Rela

tive a

cti

vit

y (

%)

120

100

80

60

40

20

0 0 500 1000 1500 2000 2500

NaCl concentration (mM)

10

J. Biol. Chem. 291 (2016) 14120–

Enzyme Microb. Technol. 104 (2017) 22–2

Page 11: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

AxyAgu115A displayed higher activity towards complex xylans compared to SdeAgu115A

Polymeric substrates

Activity (μmol product/min/μmol enzyme)

SdeAgu115A AxyAgu115A

Beechwood glucuronoxylan

2470 ± 704700 ± 100

Spruce arabinoglucuronoxylan

917 ± 65630 ± 60

Oat spelt glucuronoarabinoxylan

24 ± 1 501 ± 12

(C) 6.5 Å -14.0 kcal/mol

• Accommodation of complex xylans was consistent with docking analysis that predicted accessibility of AxyAgu115A to branched xylo-oligosaccharides.

(D) 11.2 Å -11.7 kcal/mol

11Enzyme Microb. Technol. 104 (2017) 22–28.

Page 12: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

12

Thin layer chromatography

Scanning transmission X-ray microscopy

LC-MS/MSColorimetric analysis

Glucuronoxylan + H2O GH115

Xylan + GlcA

GlcA + NAD+ + H2O

Uronate dehydrogenase Glucarate + NADH + H+

NADH is measured by the increase in absorbance at 340 nm.

Standard enzyme assay used to measure GH115 activity: Enzyme coupling method

https://www.spectrumchemical.comhttp://unicorn.mcmaster.ca/research/stxm-intro/polySTXMintro-all.html

library/pierce-protein-methods/overview-mass-spectrometry.html

Page 13: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

Pros and cons of existing techniques

13

Separation techniques

Pros Cons Ref

Size exclusion

Identified analytes above 9 kDa

Compounds with the same size co-elute

1

Ion chromatography

Separated neutral and acidic oligomers

Substitution position of MeGlcA was not confirmed

2

HPAEC-PAD Effective separation of oligomers

Poor compatibility with mass spectrometry due to high salt concentration

3

Capillary electrophoresis

High resolution

Samples typically need derivitation

4

• Differentiate diverse characteristics.

• Be label-free.

• Require minimum sample pretreatment.

• Be compatible with mass spectrometry.

• Establish an in-house ms/ms library.

• Be capable of analyzing industrially relevant samples containing a mixture of sugars.

• Be high throughput.

My goal was to develop a LC-MS/MS method that could

Page 14: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

An LC-MS/MS method was developed; over 70 sugars derived from lignocellulose with different sizes,

polarity, acidity and linkage positions were identified.

Total ion/mass chromatogram

Total ion/mass chromatogram

Relative abundance distribution

Relative abundance distribution

In-house libraryIn-house library Quantitative assessment

Quantitative assessment

X1

X2

X3

X4

X5

X6

X1

X2 X3 X4

X6

X5

Retentiontime(min)

Retentiontime(min)

Inte

nsi

tyIn

ten

sity

Compound

Prediction of precursor ions

Posi vemode[M+Na]+

[M+H]+

[2M+Na]+

[M+K]+

[M+NH4]+

[M-OH-]+

Nega vemode[M+COOH]-

[M-H+]-

[2M-H+]-

[M+Cl]-

Calculate exact mass

Extract mass chromatograms

Calculate peak area

Calculate relative abundance

h c d $4 0 $( ++) $

h c d $3 0 $( +) $

h c d $1 5 $(- ) $

( A ) $ ( B ) $ ( C ) $N C E 4 0 % + +

N C E 3 0 % +

N C E 1 5 % -

MS/MSspectraof U4m2XXtarge ngprecursor [M-H]- withvaryingcollisionenergy

Mode Precursors NCE Arb X1 X2 X3 X4 X5 X6 GlcA U4m2XX XU4m2XX

Positive

[M+Na]+

30% - - - - -

40% ND2. + +

50% + + + + + + + ND1 ++ ++

[M+H]+

10% + +

15% ND + + + + + + ND ++ ++

30% ND ++ ++ ++ ++ ++ ++ ND

[M-OH]+ 10% + +

15% ND1 ++ ++

Negative

[M+COOH]- 15% + + + + + + + ND ND ND

[M-H+]-

15% + + + + + + + - -

30% + + +

40% ++ ++ ++

0

1E+09

2E+09

3E+09

4E+09

5E+09

6E+09

7E+09

8E+09

0.2 0.4 0.6 0.8 1

Peak

are

aofm

ass

chro

mato

gra

m

Concentra on(mM)

xylose

xylobiose

xylotriose

xylotetraose

xylopentaose

xylohexaose

Xylose (R2=0.988) Xylobiose (R2=0.985) Xylotriose (R2=0.957) Xylotetraose (R2=0.934) Xylopentaose (R2=0.955) Xylohexaose (R2=0.997)

(A)

14

Page 15: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

Application of the established LC-MS/MS method to characterize

industrial sample before and after enzyme treatment

Compound UntreatedEnzymes cocktail containing AxyAgu115A

Xylose ++ +++Xylobiose ++ ++Xylotriose +++ ++++

Xylotetraose +++ ++++

Xylopentaose +++ ++++

Xylohexaose +++ ++++

MeGlcA + ++++U4m2X +++ ++U4m2XX +++ ++XU4m2XX +++ ++

15

Page 16: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

Engineering significance

• SdeAgu115A and AxyAgu115A have the potential to tailor xylans with high molecular weight.

• The unique tolerance properties of these two enzymes can benefit industrial applications with corresponding conditions.

• AxyAgu115A was active on xylan recovered from liquid hot water extraction of mixed hardwood.

• An in-house LC-MS/MS method was developed to characterize the hemicellulosic fraction before and after enzyme treatment, which has the potential to be applied to other plant fibers.

16

Page 17: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

17

University of Toronto, Canada

A. Gaona, Prof. B. Saville, T. Oakes, F. Razeq, Prof. E. Master, Dr. A. Starostine

University of Helsinki, Finland Prof. M. Tenkanen

University of Toronto, Canada

Dr. W. Wang, Prof. E. Master Chalmers University of Technology,

Sweden Prof P. Gatenholm

University of Guadalajara, Mexico

Prof G, Toriz University of Helsinki, Finland

Prof. M. Tenkanen

University of Toronto, Canada

Dr. W. Wang, Dr. T. Vuong, R. Leo, X. Xu, Prof. A. Savchenko, Prof. E. Master

The Structure Biology Center, United States

Dr. B. Nocek Chalmers University of Technology,

Sweden

Prof. P. Gatenholm University of Guadalajara, Mexico

Prof. G, Toriz University of Helsinki, Finland

Prof. M. Tenkanen

Enzyme characterization

Synergy

Application

Action of a GH115 α-glucuronidase from Amphibacillus xylanus at alkaline condition promotes release of 4-O-methylglucopyranosyluronic acid subunits of glucuronoxylan and arabinoglucuronoxylan

Publication Biochemical and structural characterization of a five-domain GH115 alpha-glucuronidase from the marine bacterium Saccharophagus degradans

2-40T

Manuscript to be submitted Synergistic action of accessory hemicellulases from glycoside hydrolase families GH115, GH62, and GH51 towards spruce arabinoglucuronoxylan

Ongoing Project LC-MS/MS method development and application of GH115 α-glucuronidase AxyAgu115A, to process glucuronoxylan released through hot-water extraction of mixed hardwood prior to steam-explosion

University of Toronto, Canada

Prof. E. Master Mississippi State University, United

States Prof. D. Jeremic

Niagara University, United States

Prof. R. Goacher Canadian Light Source Inc, Canada

C. Karunakaran (Science manager)

Fiber characterization

Publication Direct and up-close views of plant cell walls show a leading role for lignin-modifying enzymes on ensuing xylanases

Professor E. Master Department of Chemical

Engineering, University of

Toronto, Canada

Professor D. J eremic Department of Sustainable

Bioproducts, Mississippi State

University, United States

Professor P. Gatenholm Department of Chemical and

Biological Engineering, Chalmers

University of Technology, Sweden

Professor B. Saville Department of Chemical

Engineering, University of

Toronto, Canada

Dr. T. Vuong Research associate, BioZone,

University of Toronto, Canada

Dr. W. Wang Research associate, BioZone,

University of Toronto, Canada

Ms. C. Hong Research technician, BioZone,

University of Toronto, Canada

Dr. A. Starostine, Manager of Mass Spec Facility,

BioZone,

University of Toronto, Canada

Professor R. Goacher Department of Biochemistry,

Chemistry & Physics at Niagara

University, United States

Professor M. Tenkanen Department of Food and

Environmental Sciences, University

of Helsinki, Finland

Ms. F. Razeq MSc. candidate, Department of

Chemical Engineering,

University of Toronto, Canada

Ms. A. Goana PhD candidate, Department of

Chemical Engineering,

University of Toronto, Canada

Prof. G. Toriz University of Guadalajara,

Mexico

Prof. A. Savchenko Department of Chemical

Engineering, University of

Toronto, Canada

C. Karunakaran Science manager

Canadian Light Source Inc,

Canada

AcknowledgementsAcknowledgements

Page 18: Enzyme tools and carbohydrate tools for bioproducts ... tools and carbohydrate tools for bioproducts development Ruoyu Yan, PhD Department of Chemical Engineering and Applied Chemistry

References

1. M.J. Deery, E. Stimson, C.G. Chappell, Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides, Rapid Commun. Mass Spectrom. 15 (2001) 2273–83. doi:10.1002/rcm.458.

2. T. Ohbuchi, T. Takahashi, N. Azumi, M. Sakaino, Structual analysis of neutral and acidic xylooligosaccharides from hardwood kraft pulp, and their utilization by intestinal bacteria in vitro, Biosci. Biotechnol. Biochem. 73 (2009) 2070–6. doi:10.1271/bbb.90260.

3. M.A. Kabel, W.H. Heijnis, E.J. Bakx, R. Kuijpers, A.G.J. Voragen, H.A. Schols, Capillary electrophoresis fingerprinting, quantification and mass-identification of various 9-aminopyrene-1,4,6-trisulfonate-derivatized oligomers derived from plant polysaccharides, J. Chromatogr. A. 1137 (2006) 119–26. doi:10.1016/j.chroma.2006.10.058.

4. J. Khandurina, A. Guttman, High resolution capillary electrophoresis of oligosaccharide structural Isomers, Chromatographia. 62 (2005) s37–s41. doi:10.1365/s10337-005-0606- 8.

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