Applications of Gluco-oligosaccharide Oxidase

By

Ben MacCormick

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science

Department of Chemical Engineering University of Toronto

© Copyright by Ben MacCormick (2016)

ii

Applications of Gluco-oligosaccharide Oxidase

By

Ben MacCormick

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science

Department of Chemical Engineering University of Toronto, 2016

Abstract: The nascent study of carbohydrate-active enzymes is now influencing a variety of

industries at the commercial scale, particularly in processing lignocellulosic biomass for

the production of fuels, materials, and other commodities. These enzymes are highly

specific and can modify sugars with selectivity unmatched by chemical processes. Our

group has worked extensively with gluco-oligosaccharide oxidase (GOOX), and feel the

enzyme’s oxidative activity would prove desirable in many new and existing applications

where other commercialized carbohydrate oxidases are employed. The work presented

here has demonstrated the improved characteristics of an immobilized fusion enzyme,

CtCBM22A_GOOX-Y300A, on insoluble polysaccharide fibre, with potential

applications in food additives, protein purification, and aldonic acid production. A second

study has successfully employed GOOX oxidation as part of a synthetic pathway to

generate a clickable monomer from glucuronoxylan capable of forming a carbohydrate-

based polytriazole polymer; a process which could be extended to include neutral

hemicelluloses such as galactoglucomannan.

iii

Acknowledgements I would first like to thank my supervisor Dr. Emma Master for her constant support,

advice, and attention. I could not have found a better person to oversee my graduate

work, and your style of open communication and eagerness to collaborate is something I

will always strive to emulate in the future.

I would also like to thank all the members of the Master lab for sharing their knowledge

and friendship with me, you are an extremely smart group of people. In particular, I want

to express my most sincere gratitude to Dr. Thu Vuong, who has guided and mentored me

since my first day, and has since fielded my endless questions with great patience and

wisdom. In addition, I must thank all BioZone staff and students for creating a fantastic

environment to learn and share ideas, especially Susie – I’ve always admired your ability

to handle so many things while finding the time to have a laugh and enjoy yourself.

To my parents, Joanne and Keith, I will be eternally in debt (hopefully only figuratively)

for the last 26 years of encouragement, inspiration, and love. To my sister, Hilary, I will

always look up to you as an icon of hard work and dedication; you have set the bar pretty

high, thanks a lot. Finally to my girlfriend, Sarah, what can I say - you are my second half

and without you I would fall apart, and your entire family is an unending source of

happiness and generosity. You are all at the centre of my life, my thoughts, and my

future, and for that I love you.

iv

Table of Contents 1.0 Introduction .............................................................................................................................. 1  

1.1 Biomass to bioproducts: harnessing plant made polymers .................................................... 1  1.2 Canadian “bio-based” market ............................................................................................... 3  1.3 Research hypotheses .............................................................................................................. 5  1.4 Research objectives ................................................................................................................ 6  

2.0 Overview and Literature Survey ............................................................................................. 7  2.1 Plant products ........................................................................................................................ 7  

2.1.1 Cell wall components ...................................................................................................... 7  2.1.1.1 Cellulose ................................................................................................................................ 8  2.1.1.2 Hemicellulose ........................................................................................................................ 8  2.1.1.3 Lignin .................................................................................................................................. 10  

2.1.2 Biomass processing ....................................................................................................... 12  2.1.2.1 Pretreatment ......................................................................................................................... 12  2.1.2.2 Fractionation ........................................................................................................................ 14  

2.1.3 Major applications of plant products ............................................................................ 16  2.1.3.1 Carbohydrate based products .............................................................................................. 16  2.1.3.2 Lignin based products ......................................................................................................... 17  

2.2 Carbohydrate active enzymes (CAZymes) ............................................................................ 18  2.2.1 Auxiliary activity (AA) class ........................................................................................ 19  

2.2.1.1 FAD cofactor ....................................................................................................................... 20  2.2.1.2 FAD/heme cofactor ............................................................................................................. 22  2.2.1.3 Copper radical cofactor ....................................................................................................... 22  

2.2.2 Carbohydrate binding modules (CBMs) ....................................................................... 24  2.3 Applications of carbohydrate oxidases ................................................................................ 25  

2.3.1 Biosensors ..................................................................................................................... 25  2.3.2 Synthesis ....................................................................................................................... 25  2.3.3 Targeted binding via CBMs .......................................................................................... 26  2.3.4 Food applications .......................................................................................................... 26  

2.4 Green synthetic chemistry .................................................................................................... 27  2.4.1 Amide bond synthesis ................................................................................................... 27  2.4.2 Copper catalyzed azide-alkyne cycloaddition click chemistry (CuAAC) .................... 30  2.4.3 Alternative solvents for sugar chemistry ...................................................................... 31  

3.0 Chapter I - CBM-GOOX fusion production and studies .................................................... 33  3.1 Background .......................................................................................................................... 33  3.2 Methods ................................................................................................................................ 34  

3.2.1 Production and purification of CtCBM22A_GOOX-Y300A ....................................... 34  3.2.1.1 Enzyme production .............................................................................................................. 34  3.2.1.2 Ni-NTA purification of CtCBM22A_GOOX-Y300A ........................................................ 35  3.2.1.3 OSX binding of CtCBM22A_GOOX-Y300A .................................................................... 35  

3.2.2 Assessment of OSX binding stability ........................................................................... 36  3.2.2.1 Salt elution of OSX-bound protein ...................................................................................... 36  3.2.2.2 Long term binding stability ................................................................................................. 36  3.2.2.3 Effect of Ca2+ on binding .................................................................................................... 37  

3.2.3 Effects of CBM fusion and immobilization on enzyme performance .......................... 37  3.2.3.1 Enzyme activity assay ......................................................................................................... 37  3.2.3.2 Thermal stability .................................................................................................................. 37  3.2.3.3 H2O2 inhibition and stability ............................................................................................... 38  

3.3 Results and Discussion ......................................................................................................... 38  

v

3.3.1 Stable enzyme immobilization on insoluble hemicellulose .......................................... 38  3.3.2 Enhanced stability through CBM fusion and immobilization ...................................... 41  3.3.3 Immobilized CtCBM22A_GOOX-Y300A for aldonic acid production ...................... 43  

3.4 Conclusion ............................................................................................................................ 44  4.0 Chapter II - Carbohydrate-based polymer synthesis .......................................................... 45  

4.1 Background .......................................................................................................................... 45  4.2 Methods ................................................................................................................................ 46  

4.2.1 Analytical separation and detection of sugars .............................................................. 46  4.2.1.1 Thin layer chromatography (TLC) analysis ........................................................................ 46  4.2.1.2   High pressure liquid chromatography (HPLC) analysis .................................................. 46  

4.2.2 Preparative separation and purification techniques ...................................................... 47  4.2.2.1 Anion-exchange chromatography (AEC) ............................................................................ 47  4.2.2.2 Size exclusion chromatography (SEC) ................................................................................ 47  

4.2.3 Production of acidic xylo-oligosaccharides .................................................................. 47  4.2.4 Sugar oxidation ............................................................................................................. 48  

4.2.4.1 GOOX oxidation ................................................................................................................. 48  4.2.5 Amidation of carboxylic acids ...................................................................................... 48  

4.2.5.1 EDAC coupling ................................................................................................................... 48  4.2.5.2 DMT-MM coupling ............................................................................................................. 49  

4.2.6 CuAAC click reactions ................................................................................................. 49  4.3 Results and Discussion ......................................................................................................... 49  

4.3.1 Production of acidic xylo-oligosaccharides from BWX ............................................... 49  4.3.2 Amidation of sugar acids .............................................................................................. 52  

4.3.2.1 EDAC amide synthesis ........................................................................................................ 52  4.3.2.2 DMT-MM amide synthesis ................................................................................................. 54  

4.3.3 GOOX oxidation of modified oligosaccharides ........................................................... 60  4.3.4 Polymerization of bifunctional monomers .................................................................... 62  

4.4 Conclusion ............................................................................................................................ 64  5.0 Conclusions .............................................................................................................................. 66  6.0 Future Work ........................................................................................................................... 67  

6.1 Aldonic acid production via GOOX ..................................................................................... 67  6.2 Improved amide synthesis .................................................................................................... 68  6.3 Polytriazole characterization ............................................................................................... 69  6.4 Alternate monomer formats and linker chemistry ................................................................ 69  

APPENDIX A ................................................................................................................................... i  I. Polyamide synthesis ................................................................................................................... i  II. Functionalization of neutral sugar fragments ......................................................................... ii  

References ........................................................................................................................................ v  

vi

List of Tables Table 1: Summary of relevant AA family enzymes ........................................................ 20  Table 2: Summary of GOOX variants used in experiments ............................................. 35   List of Figures Figure 1: Chemical structure of cellulose .......................................................................... 8  Figure 2: Chemical structure of glucuronoarabinoxylan (GAX) ....................................... 9  Figure 3: Chemical structure of galactoglucomannan (GGM) ........................................ 10  Figure 4: Chemical structure of glucuronoxylan (GX) .................................................... 10  Figure 5: Chemical structures of primary monolignols ................................................... 11  Figure 6: General reaction mechanism for GOOX oxidation .......................................... 21  Figure 7: Amide synthesis through acid-amine condensation ......................................... 28  Figure 8: Water soluble coupling reagents ...................................................................... 29  Figure 9: CuAAC click reaction ...................................................................................... 30  Figure 10: NaCl elution test on OSX-bound GOOX ....................................................... 40  Figure 11: SDS-PAGE of bound and unbound fractions after Ca2+ exposure. .............. 41  Figure 12: Effects of CBM fusion and immobilization on GOOX thermostability ......... 42  Figure 13: H2O2 inactivation of GO and GOOX variants (free and immobilized) .......... 43  Figure 14: Model substrate for acidic hemicellulose fragments ...................................... 45  Figure 15: TLC monitoring of BWX digestion with GH10 ............................................. 50  Figure 16: TLC of SEC fractions from acidic BWX fragments after GH10 digest ......... 50  Figure 17: Confirmation of UXX product ....................................................................... 51  Figure 18: TLC analysis of EDAC coupling ................................................................... 53  Figure 19: EDAC intermediate and side product ............................................................. 54  Figure 20: TLC monitoring of pro-UXX synthesis via DMT-MM ................................. 55  Figure 21: By-Products of DMT-MM coupling .............................................................. 56  Figure 22: DMT-MM mediated coupling of pro-UXX-ox and azidopropylamine (AzP) 57  Figure 23: Formation of triazine adduct in DMT-MM coupling ..................................... 57  Figure 24: MS analysis of DMT-MM mediated C1 amidation ....................................... 59  Figure 25: MS/MS analysis of pro-UXX-azide (2) + [O] ................................................ 60  Figure 26: Relative activity of wt-GOOX on modified substrates .................................. 61  Figure 27: TLC analysis of pro-UXX oxidation via GOOX ........................................... 62  Figure 28: Preliminary TLC results for click polymerization ......................................... 63  Figure 29: Proposed synthetic pathway to clickable monomers from UXX ................... 65  Figure 30: TLC analysis of ox-UXX and EDA coupling ................................................... i  Figure 31: Pro-lactose synthesis and AEC of products .................................................... iii  Figure 32: Products from C1 amidation of lactobionic acid ............................................. iv  

vii

List of Abbreviations AA Auxiliary activity

AEC Anion exchange chromatography

AFEX Ammonia fiber explosion

Araf α-L-arabinofuranose

BMGY Buffered glycerol-complex media

BMMY Buffered methanol-complex media

BWX Beechwood xylan

BX Buffered exchange

CBM Carbohydrate binding module

CDH Cellobiose dehydrogenase

CS Culture supernatant

CuAAC Copper catalyzed azide-alkyne

cycloaddition

DES Deep-eutectic solvent

DMT Dimethoxytriazine

DMT-MM 4-(4,6-Dimethoxy-1,3,5-triazin-2-

yl)-4-methylmorpholinium chloride

DP Degree of polymerization

EDA Ethylenediamine

EDAC 1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide

FAD Flavin adenine dinucleotide

FPLC Fast protein liquid chromatography

Galp D-galactopyranose

GaO Galactose oxidase

GAX Glucoarabinoxylan

GGM Galactoglucomannan

GH Glycoside hydrolase

Glcp D-glucopyranose

GO Glucose oxidase

GOOX Gluco-oligosaccharide oxidase

GX Glucuronoxylan

HMF Hydroxymethylfurfural

HPAEC-

PAD

High pressure anion exchange

chromatography with pulsed amperometric

detection

HPLC High pressure liquid chromatography

IL Ionic liquid

LBA Lactobionic acid

LCC Lignin-carbohydrate complex

LPMO Lytic polysaccharide mono-oxygenase

Manp D-mannopyranose

MeGlcpA 4-O-methylglucuronic acid

MES 2-(N-morpholino)ethanesulfonic acid

MnCO Carbohydrate oxidase from Microdochium

nivale

NMM N-methylmorpholine

OSX Oat spelt xylan

P2Ox Pyranose-2-oxidase

PDH Pyranose dehydrogenase

pro-UXX Propargyl-UXX

pro-UXX-ox C1 oxidized propargyl-UXX

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

SEC Size exclusion chromatography

SME Small to medium sized enterprise

sulfo-NHS N-hydroxysulfosuccinimide

TLC Thin layer chromatography

Tris-HCl Tris(hydroxymethyl)aminomethane

UXX 4-O-methyl α-GlcpA (1,2)-β-Xylp (1,4)-β-

Xylp (1,4)-Xylp

X3 Xylotriose

Xylp D-xylopyranose

1

1.0 Introduction

1.1 Biomass to bioproducts: harnessing plant made polymers

Throughout our history, humans have maintained an extremely close relationship with

wood. While it may seem like a trivial connection, our survival and success as a species

has often hinged on the creative use of the materials around us. Wood fueled the fires of

our early ancestors, it provided spears and arrows for hunting, and materials to build

shelter. As our species advanced, so too did our application of wood; we discovered the

many unique properties of different tree species, some lent themselves to building ships,

while others were more suited to making paper. Hyperbole or not, one might say that

wood-based bioproducts have been at the center of some of the greatest cultural

revolutions in our history.

The bioproducts of today have taken on a much different and more diverse role in

society; we are now realizing the true value of biomass beyond its utility as a raw

material. The intricate network of polysaccharides and lignin that comprise the plant cell

wall can now be deconstructed and repurposed into fuels, platform chemicals, foods, and

new materials. These modern bioproducts have vastly expanded the potential uses of

biomass feedstocks at a time when the world is searching for more sustainable,

environmentally friendly ways to make the products we need, without relying on

petrochemicals.

Lignocellulosic biomass is a promising new feedstock entering the bioproducts

industry, and holds several advantages over traditional starch-based feedstocks. Since it is

generally inedible, lignocellulosic biomass does not compete with food production; an

2

obstacle that has greatly impeded the adoption of corn or sugarcane based bioethanol. It is

also available in enormous supply; the US alone produces approximately 1.3 billion dry

tons/yr of lignocellulosic biomass consisting mainly of forest woody biomass and

agricultural residues (Limayem & Ricke 2012). In addition to biofuels, the three major

constituents of lignocellulosic material are each being used in exciting new ways:

cellulose based products such as cellulose nanofibrils and nanocrystals (Deepa et al.

2015), hemicellulose products including xylitol (Roberto et al. 1991) and packaging films

(Ibn Yaich et al. 2015), and lignin based products such as vanillin and ferulic acid (Fache

et al. 2016), to name a few.

Despite the many advantageous aspects of lignocellulosic bioproducts, commercial

development has been relatively slow due to the challenges posed by the highly

recalcitrant property of the material. Nature has created an incredibly resilient matrix of

cellulose, hemicelluloses and lignin in the cell wall to maintain structural integrity and

prevent degradation. Fortunately for us, other organisms have been simultaneously

evolving equally complex techniques for attacking the plant cell wall, which we are now

beginning to harness for use in the precise modification or depolymerization of the cell

wall matrix. The identification, characterization, and engineering of cell wall degrading

enzymes is rapidly progressing, generating a diverse toolbox for researchers and industry

to use in the treatment of lignocellulosic materials. The Carbohydrate-Active Enzyme

Database (CAZy, www.cazy.org) has become a critical resource to the field, cataloging

over 100,000 protein sequences with activity on carbohydrate substrates. Using these

enzymes, we are now able to cut, join, debranch, or otherwise modify sugars with

precision and selectivity far superior to any chemical process.

3

The research presented here focuses on the Auxiliary Activity (AA) 7 family of the

CAZy database, namely gluco-oligosaccharide oxidase (GOOX), which catalyzes the

reducing-end oxidation of a variety of mono- and oligo-saccharides (Lin et al. 1991). This

enzyme has been extensively studied in our lab, and members of our group have

produced several mutants with enhanced substrate specificity and activity (Foumani et al.

2011). As examples of industrial applications of CAZymes continue to appear, there is an

increasing number of potential applications for GOOX across a range of industries. The

overall aim of my research was to identify and explore novel and promising applications

for GOOX, specifically to assess the feasibility of using GOOX for in situ applications

and in bioproduct synthesis pathways.

1.2 Canadian “bio-based” market

Canada has had a long history with the forestry and wood products industry,

beginning with the production and sale of timber, which eventually gave rise to a strong

pulp and paper industry in the mid-late 20th century. While these markets still exist today,

the industry as a whole is now attempting to respond to numerous national and global

demands for new high-value biobased products to be made from our rich biomass

resources. This global trend to develop safer, greener products from sustainable resources

is driven largely by the need to decrease fossil fuel dependence and reduce greenhouse

gas emissions, and with some of the largest biomass resources on Earth and a positive

political climate, Canada is well positioned to be at the leading edge of this industrial

evolution.

4

The Canadian bioproducts industry is currently estimated to be valued at

approximately $1.3 billion USD, of which about $228 million USD comes from non-

energy biobased chemical production (Johnston 2015). Globally, these markets are

rapidly expanding; with bioplastics and platform chemicals leading the growth at 23.7%

and 12.6% compound annual growth rates from 2009-2015, respectively (Natural

Resources Canada 2016).

Although the industry is growing quickly, the majority of participants are classified as

small to medium-sized enterprises (SME), which exist at the pilot or demonstration scale

of implementation. While segments such as bioethanol are closer to full-scale

commercialization, there remains a gap between development of high-value products and

technologies, and uptake by larger existing industry participants. To help address this

issue, the Canadian National Research Council (NRC) has developed several programs to

help facilitate partnerships and technology transfer between companies at the R&D stages

of development and large commercial corporations. For example, the Bio-based Specialty

Chemicals (BSC) program is designed to assist in technology improvement and

commercialization of the fastest growing bioproduct sectors, which account for 90% of

non-energy bioproduct revenues (Johnston 2015). Products from this segment are

generally high-value with niche applications, and include industrial chemicals, health and

cosmetic products, household products, and agricultural chemicals.

Despite commercialization challenges in this sector, there are several encouraging

examples of companies leading the way in the market. EcoSynthetix Inc. based in

Burlington, ON, has become a major manufacturer of starch-based polymers used to

replace existing petroleum based products, as has several lignin-based products in

5

development. With a current market capitalization of $91 million CAD, the company has

successfully made the leap to a large commercial enterprise. Bioamber Inc. is another

example of a fully commercialized (~$100 million USD) enterprise based in Montreal,

QC, specializing in the production of platform chemicals for polymer synthesis using

fermentation of C5 and C6 sugars from a variety of biomass feedstocks. Finally, Quebec

based CelluForce has begun large scale manufacturing of cellulose nanocrystals at their

plant in Windsor, QC, with a production capacity up to 300 ton/yr.

With the growing momentum of the bioproducts industry in Canada, and successful

development of several companies in the biomaterials and platform chemicals sector,

there is strong motivation to develop new technologies that can find a place in the

bioproducts mix in Canada. To this end, this project aims to develop new and improved

enzymatic and chemical processes for the modification and upgrading of biomass-derived

sugars, with end-point applications including materials, adhesives, enzyme screening, and

platform chemicals.

1.3 Research hypotheses

A. Fusion of Carbohydrate Binding Modules (CBMs) to GOOX

The activity and stability of GOOX can be improved through fusion to a CBM

domain, while allowing for selective immobilization on polysaccharide fibers.

These improvements to GOOX performance will strengthen its standing as a

superior replacement to glucose oxidase in existing and novel applications. The

ability to immobilize the enzyme on insoluble fiber can be exploited for protein

purification or fixed-bed reactors for oxidized sugar production.

6

B. Production of functionalized monomers via GOOX

Carboxylic acids can be generated at the reducing end of a variety of native or

chemically modified mono- and oligo-saccharides using GOOX. The respective

aldonic acid products can then be specifically modified to introduce new chemical

functionalities for use in polymerization, tagging, or the synthesis of glyco-

conjugates.

C. Bifunctional monomers from glucuronoxylan

The hydrolysis of glucuronoxylan generates a range of acidic sugar fragments

containing 4-O-methyl glucuronic acid units, which can be oxidized using GOOX

to generate diacids. Diacidic oligosaccharides can then be further functionalized

to generate monomers for polymerization.

D. Bifunctional monomers from galactoglucomannan

The hydrolysis of galactoglucomannan generates a range of neutral sugar

fragments carrying pendant galactose units. C6 hydroxyl groups of galactose can

be oxidized to carboxylic acids using galactose oxidase and chemical oxidation,

and C1 acids can be generated using GOOX. Diacidic oligosaccharides can then

be further functionalized to generate monomers for polymerization.

1.4 Research objectives

Assess effects of CBM fusion on GOOX performance and utility

I. Determine the impact of CBM fusion on GOOX activity and resistance to H2O2

and compare CBM-GOOX fusion performance to glucose oxidase

II. Investigate applicability of OSX-immobilization of CBM-GOOX fusion for

protein purification and oxidized sugar production

7

Determine effective pathways to generate bifunctional oligosaccharide monomers

from hemicellulose fractions

III. Synthesize bifunctional oligosaccharides capable of polymerization via click

chemistry using UXX as a model substrate for glucuronoxylan fragments.

IV. Synthesize bifunctional oligosaccharides capable of polymerization via click

chemistry using lactose as a model substrate for galactoglucomannan fragments.

2.0 Overview and Literature Survey

2.1 Plant products

2.1.1 Cell wall components

The composition of dry lignocellulosic materials can be generalized to the three

major constituents of the plant cell wall: cellulose (45-55%), hemicellulose (25-35%)

and lignin (20-30%) (Deutschmann & Dekker 2012). These proportions do not include a

small fraction of other compounds such as lipids, protein and trace minerals, and can vary

significantly between species. For example, woody biomass tends to have higher levels of

lignin and cellulose compared to most grass species which generally have elevated levels

of hemicellulose (Zhao et al. 2012). These components are arranged in a dense, tightly

packed matrix in the secondary cell wall, forming a composite material with extremely

high resistance to chemical and enzymatic degradation.

8

2.1.1.1 Cellulose

As the main load bearing component of the cell wall, cellulose exists as a highly

ordered linear polymer of β-1,4-linked D-glucopyranose units with a degree of

polymerization (DP) ranging from approximately 9,000 – 15,000 units (Pettersen 1984).

Figure 1: Chemical structure of cellulose

These long homopolymeric chains self-associate through hydrogen bonding to form

strong, highly crystalline fibers containing 30-36 individual chains (Vorwerk et al. 2004).

At the intermolecular scale, native cellulose typically contains both crystalline regions as

well as amorphous regions where the packing between β-glucan chains is less ordered.

This amorphous structure allows for increased water absorption, as well as increased

chemical and enzymatic penetration, and is therefore the desired state for biomass

processing. Several additional models of cellulose crystal structure have been designed

(cellulose I-1V) which can be generated through various chemical or physical treatment

and reflect slight changes in the chain organization within the fibrils (Karimi &

Taherzadeh 2016).

2.1.1.2 Hemicellulose

In the cell wall, hemicellulose acts as a comparatively flexible cross-linking

polymer, holding cellulose microfibrils in an ordered, yet elastic, matrix. This feature

allows for a high degree of control over the physical properties of the cell wall composite,

O

O

O

OO

OH

OH

OH

OOHO

OH

HOOH

HOOH

n

9

which explains the large amount of variability in hemicellulose structure across different

plant species, due to the many different physical and environmental demands facing the

organism.

Generally speaking, hemicellulose is a lower molecular weight heteropolymer

(average DP of 80-200) consisting of a β -1,4 linked backbone of pentose or hexose

sugars with various degrees of branching and/or acetylation. While there are many

variations, there are several predominant types of hemicellulose, each enriched in specific

plant groups. In grasses and other non-woody species, the main hemicellulose constituent

is glucuronoarabinoxylan (GAX, Figure 2), a branched polymer with a β-1,4-D-

xylopyranose backbone and branches of 4-O-methylglucuronic acid (MeGlcpA) and α-L-

arabinofuranosyl (Araf) units at the C2 and C3 positions, respectively (Peng et al. 2012).

Figure 2: Chemical structure of glucuronoarabinoxylan (GAX)

In contrast, the primary hemicellulose component in softwoods like pine and

spruce is galactoglucomannan (GGM, Figure 3), a branched heteropolymer with a β -1,4

linked backbone consisting of both D-glucopyranosyl (Glcp) and D-mannopyranosyl

(Manp) units and α-1,6 linked galactopyranosyl (Galp) branches on some Manp units.

GGM is often acetylated at the C2 or C3 positions of the Manp units, with the degree of

acetylation varying significantly between species (Song et al. 2013).

O

OO

OHHOO HO

O

OHO

O

O

OHO

OHHOO

OHO

O

OHO

O

nO

OH

OH

HO

10

Figure 3: Chemical structure of galactoglucomannan (GGM)

Hardwoods, such as beech and maple, possess yet another main hemicellulose

type, glucuronoxylan (GX, Figure 4). This structure is similar to GAX, except it lacks the

Araf branches and possesses some degree of acetylation on the C2 and/or C3 position of

the Xylp units. While the spacing of MeGlcpA branching is irregular on the xylan chain,

molar ratios of MeGlcpA:Xylp have been reported to range from 1:8 (Teleman et al.

2000) to 1:15 (Rivas et al. 2016).

Figure 4: Chemical structure of glucuronoxylan (GX)

2.1.1.3 Lignin

A key contributor to the structural integrity and impervious nature of the cell wall

matrix is a complex network of lignin surrounding the carbohydrate fibers. Lignin exists

O

OO

OHOO HO

O

OHHOO

OH

OHO

O

OHHO

OH OH

OHO

O

O

OO

OHOO O

O

OHO

O

O

OHO

OHHOO

OH

O

O

O

11

as a highly diverse cross-linked polymer (C-O and C-C bonds) of three primary

monolignols (Figure 5), which vary greatly in proportion between species and cell type.

Phenylalanine in the cell is carried through various biosynthetic pathways to generate p-

coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Figure 5), which are cross-

linked by enzyme initiated radical polymerization to form the hydroxyphenyl (H),

guaiacyl (G), and syringyl (S) subunits of lignin, respectively (Boerjan et al. 2003;

Ragauskas et al. 2014).

Figure 5: Chemical structures of primary monolignols

As with hemicellulose, the composition of lignin varies significantly between

plant species. Typically, softwoods contain a high proportion of G lignin, hardwoods and

other dicotyledonous angiosperms have various ratios of G/S lignin, while grasses often

have all three H/G/S lignin types (Suhas et al. 2007). In certain cases, modified lignin

subunits, 5-hydroxyguaiacyl (5HG) and catechyl (C), can occur. For example, the seed

coatings of a range of monocots and dicots were recently shown to contain linear

homopolymers of only C lignin units (Ragauskas et al. 2014).

Due to the high degree of variability and complexity within the lignin network,

there is no definitive model for the macromolecular structure of lignin, however several

commonalities can be seen between various models. The lignin matrix is covalently

linked to polysaccharides in the cell wall, forming a lignin-carbohydrate complex (LCC),

HO

OH

coumaryl alcohol

OHO

OOH

sinapyl alcoholO

HO

OH

coniferyl alcohol

12

which has proven elusive in efforts to extract and isolate in its native state. These

linkages are generally thought to consist of benzyl ethers, benzyl esters, and phenyl

glycosidic bonds (Giummarella & Lawoko 2016). The molecular weight of lignin has

also been challenging to analyze due to the inevitable breakdown of native lignin during

the extraction process, but various reports have ranged from 1,000 – 20,000 g/mol

(Thakur et al. 2014).

2.1.2 Biomass processing

The treatment of biomass from start to finish entails an extensive number of steps

and is generally considered to pose one of the greatest obstacles to large-scale

implementation of lignocellulosic technologies. Whether the feedstock comes from

forestry, farming, or municipal waste streams, the myriad logistical challenges associated

with harvest, transportation and pre-processing are complex and will not be considered in

the scope of this thesis. Instead, the various processing practices that are employed once

the feedstock reaches the mill/biorefinery will be examined.

2.1.2.1 Pretreatment

In the majority of cases, the ultimate goal of biomass pretreatment is to

deconstruct the cell wall matrix and increase enzymatic or chemical access to the

cellulose fraction. This typically involves the removal of lignin and hemicellulose

fractions, as well as physico-chemical activation of the cellulose fibers. This is

accomplished using a range of techniques which can be broadly categorized as either

physical, chemical, physico-chemical, and biological (Haghighi Mood et al. 2013).

13

Currently, some of the most commonly applied techniques in industry include steam

explosion, acid treatment, and alkaline treatment (Singh et al. 2015).

In steam explosion, biomass is exposed to high pressure steam at around 180 –

220 C (1 – 2.3 MPa) for a period of time typically ranging from 2 – 10 min, followed by

a sudden exposure to atmospheric pressure causing an explosive decompression in the

reactor (Chiaramonti et al. 2012). This process has a two-fold effect on the cell wall

structure; lignin and hemicellulose are hydrolyzed and degraded, and the crystalline

structure of the cellulose fibers is disrupted. While the material is under high temperature

and pressure, lignin linkages are broken, and the hydrolysis of acetyl groups and other

organic esters lead to the generation of organic acids, which then cause depolymerization

of xylan chains (Singh et al. 2015). The extent of hemicellulose hydrolysis leading to

mono- and oligosaccharides is correlated to the reaction temperature and retention time,

and sufficient increases in both can lead to the decomposition of hemicellulose to

furfurals, formic acid, and other byproducts that can interfere with subsequent enzyme

hydrolysis and/or microbial fermentations. Additional advantages of steam explosion

include low energy consumption and no requirements for added chemicals, although

some techniques include the addition of acid or base to increase hydrolysis (Jacquet et al.

2015).

Acid and alkaline treatment are both performed under similar conditions (elevated

temperature and pressure) but produce significantly different final products. Alkaline

treatment is usually performed using ammonia, NaOH, or Ca(OH)2, and results in higher

lignin removal relative to hemicellulose. The main mode of action for alkaline treatment

is the saponification of ester linkages, and hydrolysis of ether linkages in lignin, although

14

other groups in the LCC are also attacked to a lesser extent (Kim et al. 2016). One form

of alkaline pretreatment, ammonia fiber explosion/expansion (AFEX), is performed by

the addition of a pressurized mix of ammonia and steam followed by rapid

decompression and vaporization. Base-soluble components are dissolved in the liquid

ammonia and re-deposited on the surface of the material upon ammonia vaporization,

resulting in drastic changes to the porosity and macro-structure of the material without

the requirement of separating lignin and hemicellulose fractions into a second stream

(Chundawat et al. 2011).

In acid pretreatment, dilute sulfuric acid is most commonly used, while

hydrochloric, phosphoric, and nitric acids have also been investigated. In contrast to

alkaline treatment, acid treatment more so solubilizes the hemicellulose fraction, and

results in higher monosaccharide release (Alvira et al. 2010). The acid-catalyzed

hydrolysis of the sugar fraction to monosaccharides can sometimes eliminate the need for

subsequent enzymatic saccharification if fermentable sugars are the desired product;

however acid treatment can also cause further unwanted degradation of sugars to furfural

and hydroxymethylfurfural (HMF) which are detrimental to downstream processing

(Brodeur et al. 2011).

2.1.2.2 Fractionation

As more value-added applications are identified for biomass processing streams,

increasing attention is being paid to techniques for fractionating lignocellulose

components in ways that minimize degradation of the molecular structure and allow for

recovery of a relatively pure product. Development of such processes is core to the

success of modern biorefineries, which seek to make use of all cell wall components as

15

opposed to targeting only the cellulose fraction. To this end, processes such as hot water

extraction for hemicellulose recovery and lignin precipitation methods like LignoBoost

and LignoForce are gaining popularity in biomass processing.

Hot water extraction (autohydrolysis) is performed under relatively mild

conditions (160-180 C water) to solubilize the hemicellulose fraction without significant

molecular degradation. As a result, hot water extract tends to contain a high level of

oligosaccharides, as well as some water-soluble lignin, monosaccharides, furfurals and

MeOH (Chen et al. 2014). Although subsequent purification of oligosaccharides from hot

water extracts remains a challenge, the development of products from hemicelluloses,

including some research presented in this report, is advancing quickly with various end

uses including materials, food additives and fuels (Borrega et al. 2013).

Recovery of high purity lignin during biomass processing is also becoming more

popular since it can be used to create additional revenue streams as well as improve

general process efficiency. An effective method for purifying lignin is through

precipitation from the black liquor stream generated during alkaline pretreatment. This

can be accomplished by acidification of the strongly basic black liquor using carbon

dioxide or sulfuric acid, causing lignin to precipitate from solution so it can be collected

by filtration (Zhu et al. 2014). The LignoBoost process, now owned by Valmet, was

developed to improve the efficiency of this process and aid in large-scale implementation.

This process works by re-dispersing the lignin filter cake into a slurry before washing,

reducing the amount of acid and wash water required to clean the precipitate, and

improving the purity of the final product (Tomani 2010). Another advancement in lignin

recovery is the LignoForce process developed by FPInnovations in Thunder Bay, ON.

16

This process involves the oxidation of black liquor prior to acidification, leading to

improved lignin precipitation and larger particle sizes, which greatly enhances filtration

efficiency and affords a higher quality final product (Kouisni et al. 2012).

These methods and others are allowing mills and biorefineries to capture more

value from incoming feedstocks, while also reducing operating costs and improving

overall efficiency. Together with cellulose, the hemicellulose and lignin process streams

present many opportunities to create novel value-added products with applications across

a wide range of industries.

2.1.3 Major applications of plant products

2.1.3.1 Carbohydrate based products

Of the many polysaccharide-derived products that are currently produced from

lignocellulosic feedstocks, bioethanol, pulp-based products (paper, cardboard, tissue,

etc.), and textiles account for the vast majority of output volumes and are by far the most

commercialized products. Both product categories are generally focused on the extraction

of cellulose or hexose sugars, with hemicellulose and pentose sugars only beginning to

gain industrial relevance.

Cellulosic ethanol production has been viewed as a sustainable alternative to

starch based ethanol production for several decades now, but is still lagging in production

volumes and industry uptake. Despite scale up challenges, the US Energy Independence

and Security Act of 2007 has set forth ambitious production targets for cellulosic ethanol

of almost 40 billion l yr-1 by 2020 (U.S. Congress 2007; Scown et al. 2012). The world’s

largest cellulosic ethanol plant, opened by DuPont in 2015 in Nevada, Iowa, has a

production capacity of just over 110 million l yr-1 using corn stover feedstock, and is

17

joined by two similar plants in Iowa and Kansas owned by POET-DSM and Abengoa,

respectively (Peplow 2014). Clearly, there is still a long way to go to meet US

government targets, but new technologies such as genetic engineering of yeasts to

improve pentose sugar conversion to ethanol could significantly enhance overall output in

the near future (Koppram et al. 2013).

Cellulose-based textiles, on the other hand, represent a very mature industry that

has existed for over a century. Pulp and paper mills have established reliable methods of

isolating cellulose fibers, which have gone on to be used for a surprising number of

products aside from paper. The global consumption of natural and synthetic cellulose

fibers was approximately 72.5 Mt in 2010 and the textiles industry is expected to grow

rapidly in coming decades, with synthetic fibers largely outpacing natural fibers such as

cotton (Hämmerle 2011). The production of high purity dissolving pulps is increasing and

becoming more efficient, with 2014 production volumes of approximately 6.06 Mt

(Chen et al. 2016), and has given rise to many synthetic cellulose products such as rayon,

Tencel, cellulose acetate, nitrocellulose, and cellophane.

2.1.3.2 Lignin based products

Traditionally, lignin produced as a byproduct of biomass processing was burned

to supply additional heat and electricity to the processing facility. Only recently has the

potential to refine lignin to various chemicals and materials become a realistic

proposition in industrial settings. With the rate of growth in bioprocessing volumes, the

amount of available lignin for further usage will greatly outstrip current demand. That

being said, the implementation of lignin upgrading on a commercial scale is extremely

18

limited, but products such as carbon fiber, foams and plastics, phenolic platform

chemicals, and fuels are being developed (Ragauskas et al. 2014).

Most current applications of lignin require sulfonation or carboxylation to

increase solubility, allowing them to be used as dispersants or emulsifying agents in a

variety of products (E. Adler 1954). These processes often produce relatively low quality

lignin derivatives, which are frequently used as plasticizers or stabilizers in asphalt,

cement, and concrete mixtures. Conversely, high quality extracted lignin can be used to

generate a wide variety of aromatic platform chemicals, although this currently accounts

for a very small fraction of the total lignin produced. For example lignin-based vanillin,

which as of 2006 accounted for about 2,000 ton yr-1 or about 8% of the total vanillin

market, has been manufactured by a handful of companies such as Borregaard (Norway),

and is performed through the oxidation of lignosulfonates (Baskar et al. 2012).

2.2 Carbohydrate active enzymes (CAZymes)

As mentioned in Section 1.1, the CAZy database is a cornerstone of the scientific

community involved in enzymatic research on lignocellulose degradation and other sugar

biochemistry. This resource provides information on the substrate preference, reaction

mechanism, protein sequence and host organism, as well as any published crystal

structures for the enzyme of interest. Needless to say, any researcher in industry or

academia seeking specific enzyme activities on sugar would find this database to be

invaluable in their search.

The CAZy database lists 5 classes of enzymes based on the nature of the reaction

they catalyze: glycoside hydrolases, glycosyltransferases, polysaccharide lyases,

carbohydrate esterases, and auxiliary activities (AA). One non-catalytic class of protein,

19

the carbohydrate binding modules (CBMs), is also listed. The variety of functions

covered in these 6 classes is extensive; however, this report will focus specifically on the

AA and CBM classes.

2.2.1 Auxiliary activity (AA) class

The AA class is the newest addition to the CAZy database, first created in

response to the finding that former GH61 enzymes acted as lytic polysaccharides mono-

oxygenases (LPMOs) to enhance cellulase activity through oxidative cleavage of the

cellulose chain using small electron donating molecules such as ascorbate, gallate, and

lignin fragments (Quinlan et al. 2011). An objective of the AA class was to include

enzymes that are involved in cooperative breakdown of both carbohydrates and lignin,

since many of the enzymes in this group display synergistic activity with lignin degrading

enzymes as well as other CAZyme classes (Levasseur et al. 2013). The AA class is

broadly defined as carbohydrate oxidases, and now includes 13 families with a diverse set

of substrate preferences, cofactors, and reaction mechanisms, some of which fall outside

the scope of lignocellulose degradation and will therefore not be considered here. Some

of the main AA class enzymes relevant to this report are summarized in Table 1, and

details of each are presented in this section according to the type of cofactor used for

catalysis.

20

Table 1: Summary of relevant AA family enzymes

Family Number Enzyme Name Cofactor Activity

AA3 Glucose oxidase (GO) FAD (noncovalent) C1 oxidation

Pyranose dehydrogenase (PDH) FAD (covalent) C2 and/or C3 oxidation

Pyranose 2 oxidase (P2Ox) FAD (covalent) C2 and/or C3 oxidation

Cellobiose dehydrogenase (CDH) FAD/Heme C1 oxidation

AA5 Galactose oxidase (GaO) Copper radical C6 oxidation

AA7 Gluco-oligosaccharide oxidase (GOOX) FAD (bicovalent) C1 oxidation

AA9 Lytic polysaccharide mono-oxygenase (LPMO) Copper radical Cellulose cleavage - C1/C4 oxidation

2.2.1.1 FAD cofactor

The AA3 and AA7 families all contain an FAD (flavin adenine dinucleotide)

cofactor in their active site, and can differ in substrate preference, cofactor linkage, and

type of electron acceptor. The most notable enzymes included in this group are GOOX in

AA7, and glucose oxidase (GO), pyranose dehydrogenase (PDH), and pyranose-2-

oxidase (P2Ox) in AA3.

GO oxidizes the C1 position of D-glucose with the concomitant reduction of

molecular oxygen to H2O2, although alternative electron acceptors can be used. GO is

composed of two protein subunits each holding a non-covalently bound FAD molecule,

21

which are tightly associated but can be lost upon protein denaturation (Hecht et al. 1993).

These FAD cofactors mediate electron transfer from the sugar substrate to the electron

acceptor, which can proceed through a one- or two-electron transfer process depending

on the electron acceptor used (Leskovac et al. 2005).

Both P2Ox and PDH possess a covalently bound FAD cofactor and are capable of

oxidizing the C2 or C2/C3 position, respectively, of various pyranose sugars to the

corresponding ketones (Martin Hallberg et al. 2004; Sygmund et al. 2008). Whereas

P2Ox is capable of reducing oxygen to H2O2 as well as transferring electrons to alternate

acceptors, PDH is less reactive towards oxygen and prefers alternate acceptors such as

quinones, a function which could be used to help remove reactive oxidized quinones and

radicals generated during lignin breakdown (Tan et al. 2013).

Finally, GOOX in family AA7 possesses a doubly bound FAD cofactor and

catalyzes the C1 oxidation of a wide range of mono-and oligosaccharides using molecular

oxygen as an electron acceptor, resulting in the production of H2O2 as shown in Figure 6

(Lin et al. 1991; Huang et al. 2005).

Figure 6: General reaction mechanism for GOOX oxidation

The unusual double covalent linkages to the FAD cofactor have been shown to

not only alter flavin redox potential, but also to improve the structural stability of the

enzyme (Huang et al. 2008). GOOX displays an extremely broad substrate profile, and

O

OHHO

RO

OH

O

OHHO

RO

O

OH

OHHO

RO

O

OH

O2 H2O2

GOOX

H2O

Spontaneous hydrolysisR= H, OH, CH2OH

22

recent studies in our group have not only further expanded substrate preference (Foumani

et al. 2011), but have also created mutants with significantly reduced substrate inhibition

(Vuong et al. 2013) which are more suited to the high substrate concentrations found in

industrial settings. In comparison to GO, whose high specificity for glucose is caused, in

part, by a deeply buried active site (Hecht et al. 1993), the binding cleft of GOOX is

much more open, allowing larger oligosaccharides to access the active site (Huang et al.

2005).

2.2.1.2 FAD/heme cofactor

Cellobiose dehydrogenase (CDH, AA3 family) is a flavocytochrome containing a

190 residue heme-binding cytochrome domain linked to a flavoprotein, and catalyzes the

C1 oxidation of cellobiose as well as a range of other oligosaccharides. While oxygen can

be used as an electron acceptor, it is thought most electrons are transferred to the heme

iron, generating Fe(II) which can either produce hydroxyl radicals via Fenton reactions

with H2O2 (Hallberg et al. 2000), or directly reduce LPMOs (Tan et al. 2015) to assist in

cellulose depolymerization.

2.2.1.3 Copper radical cofactor

The most notable carbohydrate active enzymes containing a copper cofactor

include galactose oxidase (GaO, AA5), as well as two LPMOs in family AA9 and AA10,

formerly known as GH61 and CBM33, respectively. In general, these three families share

the trait of acting on polysaccharide substrates, albeit in significantly different ways.

Galactose oxidase catalyzes the oxidation of a variety of primary alcohols,

including non-carbohydrate substrates, to their respective aldehydes with molecular

oxygen acting as the electron acceptor to form H2O2, although other oxidants such as

23

potassium ferricyanide are also compatible. While the enzyme can accept a diverse range

of substrates, it is highly stereospecific, for instance in sugar substrates an axial position

in the C4 hydroxyl is required, hence glucose is not an accepted substrate (Ito et al.

1991). In addition to a coordinated copper ion, the active site has also been shown to

include a stabilized tyrosine free radical, which is responsible for the second electron

transfer during substrate oxidation (Ito et al. 1994).

LPMOs are a relatively new group of enzymes gaining significant attention in the

biofuels community due to their ability to significantly enhance cellulose degradation.

AA9 LPMOs, fungal proteins initially characterized as GH61 enzymes, were first thought

to exhibit weak endo-glucanase activity, when new research (Harris et al. 2010)

demonstrated that the addition of divalent metal ions with the enzyme allowed for a 2

fold reduction in cellulase loading during cellulose digestion. AA9 LPMOs were later

shown to be copper dependent (Westereng et al. 2011) and able to cleave crystalline

cellulose chains at the glycosidic linkage producing oxidized C1 and/or C4 fragments.

The ability to bind and attack crystalline cellulose is attributed to a planar face on the

enzyme which contains the exposed copper cofactor; this planar region contains several

hydrophobic residues which can associate with crystalline cellulose through stacking

interactions (Bennati-Granier et al. 2015). AA10 LPMOs share a similar catalytic

mechanism to AA9 LPMOs, and comprise a group of bacterial enzymes originally

classified as CBM33. These enzymes were first shown to act on crystalline chitin (Vaaje-

kolstad et al. 2010) and shortly after were shown to also cleave cellulose (Forsberg et al.

2011) in an oxidative manner very similar to AA9 LPMOs. Whereas LPMOs have also

24

been classified into families AA11 and AA13, comparatively few have been functionally

characterized.

2.2.2 Carbohydrate binding modules (CBMs)

Many carbohydrate active enzymes have evolved to contain CBMs to assist in the

targeting and binding of their respective substrate. These modules are non-catalytic

proteins which act to bring their associated degradative enzymes into close contact with

the substrate, effectively increasing enzyme concentration at the surface of the substrate,

resulting in higher efficiency (Bolam et al. 1998). Currently, there are 80 CBM families

listed in the CAZy database capable of binding all common polysaccharides, but they can

be generalized to three main Types; A, B and C. These groups are divided based on the

structure of the binding surface of the protein. Type A CBMs contain a planar binding

face with several hydrophobic residues, allowing them to bind to the flat surface of

crystalline cellulose. Type B CBMs contain a binding cleft or groove with several

recognition sites for sugar units in a polymer chain, and are more suited to binding

individual polysaccharide chains as well as branched polysaccharides including

hemicellulose. Finally, Type C CBMs are characterized by shorter binding grooves

compared to Type B, and were initially described as “small sugar binding CBMs”

(Boraston et al. 2004) but have since been more accurately described as exo-acting CBMs

(binding to chain termini), while Type B have been redefined as endo-acting CBMs

(binding internally on chain) (Gilbert et al. 2013).

25

2.3 Applications of carbohydrate oxidases

The diverse properties of CAZymes have led to their application in a wide variety

of commercial products and processes outside of biofuel production. Some of the main

applications in use today will be explored here.

2.3.1 Biosensors

Perhaps the earliest example of CAZyme application was the development of

amperometric biosensors that were able to measure the concentration of glucose using

GO (Clark & Lyons 1962; Updike & Hicks 1967). This was first done by measuring the

decrease in O2 concentration at the electrode during glucose oxidation, and has since

evolved through numerous iterations to involve electrical communication between GO

and the electrode using diffusible redox mediators such as ferrocene (Cass et al. 1984),

covalent linkage (aka “wiring”) between FAD and electrode (Degani & Heller 1987), or

direct electron transfer systems using carbon nanotubes (Guiseppi-Elie et al. 2002). The

intense research on GO-based biosensors is largely driven by the need to measure blood

glucose levels in medicine, which is of particular importance for diabetic patients.

However, this same principle can be extended to other CAZymes for the measurement of

various other mono- and oligosaccharides, a concept which has been pursued for brewing

applications (Monošík et al. 2013), and has also been a suggested application for fusion

enzymes developed in our lab (Vuong & Master 2014).

2.3.2 Synthesis Many compounds used today require the selective addition of sugar moieties, a

challenging synthetic process typically requiring laborious protection/deprotection of the

multiple reactive groups found in the sugar structure. Products including pharmaceuticals,

26

antibiotics, foods, cosmetics and detergents can contain sugar structures that require high

purity and highly defined structure. CAZymes have the ability to carry out such reactions

with high specificity, and have been used to generate complex drug structures (Gantt et

al. 2011) and refine mixed oligosaccharides to structurally defined food additives (Yun

1996). Pertinent to this report, GOOX has been used for the production of various aldonic

acids, including lactobionic acid (Lin et al. 1996), which are commonly used in the food

and cosmetic industries.

2.3.3 Targeted binding via CBMs

Due to their ability to bind specific carbohydrate substrates, CBMs have received

a great deal of attention from the biotechnology community. Protein engineering can

produce recombinant CBM-fusion constructs that are capable of immobilizing a wide

variety of proteins, phages, or other compounds to a carbohydrate surface. This ability

has given rise to the development of many commercially viable products including CBM-

cellulase fusion animal feed additives (Ribeiro et al. 2008), additives for textile

processing (Ramos et al. 2007), and as affinity tags for protein purification (Kavoosi et al.

2004).

2.3.4 Food applications

As it is of particular relevance to the work presented in this report, the

applications of CAZymes in food perseveration and processing should be noted. The

ability of carbohydrate oxidases to consume O2 while producing H2O2 makes them

27

desirable for food preservation applications where sugars are available to the enzyme

(Heidebach et al. 2014). GO has been used extensively for such purposes, for example,

GO was recently incorporated in a polymeric membrane to introduce preservative

properties to food packing films (Ge et al. 2012). GO has also been used extensively as a

baking additive to improve dough consistency, and recent studies have shown similar

results using an AA7 oligosaccharide oxidase, MnCO from Microdochium nivale, which

effects dough consistency through both H2O2 and acidic sugar products (Degrand et al.

2015).

2.4 Green synthetic chemistry

The idea of green chemistry has been around for a long time, but has only become a

mainstream movement in the chemical community in the last 20 years or so. In 1999, the

journal “Green Chemistry” produced their first issue, and since then there has been a

growing number of examples of old reactions and processes being improved to become

more environmentally friendly. The key concepts of green chemistry focus on the

efficient use of materials and minimization of waste, as well as the environmental effects

of chemicals and how to minimize their release into the environment (Clark et al. 2014).

Some of the core strategies relate to atom economy, solvent usage, and improved catalyst

design.

2.4.1 Amide bond synthesis

Amide bonds are extremely prevalent in many materials (e.g., Nylon),

pharmaceuticals, pesticides, and a multitude of other chemical products. However, the

synthesis of amide bonds remains a challenge in settings where extreme heat and

temperature cannot be tolerated. In 2007, amide bond synthesis was identified as the top

28

synthetic process in need of improvement by the ACS Green Chemistry Institute together

with global pharmaceutical corporations (Constable et al. 2007).

Amides are generally synthesized through the condensation of a carboxylic acid

and primary or secondary amine with the loss of one molecule of water (Figure 7).

Figure 7: Amide synthesis through acid-amine condensation

This process does not occur spontaneously at room temperature, and the elimination of

water must be driven through the use of temperatures exceeding 200 C and/or high

pressures (Valeur & Bradley 2009). In the case of heat sensitive reactants, activation of

the carboxylic acid must be used to create a thermodynamically favorable leaving group,

this can be achieved through the formation of acyl halides, anhydrides, active esters, acyl

azides, and more (Montalbetti & Falque 2005). Coupling reagents are compounds that are

used in stoichiometric amounts to generate an activated acid prior to nucleophilic attack

by the amine, and while there is a long list of available coupling reagents, some of the

most commonly used are carbodiimides, thionyl chloride, oxalyl chloride, and T3P

(Dunetz et al. 2015). While these reagents are generally effective to some degree, they are

not ideal due to poor yields, formation of side products, poor atom economy, and high

cost. There is therefore a great deal of research focusing on new processes to synthesize

amide bonds in more effective ways; some promising techniques include boronic acid

catalysts (Ishihara & Ohara 1996), ruthenium catalysts (Zeng & Guan 2011), and lipases

(Garcia et al. 1992).

X OH

O

H2N Y -H2OX N

HY

O

29

When dealing with highly polar substrates such as carbohydrates, the challenges of

amide bond synthesis become even greater due to their poor solubility in organic

solvents, sensitivity to high temperatures, and multiple reactive groups. This dramatically

limits the number of applicable coupling reagents, the majority of which require

anhydrous conditions in organic solvents. There are several coupling reagents which are

designed for use in aqueous conditions, with N-(3-Dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride (EDAC) being the most widely used (Dunetz et al.

2015). The experiments in this project focus on the use of EDAC, as well as a lesser

known coupling reagent, 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium

chloride (DMT-MM), to perform aqueous amide synthesis (Figure 8). The use of DMT-

MM for amide bond synthesis has been very limited in the literature, although several

publications have claimed superior performance to EDAC for the functionalization of

various small molecules (Kunishima et al. 1999) and polysaccharides (Borke et al. 2015;

D’Este et al. 2014).

Figure 8: Water soluble coupling reagents

EDAC (left) and DMT-MM (right)

NC

N

NN+

O

N

N

N

O

OCl-

HCl

+

O

N NHCl

HN

R OH

O

N

N

N

O

O OR

OR

O

Active Esters

Coupling agent

30

2.4.2 Copper catalyzed azide-alkyne cycloaddition click chemistry (CuAAC)

Ever since the first publication on the topic in 2001 (Kolb et al. 2001), the field of

click chemistry has exploded with thousands of publications covering almost all fields of

biology and chemistry. The authors who coined the term click chemistry defined it as a

set of reactions which are modular, wide in scope, produce inoffensive byproducts, are

compatible with benign solvents (i.e., water), and do not require chromatographic

purification. The tenants of click chemistry fall very much in line with those of green

chemistry, and it is no surprise that click reactions have become a major tool for novel

green synthetic processes.

Of the handful of click reactions, the copper catalyzed azide-alkyne cycloaddition

(CuAAC) is by far the most commonly used (Figure 9). This reaction has existed for a

long time as the Huisgen 1,3 dipolar cycloaddition, in which a terminal azide and

terminal alkyne react to form a 1,3 disubstituted triazole ring, and became the prominent

CuAAC reaction after the finding that Cu(I) acted as a catalyst for the reaction and

increased the rate approximately 106 fold, as well as favoring the 1,4 disubstituted

triazole product (Rostovtsev et al. 2002). One of the desirable attributes of the reaction is

minimal interference or cross reactions with other common functionalities found in nature

(bioorthogonal), making this reaction extremely useful for biotechnology purposes.

Figure 9: CuAAC click reaction

NN

NR

R

R N N N

R

Cu(I)

31

Several publications of particular relevance to this report demonstrate the use of

the CuAAC reaction for the conjugation (Deepthi et al. 2013), functionalization

(Pahimanolis et al. 2011) or polymerization (Bueno-Martinez et al. 2015) of

carbohydrates. It has also been shown that the triazole ring is capable of coordinating a

number of transition metals (Suijkerbuijk et al. 2007), and polytriazole polymers have

shown promise as effective adhesives for bonding to certain metal surfaces (Díaz et al.

2004).

2.4.3 Alternative solvents for sugar chemistry

As previously mentioned, the choice of solvent presents a particular challenge

when performing chemical synthesis with carbohydrates. In cases where water causes

hydrolysis of intermediates or products, polar organic solvents such as THF, DMF,

MeOH, or DMSO must be used to achieve adequate solubilization of sugars. These

solvents are not ideal due to their toxicity, cost, and environmental effects, leaving sugar

chemists in a difficult position.

A large amount of research has been dedicated to developing appropriate solvents

for sugar chemistry, yielding several promising new technologies. Ionic liquids (ILs)

have garnered a great deal of attention in the last 25 years or so, after new formulations of

pre-existing ILs led to room-temperature liquid salts that were stable in the presence of

moisture (Wilkes & Zaworotko 1992). Modern ILs generally consist of an

asymmetrically substituted cation, such as immidazolium or pyrrolodinium, together with

an anion, often hexafluoroborate (BF6) or other complex anions containing halogens such

as Cl, Br, and I (Dai et al. 2013). These solvents have the ability to dissolve high

concentrations of polar substrates, can be made immiscible with organic solvents or water

32

(depending on formulation), and do not evaporate. These unique properties have been

exploited for numerous applications including natural product extraction from biomass

(Du et al. 2007), reaction medium for amide synthesis (Lau et al. 2000), and as a solvent

for lignocellulose degradation (Fort et al. 2007). However, ILs are not without fault, and

their high cost, complex synthesis, and potential toxicity (Thuy Pham et al. 2010) are

significant drawbacks.

In the background of the hype surrounding ILs, an alternative liquid salt system

has been developed which is only now gaining notoriety in the literature. Deep-eutectic

solvents (DESs) share many of the desirable qualities of ILs, yet are made from cheap,

renewable, and non-toxic components, making them a preferable replacement to ILs in

many green chemistry scenarios. DESs are composed of a cation, usually an ammonium

salt such as choline chloride, and a hydrogen bond donor such as urea, glycerol, or small

sugars (Durand et al. 2012). The type and ratio of cation and hydrogen bond donor

determines the freezing point of the liquid salt by altering the lattice energy of the

mixture; the commonly used choline chloride : urea (1:2) DES has a freezing point

slightly below ambient temperatures, at 12 C (Abbott et al. 2004). DESs have been used

in many similar applications as ILs, and of particular relevance to this report is the use of

DESs as reaction medium for the lipase-catalyzed synthesis of sugar esters (Pöhnlein et

al. 2015). The ability to carry out enzyme catalyzed alcoholysis or aminolysis in DESs

could dramatically improve upon existing options for synthesis of sugar esters/amides,

and open the door to exciting possibilities such as “solvent as substrate” processes in

which sugar could act as both a solvent component and substrate simultaneously.

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3.0 Chapter I - CBM-GOOX fusion production and studies

Parts of this chapter are included in a manuscript titled “Gluco-oligosaccharide oxidase

variants as substitutes of glucose oxidase in applications comprising mixed

sugars”, submitted for publication in Scientific Reports (ID# SREP-16-26730).

Contributions: Protein production and purification, immobilization studies, stability

studies, assisted in experimental design and manuscript preparation.

3.1 Background The aim of this study was to demonstrate the enhanced performance of GOOX

resulting from fusion to a CBM protein, and subsequent immobilization on insoluble

polysaccharide fiber. Since GOOX has the potential to replace GO in many applications,

enzyme performance studies were carried out using commercial GO for comparison.

Although the performance characteristics presented here are relevant to a wide range of

oxidase applications, the experiments were designed with specific focus on food and

baking applications where GO has been extensively applied in the past. For this reason,

oat spelt xylan (OSX) was chosen as the insoluble support for enzyme immobilization

due to its added benefit as a dietary fiber in food applications.

The ability to immobilize the CBM-fusion enzyme on OSX is also of interest for

protein purification, therefore, experiments were carried out using enzyme samples that

were immobilized either directly from the culture supernatant (CS) or after purification

by buffer exchange (BX).

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3.2 Methods

3.2.1 Production and purification of CtCBM22A_GOOX-Y300A

3.2.1.1 Enzyme production Variants of GOOX originating from the fungus Sarocladium strictum strain 346.70 were

expressed using a stock of Pichia pastoris KM71H cells containing a previously

constructed CBM-GOOX-His6 mutant plasmid, CtCBM22A_GOOX-Y300A, was used

to inoculate 1L of buffered glycerol-complex media (BMGY; 100 mM potassium

phosphate (pH 6.0), 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB), 4 x

10-5 % biotin, 1% glycerol) (Table 2). Cells were grown at 28 C for 2 days, after which

the culture was centrifuged at 2000 g for 5 min and cells were resuspended in 200 mL of

buffered methanol-complex media (BMMY; 100 mM potassium phosphate (pH 6.0), 1%

yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB), 4 x 10-5 % biotin, 0.5%

methanol). The culture was then incubated at 15 C with shaking, and protein production

was induced by the addition of 1 mL MeOH each day for 5 days. Cells were removed

from the culture by centrifugation (5000 g , 3 x 10 min) and the culture supernatant was

either buffer exchanged before purification or filtered and used directly for enzyme

immobilization.

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Table 2: Summary of GOOX variants used in experiments

Enzyme Name Accession Number/Description Reported In

wt-GOOX GU369974 (Lin et al, 1991)

GOOX-Y300A

Single amino acid substitution at substrate binding site

(Foumani et al, 2011)

CtCBM22A_GOOX-Y300A

Fusion of a family 22 CBM to the N-terminus of GOOX-Y300A

GOOX-W351A

Single amino acid substitution at substrate binding site

(Vuong et al, 2013)

3.2.1.2 Ni-NTA purification of CtCBM22A_GOOX-Y300A Culture supernatant was passed through a Ni-NTA resin column, and bound proteins

were eluted using 250 mM imidazole solution. The eluted protein was then buffer

exchanged into 50 mM Tris-HCl (pH 8.0) using a 30 kDa MWCO Vivaspin 20

centrifugal concentration unit.

3.2.1.3 OSX binding of CtCBM22A_GOOX-Y300A Insoluble OSX (Sigma Aldrich, US) was prepared by addition of 2 g OSX to 200 mL of

50 mM Tris-HCl (pH 8.0) buffer followed by incubation at 25 C with shaking for 48 h.

The solution was filtered using a 0.45 µm PES membrane, and the insoluble fraction was

washed three times with deionized water before drying at 60 C overnight.

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An excess of dry OSX (0.5 g) was then added to 40 mL of buffer exchanged (BX) protein

or unpurified culture supernatant (CS), and incubated at 4 C overnight with mixing.

Samples were then filtered using a 0.45 µm PES membrane, and the protein-bound OSX

was washed with 20 mL of 50 mM Tris-HCl (pH 8.0) buffer.

The proportion of bound and unbound protein was assessed by SDS-PAGE of the filtered

OSX samples and filtrates, respectively. OSX-bound protein samples (35-40 mg wet

weight) were mixed with 40 µL denaturing solution (10% SDS, 10% β-mercaptoethanol)

and heated at 100 C for 10 min. Filtrate samples were precipitated using 10% TCA and

the pellets were washed with ice-cold acetone before resuspending in 40 µL SDS loading

dye. All samples were then run on 10% SDS-PAGE gels.

3.2.2 Assessment of OSX binding stability

3.2.2.1 Salt elution of OSX-bound protein Samples of OSX-bound CtCBM22A_GOOX-Y300A from both the buffer exchanged and

crude supernatants were sequentially washed in 300 µL of 50 mM Tris-HCl (pH 8.0)

buffer containing increasing salt concentrations ranging from 0 – 2 M NaCl and filtered

using a 0.45 µm Nylon membrane centrifugal filter unit. Filtrates were tested for the

presence of enzyme using a Nanodrop ND1000 Spectrophotometer. The quantity of

remaining OSX-bound GOOX was determined using SDS-PAGE.

3.2.2.2 Long term binding stability Samples of OSX-bound CtCBM22A_GOOX-Y300A were stored in 50 mM Tris-HCl

(pH 8.0) buffer at 4 C for 12 weeks. Binding stability was assessed by monitoring the

amount of released protein in the supernatant each week using SDS-PAGE.

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3.2.2.3 Effect of Ca2+ on binding Samples OSX-bound CtCBM22A_GOOX-Y300A were incubated in 50 mM Tris-HCl

(pH 8.0) buffer with or without 5 mM CaCl2 and stored at 4 C. The effect of Ca2+ ions on

CBM binding was evaluated by measuring the concentration of bound and unbound

protein after 2 weeks of incubation using SDS-PAGE.

3.2.3 Effects of CBM fusion and immobilization on enzyme performance

3.2.3.1 Enzyme activity assay Oxidase activity was assessed by measuring H2O2 production using a chromogenic

method according to (Lin et al. 1991). Assays were performed in a 96-well microplate at

a total volume of 250 µL consisting of 50 mM Tris-HCl (pH 8.0), 0.1 mM 4-

aminoantipyrine, 1 mM phenol, 0.5 U horseradish peroxidase, and a typical enzyme

concentration of 16 nM. Reactions were incubated at 37 C, and absorbance was measured

at 500 nm using a Tecan Infinite M200 spectrophotometer.

3.2.3.2 Thermal stability The effect of CBM fusion and immobilization on enzyme thermostability was assessed

by incubating Y300A-GOOX, CtCBM22A_GOOX-Y300A, and OSX-bound

CtCBM22A_GOOX-Y300A at temperatures from 30 – 54 C for 1 h, followed by

measurement of residual activity using the colorimetric assay containing 0.5 mM

cellobiose.

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3.2.3.3 H2O2 inhibition and stability The effect of CBM fusion and immobilization on H2O2 inhibition was assessed by

incubating samples of commercial GO (Sigma), GOOX-Y300A, CtCBM22A_GOOX-

Y300A, and OSX-bound CtCBM22A_GOOX-Y300A in their optimal reaction

conditions containing 0 – 200 mM H2O2. GOOX reactions were performed in 50 mM

Tris-HCl (pH 8.0) buffer with 0.5 mM cellobiose for 2 hrs, and GO reactions were

performed in 50 mM sodium acetate (pH 5.0) buffer with 1 mM glucose for 5 hrs. After

incubation, protein was removed using a 10 K OmegaTM Nanosep centrifugal device (Pall

Corp., USA) followed by the addition of 100 U catalase and incubation at 25 C for 1 hr to

remove remaining H2O2, and finally samples were filtered again to remove catalase.

Enzyme activity was determined by measuring the amount of oxidized substrate produced

after the reactions using HPAEC-PAD (please see section 4.1.1.2).

H2O2 stability was determined by incubating each enzyme (16 nM) with 200 mM H2O2

for 30 min at 25 C with mixing, followed by degradation of H2O2 using catalase (2 x 100

U) before the addition of 1 mM glucose. Samples were then incubated at 37 C for 2 hrs

before filtering using a 10 K OmegaTM Nanosep centrifugal device (Pall Corp., USA) and

residual activity was determined by measuring the amount of oxidized glucose using

HPAEC-PAD.

3.3 Results and Discussion

3.3.1 Stable enzyme immobilization on insoluble hemicellulose

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The ability to immobilize enzymes on insoluble particles can greatly enhance the

potential utility of an enzyme by simplifying purification and removal from reaction

media. In this study, insoluble OSX was chosen as the substrate for immobilization due to

its nutritional benefits as a dietary fiber (Eastwood 1992), making the immobilized

product particularly suited to food applications. The fusion of CtCBM22A to GOOX-

Y300A resulted in the ability of the fusion enzyme to bind insoluble OSX with high

affinity, and remain bound for at least 12 weeks at 4 C with minimal release from the

fiber. OSX binding was conducted in both buffer exchanged (BX) and unpurified culture

supernatant (CS) to examine the feasibility of protein recovery directly from production

media to further simplify the protein purification process.

The interaction of Type B CBMs (such as CBM22) with their respective

carbohydrate ligand is known to occur mainly through hydrophobic stacking and

hydrogen bonding (Boraston et al. 2004), which could potentially be disrupted by

exposure to high ionic strength. To test the strength of OSX binding in high salt

conditions, samples of immobilized enzyme were exposed to NaCl concentrations up to 2

M, and analysis of bound of unbound protein fractions revealed that binding was not

significantly disrupted by high ionic strength. The low levels of protein detected in the 0

and 0.1 M NaCl wash were likely residual unbound protein remaining from storage, and

subsequent washes with higher salt concentrations did not show protein concentrations

above the background noise signal. In addition, SDS-PAGE of the OSX-bound GOOX

after washing with up to 2 M NaCl revealed that a significant amount of protein remained

on the fiber (Figure 10). These results suggest that the binding of CtCBM22A_GOOX-

40

Y300A to OSX is strong, and can withstand exposure to high salt environments, a