2017

Design of high-throughput assays for the analysis of plant cell wall polysaccharides

MASTER THESIS

ELLINOR JOHNSSON

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Abstract

Plant cell walls contain a wide range of valuable polysaccharides possible to exploit for various

purposes. To profile the diverse polysaccharides of plants, high-throughput, rapid and sustainable

analytical methods are required. Currently, there are few methods designed for this purpose. An

approach has been made to develop an analytical method based on enzymatic deconstruction and

soft ionization mass spectrometry.

Primary cell wall was extracted from the cells of the well-established model plant poplar to serve as

an initial experimental subject. The cell wall was purified into alcohol insoluble residue consisting of

only insoluble polysaccharides and a small amount of protein. Pure and specific enzymes were

selected based on literature to deconstruct the polysaccharides and were evaluated on standard

sugar substrates. Enzyme activity was measured using a rapid and reliable reducing sugar assay and

achieved oligosaccharides were purified before electrospray ionization mass spectrometry. The

outcome of the thesis presents an introduction to a high-throughput, rapid and sustainable analytical

methodology for polysaccharide profiling in plant cell walls.

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Sammanfattning

Växtcellväggar innehåller en stor variation av värdefulla polysackarider möjliga att utnyttja för olika

ändamål. För att profilera de olika polysackariderna krävs snabba och hållbara analytiska metoder

med hög kapacitet. För närvarande existerar få metoder som är designade för detta syfte. Ett

tillvägagångssätt har utformats för att utveckla en analytisk metod baserad på enzymatisk

dekonstruktion och ”soft” jonisering masspektrometri.

Primär cellvägg extraherades från celler härstammande från den väl etablerade modellväxten poppel

för att verka som ett initialt testobjekt. Cellväggen renades till alkohol-olöslig residual. Rena och

specifika enzymer valdes baserat på litteratur för att dekonstruera polysackariderna samt

evaluerades med hjälp av standard socker substrat. Enzymaktivitet mättes genom användning av en

snabb och pålitlig reducerande sockeranalys. Erhållna oligosackarider renades innan elektrospray

joniserande masspektrometri. Utfallet av tesen presenterar en introduktion till en snabb och hållbar

analytisk metod med hög kapacitet för profilering av polysackarider i växtcellväggar.

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Table of contents 1. Introduction .................................................................................................................................... 5

1.1 Populus .......................................................................................................................................... 5

1.2 Plant cell wall ................................................................................................................................ 5

1.2.1 Primary cell wall ..................................................................................................................... 6

1.3 Polysaccharides ............................................................................................................................. 6

1.3.1 Cellulose ................................................................................................................................. 7

1.3.2 Hemicellulose ......................................................................................................................... 7

1.3.3 Mannan .................................................................................................................................. 9

1.3.4 Pectins .................................................................................................................................. 10

1.4 Carbohydrate-active enzymes .................................................................................................... 11

1.4.1 Glycoside hydrolases ............................................................................................................ 11

1.4.2 Polysaccharide lyases ........................................................................................................... 12

1.5 Methods and analyses ................................................................................................................ 12

2. Materials and methods ................................................................................................................. 13

2.1 Culture and harvest ..................................................................................................................... 13

2.2 AIR (Alcohol insoluble residue) preparation ............................................................................... 13

2.3 Starch removal ............................................................................................................................ 13

2.4 Sulphuric hydrolysis .................................................................................................................... 13

2.5 Methanolysis ............................................................................................................................... 14

2.6 Bradford protein assay ................................................................................................................ 14

2.7 DNS assay and enzyme incubations ............................................................................................ 14

2.8 Mass spectrometry and purification ........................................................................................... 16

3. Results ........................................................................................................................................... 17

3.1 Sugar analyses ............................................................................................................................. 17

3.2 Bradford protein assay ................................................................................................................ 18

3.3 DNS assay .................................................................................................................................... 18

3.4 Mass spectrometry ..................................................................................................................... 21

4. Discussion ...................................................................................................................................... 24

4.1 Sugar analyses ............................................................................................................................. 24

4.2 Bradford protein assay ................................................................................................................ 25

4.3 DNS assay .................................................................................................................................... 25

4.4 Mass spectrometry ..................................................................................................................... 27

5. Conclusions ................................................................................................................................... 28

6. Supplementary .............................................................................................................................. 29

6.1 Sugar analysis of standard sugar substrates ............................................................................... 29

6.2 AIR sugar concentration .............................................................................................................. 30

6.3 Bradford standard curve ............................................................................................................. 31

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6.4 Glucose standard curves ............................................................................................................. 31

6.5 Additional spectra ....................................................................................................................... 33

6.5.1 Guidelines specta interpretation ......................................................................................... 33

6.5.2 Spectra standard sugar incubations ..................................................................................... 34

6.5.3 Sequential AIR incubations .................................................................................................. 36

6.5.4 AIR incubations .................................................................................................................... 37

7. References .................................................................................................................................... 41

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1. Introduction

1.1 Populus The genus of Populus belongs to the family Salicaceae, which is one of the subgroups of the

angiosperms, also known as flowering plants. Populus includes trees as for instance poplar, aspen

and cottonwood and is widely distributed over the northern hemisphere [1].

Populus is currently a frequently used model plant in plant research along with other species, such

as Arabidopsis, maize and tobacco [2]. A few advantages of using Populus as a model plant are; the

small and sequenced genome of roughly 35 000 genes spread over 19 chromosomes, the high growth

rate and consequently fast biomass accumulation, the feasibility of growing it in cell and tissue

cultures and the possibilities for genetic transformation [3,1].

1.2 Plant cell wall Plant cell walls are complex networks consisting of polymers, which determine the shape of the plant

cell as well as provide the cells with structural stability. Plant cell walls are further regulating the rate

and direction of cell growth in living cells and are hence influencing plant development and

morphology. The composition of the plant cell wall includes several different polysaccharides,

proteins, aromatic substances and structural molecules that are acting as fibers or as a cross-linked

matrix [4].

There is generally a division of plant cell walls into two categories, the primary cell wall and the

secondary cell wall. The primary plant cell wall is created during cell division in the cell plate and

increases significantly in surface area at the time of cell expansion. The middle lamella, a pectin-rich

layer, glues the primary cell walls of neighbouring cells together [5]. On the other hand, the secondary

cell wall is created at a later stage when the growth of the primary wall has ceased, during

differentiation from the primary cell wall [4]. The secondary cell wall contains lignins, which are

complex hydrophobic molecules built from polyphenolic precursors. Lignin provides mechanical

support for the plants to stand upright as well as a water-proof protective barrier as a defence against

physical and biological attacks [6].

Plant cell walls are the primary source of renewable resources, such as cellulose, which is the most

useful and abundant biopolymer in the biosphere [7]. Due to this, they are of crucial importance for

a sustainable development and the transition from an oil-based to a bio-based economy.

Currently, different parts of plant cell walls are used in industries involving human and animal food,

textiles, wood and paper. Pectins are being used as gum and gelling agents. -glucans, which are

soluble dietary fibers, are being used for its advantages regarding obesity and metabolic syndromes.

[8] -glucans are also being exploited for their ability to cause oat and barley brans to lower serum

cholesterol and hence lower the insulin demand of people with diabetes [4].

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1.2.1 Primary cell wall The primary cell wall is often structured into different complex and interacting networks composed

of cellulose microfibrils, hemicelluloses, matrix pectic polysaccharides and structural proteins.

Throughout the years, several models have been proposed for describing how these networks are

constructed. One of the earliest models from 1973 suggests that the macromolecules of the cell wall,

such as pectic polysaccharides and structural proteins, are covalently cross-linked and there

potentially also could occur covalent linkages between cellulose and other polysaccharides. More

detailed, they suggested that xyloglucan is a link between the cellulose microfibrils and pectic

polymers, which in turn is linked to a cell wall glycosylated structural protein. [10] However, this

model was dismissed due to lack of evidence of a covalent linkage between the xyloglucan and pectic

polysaccharides. Studies providing evidence, performed at a late stage, 2000 and 2005, reported that

a proportion of the xyloglucan actually is covalently linked to acidic residues, which could be

homogalacturonan. [11, 12] Another model from 1989 propose that hemicelluloses may

spontaneously make a connection to the surfaces of the cellulose microfibrils and hence binding

neighbouring microfibrils together. [13] In 1992, it was suggested that the cellulose microfibrils are

coated with xyloglucans, which in turn adhere to other matrix polysaccharides without a direct

linkage between the microfibrils. [14] Only two years later, in 1994, it was suggested that a part of

xyloglucan could become entrapped during the formation of the microfibrils and that the other, free

part of the chain would be able to bind to other matrix polymers or to other cellulose surfaces, and

hence linking the microfibrils rigidly to each other. [15, 16] The complex interactions between the

different components of the primary cell wall are yet to be fully understood.

There are two types of primary cell wall, type I and type II. In type I, found in most dicots and

noncommelinoid monocots, there are roughly equal amounts of cellulose and xyloglucan. Type II

walls have glucuronoarabinoxylan as the crosslinking hemicellulose instead of the xyloglucan found

in type I [4].

The composition of primary cell wall isolated from cambial tissues of Populus Tremuloides was

investigated between 1968 and 1972. The polysaccharides which were found and their relative

polysaccharide percentage was determined to be as following; rhamnogalacturonan (20 %), galactan

(18 %), arabinan (9 %), arabinogalactan (13 %), xyloglucan (6 %), xylan (11 %), glucomannan (1 %) and

cellulose (22 %). In the study, the percentage of protein in the primary cell wall tissue was determined

to be 10 %, relative the total sample [9].

1.3 Polysaccharides Polysaccharides are polymers of sugars and the main component of the structural network

comprising the plant cell wall. The most common sugars used for building the polysaccharides are,

α-D-Glucose (Glc), α-L-Rhamnose (Rha), α-D-Galactose (Gal), α-D-Galacturonic acid (GalA), α-D-

Glucuronic acid (GlcA), α-D-Apiose (Api), α-D-Xylose (Xyl), β-L-Arabinose (Ara), α-D-Mannose (Man),

α-D-Mannuronic acid (ManA) and α-L-Fucose (Fuc). The sugars forms chains of various lengths by

forming glycosidic linkages, which may differ in which anomers are linked, which hydroxyl group of

each sugar participates in the binding and how many linkages are occurring. This results in the

considerable amount of variation, which exists among polysaccharides [4].

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1.3.1 Cellulose The most abundant polysaccharide in the plant cell wall is cellulose (Figure 1), which can account for

15-30% of the dry mass of primary cell walls. Cellulose is formed into microfibrils, consisting of dozens

of (1→4)-β-D-glucan chains, which are β-D-Glucose units linked by a glycosidic linkage between the

hydroxyl groups on carbons one at one unit to carbon four on the other unit. Due to the

stereochemistry of the β-(1→4) glycosidic linkage, every other Glc unit is inverted 180°. The chains

are held together by hydrogen bonds [4].

Figure 1: Cellulose, (1→4)-β-D-glucan chain [4].

1.3.2 Hemicellulose In plant cell walls, there are also cross-linking glycans who are often referred to as hemicelluloses,

which are polysaccharides with the function of forming a network by hydrogen-bonding to the

cellulose microfibrils. There are two major kinds of hemicelluloses present in flowering plants,

xyloglucans (XyGs) and glucuronoarabinoxylans (GAXs). A third hemicellulose which is common in the

order Poales (in clade commelinoid monocots), containing grasses and cereals, is the “mixed linkage”

(1→3),(1→4)β-D-glucan, and these β-glucans differentiates these species from other commelinoid

species. Other, less abundant polysaccharides in plant cell walls are glucomannan,

galactoglucomannan and galactomannan, which are likely involved in linking microfibrils in some

primary cell walls [4]. The hemicelluloses are structurally similar to cellulose, in different manners.

The xyloglucans are very similar by having the same kind of backbone and the main difference is the

substitutions described below. The major similarity of glucuronoarabinoxylans and cellulose is the β-

(1→4) linkage in the backbone, except that it occurs between Xyl units instead of Glc units, as well as

the substitutions which may occur at the xylan chain which do not exist at the cellulose. The different

kinds of mannans are similar to cellulose by having the β-(1→4) linkage in the backbone.

Glucomannan and galactoglucomannan contain segments which are identical to cellulose with the β-

(1→4) linkage between Glc units.

Xyloglucans

In dicots, such as poplar, and about half of the monocots, the xyloglucans are the major hemicellulose

to cross-linking the cellulose microfibrils. Xyloglucans have a main linear chain of (1→4)-β-D-glucan,

which has a great number of α-D-Xyl units linked at the O-6 position of the Glc units of the main

chain. Occasionally and depending on the species, further substitutions are made to the Xyl units

with α-L-Ara or β-D-Gal. The Gal unit can also be substituted by a α-D-Fuc, making it a

(fucogalacto)xyloglucan. The xyloglucans are organized in block-like structures of 6 to 11 sugars and

have three main variants of structure [4]. It is commonly found in most plant xyloglucans that every

fourth Glc unit is unsubstituted, hence the oligosaccharides from xyloglucan are often analysed in

the format of four residues. This, due to the usage of endo-(1→4)-β-glucanases who are able to

cleave the backbone only at the unsubstituted Glc unit [17].

The first variant of xyloglucans, fucogalacto-xyloglucans, is usually found in most dicots and all

noncommelinoid monocots. Fucogalacto-xyloglucans are composed of nearly equal amounts of XXXG

and XXFG (described in Table 1), but sometimes variations can occur and occasionally an α-L-Ara is

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added at a few places along the glucan chain (Figure 2A). The second variant of the xyloglucans are

the arabino- xyloglucans, found in solanaceous species and peppermint, where only two out of every

four glucosyl units contains a Xyl unit, which are further substituted with either one or two Ara units.

An acetyl group replaces the third Xyl unit in the arabino- xyloglucans. A mixture of AXGG, XAGG and

AAGG (Table 1) subunits is hence produced in the arabino- xyloglucans (Figure 2B). The third and final

variant of xyloglucans are found in the commelinoid monocots in small amounts and consists of

random additions of Xyl units to the main glucan chain and seldom with any further sugar

substitutions [4].

A compositional analysis using an endo-(1→4)-β-glucanase of xyloglucan from suspension-cultured

poplar cells from 1994 demonstrated that there are mainly four kinds of oligosaccharide repeating

units which constitutes the polysaccharide. These units were determined to be XLFG, XXFG, XXLG and

XXXG and were distributed randomly, but mainly at the same ratio among them, 0.53 : 2.08 : 0.65 :

1.0, respectively. XXXG and XXFG, the main constituents of (fucogalacto)-xyloglucans, along with

XLFG and XXLG, being common in all dicots are expected to be found [13, 18].

Table 1: Description of the single-letter designators of xyloglucan side chains, based on the non-reducing terminal sugar. Shortened versions are seen in figure 2 (A) and (B) below.

Single-letter

designator

Terminal sugar Side group on glucan chain

G D-Glucose None X D-Xylose α-D-Xyl-(1→6)- L D-Galactose β-D-Gal-(1→2)- α-D-Xyl-(1→6)- F L-Fucose α-L-Fuc-(1→2)- β-D-Gal-(1→2)- α-D-Xyl-

(1→6)- A L-Arabinose α-L-Ara-(1→2)-α-D-Xyl-(1→6)-

Figure 2: The two variants of xyloglucans; (A) Fucogalacto-xyloglucan, α-D-Xyl units, (1→6) linked to backbone on the second and third Glc units. On the fourth Glc unit a t-α-L-Fuc-(1→2)-β-D-Gal has been added to O-2 of the Xyl unit. (B)

Arabino-xyloglucan, only two α-D-Xyl units are (1→6) linked to two out of four Glc units of backbone. Additionally, one or two α-L-Ara units are attached to the Xyl units at O-2 position. The third Glc unit is occupied by an acetyl group [4].

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Glucuronoarabinoxylans

In the commelinoid monocots, such as grasses, the glucuronoarabinoxylans cross-links the majority

of the cellulose microfibrils. The glucuronoarabinoxylans have a main backbone chain of β-D-Xyl

units, linked by a (1→4) glycosidic linkage (Figure 3), with diverse substitutions depending on the

species. As for the commelinoid monocots, there is a strict addition of α-L-Ara units to the O-3

position of the Xyl units in the main chain. Further substitutions possible are feruloyl groups,

esterified to the O-5 position of the Ara units, or GlcA, which can be added to the O-2 position of the

Xyl units of the main chain. In noncommelinoid monocots and dicots there are

glucuronoarabinoxylans as well, although not in the same high amounts as the xyloglucans, and these

are substituted in the same manner as for the commelinoid monocots, with Ara units, the difference

is that the Ara units can be attached to the main chain at the O-2 position as well as the O-3 position.

Similar for both commelinoids and noncommelinoids are that the GlcA units are added to only the

O-2 position of the backbone [4].

Figure 3: Glucuronoarabinoxylan (dicot or noncommelinoid monocot); backbone of β-D-Xyl units, on the first Xyl unit of the chain is an α-D-GlcA unit linked by (1→2) glycosidic linkage. On the third and fourth Xyl units are substituted α-L-Ara units

linked to the backbone via (1→2) glycosidic linkages [4].

1.3.3 Mannan Mannan is comprised of linear or branched polymers of mannose and belongs to one of the main

constituent groups of hemicellulose in the cell wall of higher plants. The primary function of mannan

in plants is to provide structural support and crosslink the cellulose. Other functions have also been

discovered, such as acting as storage and as a signalling molecule in plant growth and development

[19, 20]. Glucomannan is a hemicellulose commonly found in softwoods and to some extent in

hardwoods with a large variety of applications, such as a weight control agent and as a preventative

against chronic disease. It has a backbone of roughly equimolar mixtures of (1→4)-β-D-Man and

(1→4)-β-D-Glc units (Figure 4A). Branching points of the polysaccharide occur at the mannose

residues by 1,6 and/or 1,3-linkages. Another common hemicellulose with mannan is

galactoglucomannan (Figure 4B) where there simply are substitutions of Gal units to the chain. It is

not unusual to find acetyl groups on mannan structures [4, 21].

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Figure 4: (A) Glucomannan; chain of (1→4)-β-D-Glc and (1→4)-β-D-Man units. (B) Galactoglucomannan; backbone of (1→4)-β-D-Glc and (1→4)-β-D-Man units and substitutions of various amounts of α-D-Gal units attached by (1→6)

glycosidic linkage to the Man units [4].

1.3.4 Pectins In the plant cell wall, there is a mixture of heterogeneous, branched and greatly hydrated

polysaccharides that are very rich in D-GalA. Examples of various functions of pectins are determining

wall porosity, govern cell-cell adhesion at the middle lamella and acting as recognition molecules

which can alert plant cells to presence of other organisms, such as symbiotic organisms, pathogens

or insects. Pectins consist mainly of homogalacturonan (HGA) (Figure 5A) and rhamnogalacturonan I

(RG I) (Figure 5B). Homogalacturonan is a polymer of (1→4)-α-D-GalA and can contain up to 200 GalA

units. [4] These smooth regions of unsubstituted homogalacturonan are often referred to as the

“linear regions” of pectin [22]. Homogalacturonan can be substituted with Xyl units at the O-3

position of approximately half of the GalA units, this polymer is referred to as xylogalacturonan.

Rhamnogalacturonan I is a polymer of repeating disaccharides of →2)-α-D-Rha-(1→4)-α-D-GalA-(1→

and about half of the Rha units can be substituted with side chains such as arabinans, galactans or

type I arabinogalactans. [4] These regions on the pectin chain with interspersed Rha units make the

so called “hairy regions” [22].

Arabinan side chains consist of (1→5)-α-L-Ara units, which are linear with occasional branches at the

O-3 position. (Figure 5C). The galactan side chains are linear, unbranched polymers of (1→4)-β-Gal

units (Figure 5D) and the third type of side chains, the type I arabinogalactans, consist of (1→4)-β-

Gal units that are substituted at the O-3 position with α-L-Ara units (Figure 5E). Rhamnogalacturonan

II is another pectin with a complex structure with four side groups containing a diversity of sugars

and sugar linkages [4].

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Figure 5: (A): Homogalacturonan (HGA); polymer of (1→4)-α-D-GalA units. (B): Rhamnogalacturonan I, (RG I); polymer of repeating disaccharides of →2)-α-D-Rha-(1→4)-α-D-GalA-(1→. (C): Arabinan side chain of RG I; polymer of (1→5)-α-L-Ara

units (D): Galactan side chain of RG I; polymer of (1→4)-β-Gal units (E): Type I arabinogalactan side chain of RG I;

polymer of (1→4)-β-Gal units substituted with α-L-Ara at the O-3 position of the Gal units [4].

1.4 Carbohydrate-active enzymes Carbohydrate-active enzymes, or CAZymes, are enzymes involved in the build-up and breakdown of

glycoconjugates and carbohydrates, for instance plant polysaccharides. The carbohydrate-active

enzymes have been assembled in a specialist, knowledge-based database known as CAZy where five

classes of enzymes are covered. The classes are glycoside hydrolases (GHs), glycosyltransferases

(GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs) and auxiliary activities (AAs), which

includes redox enzymes acting in conjugation with the carbohydrate-active enzymes [23].

1.4.1 Glycoside hydrolases The glycoside hydrolase group (EC 3.2.1.-) of the carbohydrate active enzymes is a very diverse group

of enzymes and has, as of February 2017, 135 families and more than 360 000 classified modules

[24]. The large diversity of the enzymes is a consequence of the considerable number of different

saccharides found in nature. The general mechanism of the glycoside hydrolases is hydrolysis of

glycosidic linkages between two or more carbohydrates or between a carbohydrate and a non-

carbohydrate moiety, releasing energy essential for the organism possessing the enzyme [25].

Glycoside hydrolases are currently widely used in the food industry. Examples are; α-amylases used

for liquefication and saccharification of starch in baking and preparation of high fructose corn syrup,

endoglucanases used for improving the malting of barley in wine and beer industry and β-

galactosidases used for the production of low-lactose milk [26].

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1.4.2 Polysaccharide lyases Polysaccharide lyases (EC 4.2.2.-) belongs to a group of enzymes which cleaves uronic acid-containing

polysaccharide chains to achieve an unsaturated hexenuronic acid residue and a new reducing end

via an β-elimination mechanism. Presently, February 2017, there are 24 families and on the verge of

9100 classified modules in which polysaccharide lyases can be found. The polysaccharide lyases differ

from the glycoside hydrolases in their cleaving of the glycosidic linkage by not requiring a water

molecule [27]. Applications of polysaccharide lyases are found in the food and medical industry, this

being due to the broad use of its substrates, polysaccharides containing uronic acids, in these sectors

[28]. A concrete example is the industrial use of pectin lyases in the food sector for production and

clarification of juices, fruit drinks and wines [29].

1.5 Methods and analyses Understanding the characteristics of glycans in biological functions and the advances in disease

discovery has been facilitated by mass-spectrometry-based glycomics and glycoproteomics. Both

fields, glycomics and glycoproteomics, are dynamic and are evolving at a rapid pace [30].

The term glycome refers to the complete set of glycans and glycoconjugates that cells produce under

specific conditions regarding space, time and environment. Hence, “glycomics” describes the studies

regarding the glycome. Glycomic analyses are essential for understanding how glycans relates to a

certain biological event, and since glycans participate in a large amount of biological processes, the

analyses are of great importance [31].

In glycomics, different separation techniques, such as liquid chromatography (LC), and capillary

electrophoresis are regularly combined with mass spectrometry (MS) to characterize the structural

diversity and large complexity of the glycome and its association with a number of different complex

biological systems. Further methods used for structural determination and characterization of

glycans are electron capture dissociation (ECD), collision-induced dissociation (CID) and infrared

multiphoton dissociation (IRMPD) among more [30]. Examples of soft ionization techniques used for

glycomics mass spectrometry are matrix-assisted laser desorption ionization – time of flight (MALDI-

TOF) and electrospray (ESI) where ions are produced using electrospray where a high voltage is

applied to a liquid to create an aerosol, in other words creating small charged molecules distributed

in a gas. [32].

Mass spectrometry, being a powerful tool for the sensitive and definitive glycan analysis, continues

to evolve and improve as an important tool for defining glycan structures. One of the bottlenecks of

glycobiology being the analysis of glycan structures, where the samples often come in small

quantities, MS has come to terms with [32].

The 3,5-dinitrosalicylic acid (DNSA) assay is a rapid and straightforward method for quantification of

reducing sugars. The method exploits the chemical reactivity of DNSA to detect the reducing sugars

by the oxidation occurring at the aldehyde or ketone functional group of the sugar to a carboxyl group.

As the oxidation of the sugar occur, the yellow DNSA is reduced to 3-amino, 5-nitrosalicylic acid (ANSA)

which has a red or brown colour. The colour of ANSA can be detected by absorbance measurements

at 575 nm. The method is in general simple and flexible but also very sensitive, hence it is important

to be precise in preparations of the reagent. Using the assay, a working reagent containing a small

amount of glucose is made, making it possible to extend the linearity of the response of the assay to

cover lower sample concentrations. Having glucose in the working reagent also raises the reducing

sugar concentration overall and places the concentrations in the limited linear range of concentrations

[33].

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2. Materials and methods

2.1 Culture and harvest A volume of 4 ml of poplar cells (Populus trichocarpa) in liquid shake cultures was inoculated into 45

ml of fresh MI-medium (yeast extract, lactose, sodium chloride, potassium phosphate, mono- and

dibasic, sodium laruyl sulfate, sodium deoxycholate, indoxyl-beta-d-glucuronide

cyclohexaylammonium salt, 4-methylumbelliferyl-beta-d-galactopyranoside and cefsulodin). Two

biological replicates were made. Cultures were left shaking during seven days before harvest. Cells

were harvested by an initial centrifugation (4000g, 10 minutes) were the supernatant was discarded.

Following centrifugation, were three water washes of the cells. Cells were freeze-dried and disrupted

by ball milling using small steel balls (30 Hz, 2x30 seconds).

2.2 AIR (Alcohol insoluble residue) preparation Once the cells had been disrupted the AIR preparation was performed by initially shaking the biomass

in a rotating wheel for two hours with 80 % ethanol, using a volume of approximately five times the

amount of biomass. Next, the biomass was centrifuged (3500g, 10 minutes) and the supernatant

discarded. The ethanol wash was repeated and left in the rotating wheel overnight, twice. A second

kind of wash of the biomass was performed three times using acetone where the samples were left in

the rotating wheel for only 10-20 minutes. After each wash, the samples were centrifuged (4000g, 10

minutes) and the supernatant discarded. The third and final wash consisted of using methanol three

times, again left in rotating wheel 10-20 minutes and centrifuged (4000g, 10 minutes) after each wash.

The AIR samples were freeze-dried and stored in a freezer.

2.3 Starch removal Starch was removed from the AIR samples using a porcine pancreatic α-amylase (Sigma Aldrich). The

AIR samples were soaked in a small amount of 0.01 M phosphate buffer, pH 7, with 100 mM KCl2.

Samples were boiled in a water bath for five minutes, gelatinizing the starch granules. Following, the

temperature of the water bath was equilibrated to 40°C and a small amount, approximately one tenth

of the weight of the carbohydrate, of α-amylase was added to the samples which were incubated for

one hour in the water bath. Next, a full dose of α-amylase was added and further incubation during

30 minutes was performed. Following, four volumes of cold, absolute ethanol were added to the

samples and polysaccharides was precipitated at -20°C overnight. Afterwards precipitation, the

samples were centrifuged (1500g, 5 minutes) and the supernatant was discarded. Three further

washes using cold absolute ethanol were performed with centrifugation between each wash. Finally,

the samples were air-dried. To confirm the digestion of the starch, a small amount of AIR residue was

placed on a glass slide with one drop of water and one drop of 1% iodine solution. The slide was

studied under light microscopy where starch granules appear purple.

2.4 Sulphuric hydrolysis Sugar analysis by sulphuric acid hydrolysis was performed to determine which monosaccharides were

present in the AIR samples and in which concentration. Sulphuric acid hydrolysis was necessary to

perform in addition to methanolysis, described below, since it can hydrolyse crystalline cellulose, and

methanolysis cannot. Standards were used containing nine monosaccharides; fucose, arabinose,

rhamnose, galactose, glucose, xylose, mannose, glucuronic acid and galacturonic acid. The amount of

each monosaccharide used was 500 μg. The standards were treated as the samples. Approximately 4

mg of AIR was incubated for one hour at room temperature in 125 μl of 72 % H2SO4. Following, 1375

14

μl of water was added and the tubes were sealed tightly for further hydrolysis during three hours at

100°C. Once hydrolysis had finished, 500 μg of inositol (internal standard), 50 μl of 10 mg ml-1, was

added to the samples. The samples were filtered using 0.45 μm filters, diluted 1:50 with water and

run in high-performance anion-exchange chromatography coupled with pulsed electrochemical

detection (HPAEC-PAD) with a ICS-3000 system (Dionex) equipped with a CarboPac PA1 column (4x250

mm, Dionex). For the analysis of the sugars, an isocratic program in water at 1 ml min-1 was used,

which included a conditioning step before each run with concentrated sodium acetate and sodium

hydroxide. Post-column addition of sodium hydroxide (pump 2; 0.5 ml min-1) was necessary to ensure

a stable and accurate signal throughout the run.

2.5 Methanolysis Sugar analysis by methanolysis was performed in order to determine the uronic acid content of the

AIR samples. As in the sulphuric acid hydrolysis, standards were used with the same nine

monosaccharides with a total amount of 500 μg of each and were put through the same protocol as

the samples. 1 ml of 2M HCl was added to approximately 1 mg of AIR. Tubes were flushed with argon

to avoid humidity and tightly sealed. Samples were incubated for five hours at 100°C in a heating block.

Neutralization using 200 μl pyridine was performed and tested using pH strips. Samples were cooled

to room temperature and dried. Methoxyl groups were hydrolyzed using 1 ml of 2M TFA

(trifluoroacetic acid), incubating for one hour at 120°C. The samples were filtered through 0.45 μm

filters and 100 μl was air-dried. The dried sample was resuspended in 1 ml of water and run in high-

performance anion-exchange chromatography coupled with pulsed electrochemical detection

(HPAEC-PAD) with a ICS-3000 system (Dionex) equipped with a CarboPac PA1 column (4x250 mm,

Dionex). The uronic acids were separated using 30mM sodium hydroxide and a gradient of increasing

sodium acetate, from 0 to 0.5M, over 25 minutes. No post-column addition was necessary in this case

as there was a small amount of sodium hydroxide running all the time.

2.6 Bradford protein assay Bradford protein assay was used for determination of the protein content of the AIRs. To be able to

quantify the protein content, a standard curve using bovine serum albumin was used. A Bradford

working reagent was created from the Bio-Rad Protein Assay Dye (Bradford) Reagent Concentrate by

mixing reagent concentrate and water in a ratio of 2:7. The assay was performed by adding 900 μl of

working reagent to 100 μl of sample, which consisted of AIR dissolved in water, followed by a five-

minute incubation in room temperature. The AIR concentration used for the protein assay was 10 and

20 g l-1. The absorbance of the samples was measured at 595 nm and as a blank for the measurements,

100 μl water in 900 μl working reagent was used.

2.7 DNS assay and enzyme incubations The DNS (3,5-dinitrosalicylic acid) assay was used for fast quantification of reducing sugars. As the

specific endo-enzymes cleaves the polysaccharides in the AIR, creating oligosaccharides, the amount

of reducing sugars increase, hence being able to quantify using the DNS assay where differences

measuring absorbance can be observed. The enzyme incubations consisted of enzyme, buffer (varying

due to optimal conditions for the enzymes) and substrate. The incubations had incubation times of

two kinds, overnight and one hour. The enzymes (Nzytech) used in the assay are presented in table 2

below along with volume of enzyme used in a 250 μl incubation, optimal buffer conditions used during

specificity determinations and origin of the enzyme.

15

Table 2: Enzymes used in assay, their corresponding GH-family (Glycoside hydrolase family), the organism of origin, optimal buffer conditions used during specificity determination and volume of enzyme used in a 250 μl incubation.

Enzyme GH-family

Organism Optimal buffer conditions

Enzyme volume in a 250 μl incubation

Cellulase 6A GH6 Podospora anserina

50 mM sodium phosphate, pH 7

5 μl

β-Mannanase 26A GH26 Cellvibrio japonicus

50 mM sodium phosphate, pH 7

5 μl

Xylanase 10A GH10 Cellvibrio japonicus

12 mM citrate, pH 6.5/50 mM sodium

phosphate, pH 7

5 μl

Xyloglucanase 74A GH74 Clostridium thermocellum

20 mM sodium phosphate, pH 7

5 μl

Galactanase 53A GH53 Cellvibrio japonicus

50 mM sodium phosphate, pH 7

5 μl

Unsaturated rhamnogalacturonyl

hydrolase 105A (RGase)

GH105 Bacillus subtilis 50 mM sodium acetate, pH 4

10 μl

Arabinanase 43A GH43 Bacteroides thetaiotaomicron

50 mM sodium phosphate, pH 7

15 μl

Endopolygalacturonase 28A

GH28 Dickeya dadantii 100 mM sodium acetate, pH 6.5

5 μl

For all enzyme incubations, 5 μl of enzyme was enough to see activity, with the exception of the

arabinanase and unsaturated rhamnogalacturonyl hydrolase where higher enzyme concentration was

required to see any activity at all. Initially, standard sugar substrates were used to determine whether

the enzyme had activity or not for certain polysaccharides. All enzymes were incubated with all of the

standard sugar substrates except for Avicel, which was replaced with carboxymethyl cellulose due to

low solubility in water. For all incubations with standard sugar substrates a concentration of 1 g l-1 of

substrates was used. All standard sugar substrates had an addition of a small amount of 2 % azide to

prevent any growth. The standard sugar substrates used in this thesis were chosen due to them being

commonly found in plants. The standard sugar substrates used are presented in table 3 below.

Table 3: Standard sugar substrates summarized with corresponding abbreviation and source.

Standard sugar Abbreviation Source

Carboxymethyl cellulose CMC Sigma

Cellulose, Avicel Avi Fluka

Konjac Glucomannan KGM Megazyme

Carob Galactomannan CGM Megazyme

Xyloglucan (from tamarind) XG Megazyme

Wheat arabinoxylan WAX Megazyme

Larch Arabinogalactan LAG Megazyme

Lupin Galactan LG Megazyme

Pectin from citrus fruits CPe Sigma

Pectin from apples APe Fluka

Sugar beet Arabinan SA Megazyme

Rhamnogalacturonan I from potato pectic fibre

RG1 Megazyme

Barley Beta Glucan BBG Megazyme

Birchwood Xylan BX Sigma

16

Once the specificity for certain polysaccharides had been determined, the substrate for the

incubations was the AIR. The concentration of AIR used in the incubations varied between 1 g l-1 (AIR2)

and 2 g l-1 (AIR1). All of the final incubations with AIR contained only a buffer of 20 mM sodium acetate,

pH 6.5, due to limitations of the ESI-MS (electrospray ionization-mass spectrometry) where large ions

(e.g. phosphate) would have interfered with the obtained oligosaccharides. A few tries of sequential

incubations were made as one-hour incubations but for two enzymes. An example was a one-hour

incubation of AIR with cellulase, followed by boiling of sample to inactivate the cellulase. Sample was

then incubated with xyloglucanase for one hour.

For the DNS assay, a DNSA stock reagent was prepared containing 1 % (w/v) DNSA, 0.2 % (v/v) phenol,

0.05 % (w/v) Na2SO3 and 1 % NaOH. From the stock reagent, a working reagent was prepared. The

working reagent varied between two types; DNSA-10 and DNSA-20. DNSA-10 consisted of 20 ml of

stock reagent with 10 μl of 20 % glucose solution and DNSA-20 consisted of 20 ml of stock reagent

with 20 μl of 20 % glucose solution. The addition of glucose in the working reagent raises the overall

reducing sugar concentration in the limited linear range of concentrations. For quantification of

reducing ends, standard curves were made using increasing glucose concentrations from 0 to 1 g L-1.

Performing the DNS assay on the finished enzyme incubations started with addition of an equal

amount of volume of DNSA working reagent to the samples. Next, the samples were heated for seven

minutes in 95°C where the colour change developed. The samples were cooled to room temperature

using a cold centrifuge (11 000 rpm, 3 minutes, 4°C), which also spun down solids. The supernatant

was read in a 1 cm polystyrene cuvette in a UV/visible spectrophotometer at a wavelength of 575 nm.

Incubations containing water instead of enzyme, treated as the ordinary incubations, were used as

blanks for the reading.

2.8 Mass spectrometry and purification The initial runs in the mass spectrometer did not provide any readable results which led to the theory

of the samples not being pure enough. Trial and error led to the follow purification steps necessary to

see results. The AIRs were dialysed using Spectra/Por molecular porous membrane tubing with a

molecular weight cut-off of 6-8 kDa to obtain only oligo- and polysaccharides. The dialysis proceeded

two nights before the water was exchanged for another night. The dialysed AIRs were freeze-dried

and stored in freezer. To remove the remaining enzyme, the samples were boiled for five minutes

followed by being filtered in Amicon Ultra 0.5 ml centrifugal filters (11 000 rpm, 15 minutes). The

supernatant was collected and put through solid phase extraction, HyperSep™ Hypercarb™ cartridges

(Thermofischer, UK), where it was desalted. The solid phase extraction was initially conditioned by

passing 2 ml of conditioning solvent (90 % acetonitrile in milliQ water with 0.1 % formic acid) through

the cartridge bed. An additional 2 ml of milliQ water was passed before the samples were loaded at a

slow pace of approximately one drop per second. Another 2 ml of milliQ water was passed through

the bed for cleaning of the absorbed sample. Elution was made by adding 0.5 ml of elution solvent (50

% acetonitrile in milliQ water with 0.1 % formic acid). Oligomeric mass profiling (OLIMP) was

performed by electrospray ionization mass spectrometry (ESI-MS) using a Q-TOF2 mass spectrometer

(Micromass, UK). The samples were then infused into the positive mode operated Q-TOF through the

autosampler at a rate of 8 µl min-1. Capillary and cone voltage were set to 3.3kV and 80V, respectively.

17

3. Results

3.1 Sugar analyses The results of the sulphuric hydrolysis and the methanolysis can be seen in figures 6 and 7 below,

showing the sugar percentage of the sugars fucose, arabinose, rhamnose, galactose, glucose, xylose,

mannose, galacturonic acid and glucuronic acid of AIR1 and AIR2, respectively. As seen in the figures,

the major type sugar found in both AIRs is glucose. After glucose follows galactose, galacturonic acid

and arabinose, and these four sugars constitutes over 90 % of the tested types of sugars in both AIRs.

Figure 6: Percentage of sugars fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid established by sulphuric acid hydrolysis (blue) and methanolysis (blue) achieved in AIR1.

18

Figure 7: Percentage of sugars fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid established by sulphuric acid hydrolysis (blue) and methanolysis (blue) achieved in AIR2.

3.2 Bradford protein assay The protein concentrations of the AIRs were determined to be 13.4 mg protein per 1 g of AIR1,

constituting approximately 1.3 % of AIR1 content, and 16.1 mg protein per 1 g of AIR2, constituting

approximately 1.6 % of AIR2 content.

3.3 DNS assay The results of the DNS assay for the enzyme incubations with all standard sugar substrates, incubated

overnight, are presented in table 4 below. The activity of the enzymes for the substrates are colour-

coded where red colour represents no activity at all or too low to measure, yellow colour represents

medium activity and green colour represents the preferred substrate where the activity is the highest.

All incubations have been measured using the DNSA-10 working reagent except for the incubations

with xylanase, where extra incubations were made using the DNSA-20 working reagent due to low

activity. The abbreviations of the substrates are found in table 2 in the materials and methods section.

Summarized, the enzymes and their corresponding preferred substrates are; cellulase for β-glucan

and carboxymethyl cellulose, mannanase for gluco- and galactomannan, xylanase for xylan and

arabinoxylan, polygalacturonase for rhamnogalacturonan I and arabinoxylan, arabinanase for apple

pectin, galactanase for carboxymethyl cellulose, β-glucan and galactan, xyloglucanase for

glucomannan and xyloglucanase and RGase for apple and citrus pectin.

19

Table 4: The DNS assay results for all enzymes with all substrates, incubated overnight. The unit of all values are in mg g-1 of standard sugar, glucose equivalent together with the standard deviation. The red colour symbolizes no or extremely low activity, the yellow colour symbolizes medium activity and the green colour shows the substrates for which the enzymes showed the highest activity. All incubations were made with the DNSA-10 working reagent, except for the results for the xylanase within parentheses, which were made with DNSA-20 working reagent due to low absorbance values when using DNSA-10.

Cellulase Mannanase Xylanase Polygalacturonase Arabinanase Galactanase Xyloglucanase RGase

Avi - - - (-) - - - - -

CMC 55.4 ± 0.321 - 39.5 ± 0.046 (48.8 ± 2.89) - - 159.3 ± 1.58 64.9 ± 6.03 -

KGM 10.9 ± 2.45 590.1 ± 12.4 - (-) - - 13.6 ± 2.22 235.5 ± 0.344 -

CGM 9.4 ± 0.527 412.6 ± 4.83 - (-) 1.42 ± 2.84 - - - -

WAX 1.3 ± 2.66 - 574.3 ± 11.5

(440.7 ± 3.76) 47.3 ± 18.6 - 69.6 ± 1.56 - 17.3 ± 4.70

XG - - - (-) - - 78.6 ± 8.89 317.8 ± 1.86 -

RG1 - - - (10.0 ± 1.35) 73.7 ± 3.16 - - - 19.4 ± 15.5

BBG 113.4 ± 2.89 11.3 ± 0.206 30.1 ± 3.96

(19.7 ± 4.34) - - 362.8 ± 19.3 82.0 ± 3.83 9.8 ± 3.57

BX - - 424.4 ± 8.59

(428.6 ± 2.33) - 23.4 ± 9.28 26.4 ± 6.19 - 4.8 ± 5.89

CPe - - - (-) 14.0 ± 0.344 - - - 78.3 ± 3.76

APe - - - (-) 8.6 ± 3.69 43.8 ± 0.137 - - 43.4 ± 6.80

LAG - - - (-) - - - - 7.4 ± 0.504

SA - - - (8.6 ± 1.05) - - - - 22.1 ± 1.83

LG 28.7 ± 1.67 17.6 ± 1.88 5.0 ± 1.56

(12.2 ± 1.66) - 8.2 ± 8.52 253.8 ± 14.6 25.2 ± 0.848 8.7 ± 2.36

The same enzyme incubations as presented in table 4, but incubated for only one hour, are shown in

table 5 below. The colour-coding is the same as described for table 4 above. The activity is overall

lower, except for a few cases to be discussed later. In general, the one-hour incubations reflect the

theoretical, expected specificity of the enzymes since activity for only one or two substrates can be

seen, as compared to the overnight incubations.

20

Table 5: The DNS assay results for all enzymes with all substrates, incubated for one hour. The unit of all values are in mg g-1 of standard sugar, glucose equivalent together with the standard deviation. The red colour symbolizes no or extremely low activity, the yellow colour symbolizes medium activity and the green colour shows the substrates for which the enzymes showed the highest activity. All incubations were made with the DNSA-10 working reagent, except for the results for the xylanase within parentheses, which were made with DNSA-20 working reagent due to low absorbance values when using DNSA-10.

Cellulase Mannanase Xylanase Polygalacturonase Arabinanase Galactanase Xyloglucanase RGase

Avi - - - - - - - -

CMC 24.1 ± 2.06 - - - - - 2.9 ± 0.687 -

KGM - 700.3 ± 7.31 - - - - 9.0 ± 3.73 -

CGM - 528.7 ± 3.69 - - - - - -

WAX - - (882.9 ± 6.6) - - - - 5.9 ± 1.33

XG - - - - - - 92.1 ± 4.90 -

RG1 - - - 30.9 ± 13.6 - - - -

BBG - - - - - 117.8 ± 5.52 - -

BX - - (600.2 ± 10.9) - - - - -

CPe - - - - - - - 25.6 ± 1.24

APe - - - - - - - 24.2 ± 1.51

LAG - - - - - - - -

SA - - - - - - - 16.2 ± 1.26

LG - 9.48 ± 1.51 - - 82.2 ± 1.97 504.9 ± 1.63 15.3 ± 3.09 -

The results of the DNS assay of the AIR incubations are presented in table 6 below. Incubations were

made overnight and for one hour for the biological duplicates AIR1 and AIR2.

Table 6: The DNS assay results for all enzymes with the AIRs, incubated overnight and for one hour. The unit of all values are in mg g-1 of standard sugar, glucose equivalent together with the standard deviation. All incubations were made with the DNSA-10 working reagent, except for the results for the xylanase within parentheses, which were made with the DNSA-20 working reagent.

Overnight 1h

AIR 1 AIR 2 AIR 1 AIR 2

Cellulase 77.0 ± 5.44 411.5 ± 10.37 9.4 ± 1.03 59.3 ± 6.69

Mannanase 35.0 ± 3.02 48.2 ± 4.59 14.0 ± 5.75 40.7 ± 5.41

Xylanase 2.2 ± 1.49 (15.0 ± 1.68)

7.3 ± 2.34 (96.0 ± 7.31)

(20.0 ± 1.05) (98.4 ± 4.03)

Polygalacturonase 115.4 ± 6.78 91.2 ± 8.63 21.0 ± 2.27 29.1 ± 3.02

Arabinanase 56.8 ± 3.49 112.9 ± 3.05 48.4 ± 2.45 52.0 ± 4.40

Galactanase 61.7 ± 2.99 42.5 ± 4.14 23.2 ± 3.96 51.0 ± 1.97

Xyloglucanase 57.8 ± 3.01 14.8 ± 2.50 22.1 ± 5.80 62.9 ± 0.825

RGase 14.4 ± 2.04 150.6 ± 4.63 12.7 ± 2.73 40.0 ± 8.94

21

3.4 Mass spectrometry The results from the ESI-MS are shown in figures 8-12 below. The presented figures contain zoomed

in parts of few of the spectra of which results could be read.

Spectra of the enzyme incubations with the corresponding standard sugar substrate are shown in

figures 8, 9 and 10 below. Significant peaks have been marked in the figures and interpreted in red

colour. Complete spectra and full interpretation of peaks can be found in table 9, supplementary.

Figure 8: Zoomed in spectra of standard sugar substrate glucomannan incubation with mannanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in red. Full spectra is found in figure 20, supplementary.

Figure 9: Zoomed in spectra of standard sugar substrate xyloglucan incubation with xyloglucanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in red. Full spectra is found in figure 21, supplementary.

365,3 (2H)

527,4 (3H)

689,6 (4H)

731,6 (4H + Ac)

851,7 (5H)

893,7 (5H + Ac)

1 013,8 (6H)

1 055,9 (6H + Ac)

1,0

10 001,0

20 001,0

30 001,0

40 001,0

50 001,0

60 001,0

70 001,0

80 001,0

90 001,0

100 001,0

350 450 550 650 750 850 950 1050

Inte

nsi

ty

[m/z]

KGM: Mannanase

629,6 (2H+2P)791,9 (3H+2P) 954,0 (4H+2P)

1 086,2 (4H+3P) XXXG

1 248,3 (5H+3P) XXLG

1 410,5(6H+3P) XLLG

1 432,5 (6H+3P+Na) XLLG+Na

1,0

5 001,0

10 001,0

15 001,0

20 001,0

25 001,0

30 001,0

600 700 800 900 1000 1100 1200 1300 1400 1500

Inte

nsi

ty

[m/z]

XG: Xyloglucanase

22

Figure 10: Zoomed in spectra of standard sugar substrate arabinoxylan incubation with xylanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in red. Full spectra is found in figure 22, supplementary.

Figures 11 and 12, contain the spectra achieved from the purified incubations of the AIRs which were

successful and interpretable. Figure 11 contained AIR2 incubated with xylanase and figure 12 was a

sequential incubation of AIR2 where the material first was incubated with cellulase followed by

another incubation using xyloglucanase.

Figure 11: Zoomed in spectra of AIR2 incubation with xylanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in red. Full spectra is found in figure 24, supplementary.

437,4 (3P)

569,5 (4P) 701,6 (5P)

833,7 (6P)

965,9 (7P)

1 098,0 (8P)

1 230,1 (9P) 1 363,1 (10P)

1,0

5 001,0

10 001,0

15 001,0

20 001,0

25 001,0

30 001,0

425 525 625 725 825 925 1025 1125 1225 1325

Inte

nsi

ty

[m/z]

WAX: Xylanase

515,8 ("X")

754,3 ("X"+HEPES)

1 100,6 (8P)

1,0

5 001,0

10 001,0

15 001,0

20 001,0

25 001,0

30 001,0

400 500 600 700 800 900 1000 1100 1200

Inte

nsi

ty

[m/z]

AIR2: Xylanase

23

Figure 12: Zoomed in spectra of sequential AIR2 incubation with cellulase and xyloglucanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in red. Full spectra is found in figure 23, supplementary.

1 086,2 (4H+3P) XXXG

1 436,8 (6H+3P+Ac) XLLG-Ac

1,0

101,0

201,0

301,0

401,0

501,0

601,0

701,0

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500

Inte

nsi

ty

[m/z]

AIR2: Cellulase --> Xyloglucanase

24

4. Discussion

4.1 Sugar analyses The differences between the percentage values of the methanolysis and the sulphuric hydrolysis are

simply explained by the fact that sulphuric hydrolysis is capable to hydrolyze crystalline cellulose but

it degrades the uronic acids, whereas methanolysis measures the uronic acids but is incapable of

hydrolysing crystalline cellulose. If studying the percentage of the monosaccharides in figures 6 and 7,

one can see that there is approximately a 10 % units difference between all monosaccharides. From

the methanolysis, 33 % and 35 % galactose was achieved from the sulphuric hydrolysis for AIR1 and

AIR2, respectively, compared to the 25 % and 28 %, AIR1 and AIR2, respectively, from the

methanolysis. The same applies for the glucose, but not for the arabinose.

The results of the sulphuric acid hydrolysis and methanolysis showed that the absolute majority of the

AIRs are constituted of glucose. If comparing to the literature of the primary cell wall from cambial

tissues of Populus Tremuloides where only 22 % of the cell wall was cellulose, one can observe in

figures 6 (sugar analyses of AIR1) and 7 (sugar analyses of AIR2) that the AIRs examined in this thesis

have a much higher percentage of glucose, which could be interpreted as the majority of it coming

from degraded cellulose. From the sulphuric hydrolysis of AIR1, approximately 48 % consisted of

glucose and from the methanolysis, approximately 35 % was glucose. For AIR2, approximately 49 %

was glucose from the sulphuric hydrolysis and approximately 38 % glucose from the methanolysis.

This difference could be explained by the fact that the tissues from which the cell wall was extracted

simply are different, or that there are differences between the species. Cell suspension culture cells

and cambial cells may simply differ in composition.

The other monosaccharides do however match the literature of the primary cell wall from the cambial

tissues well. If comparing the results of the methanolysis in figures 6 and 7, one can see the arabinose

being at 12 % (AIR1) and 10 % (AIR2) compared to the literature reporting 9 % of arabinan. One can

also believe some of the arabinose to be originating from the arabinogalactan, which in the literature

was reported to be 13 %, hence the amount of arabinose would most likely end up close to the

achieved results from the AIRs. The same argument applies for the rhamnogalacturonan with a

reported value of 20 % compared to the achieved 23 % (AIR1) and 17 % (AIR2) of galacturonic acid,

being one of the main components of pectins. The obtained values of rhamnose do however not

match, being approximately 2 % for both AIRs. A theory could be that the cells simply had not started

to produce the rhamnose yet but simply the galacturonic acid constituting the homogalacturonan. The

literature values of 18 % galactan and 13 % arabinogalactan would together possibly match the

achieved values of 25 % (AIR1) and 28 % (AIR2) of total galactose.

If roughly comparing the concentration of monosaccharides between the two AIRs (figures 15 and 16

in supplementary), one can see that the concentration of glucose and galactose is lower in AIR1, as

compared to AIR2, this could be due to several reasons. As an example, it could be that the cultures

were placed differently in terms of distance to the light-source on the shaking table and hence being

in different phases of growth of the cells, or that there perhaps was a sort of contamination in AIR1

affecting the results in a negative manner. It could also be due to differences in crystallinity between

the cellulose populations in the different AIRs. If studying the concentrations of arabinose of both the

methanolysis and sulphuric hydrolysis, one can see that they do not differ, which means that there

are no difficulties for the methanolysis to degrade anything containing arabinose. The concentration

of arabinose is in both sugar analyses 30 μg per mg of AIR1 and 40 μg per mg of AIR2. The galactose

and glucose do differ between the analyses. The galactose values of the methanolysis was determined

to be 60 μg per mg of AIR1 and 120 μg per mg of AIR2 compared to the sulphuric hydrolysis results of

90 μg per mg of AIR1 and 140 μg per mg of AIR2. This suggests that there is some kind of crystallinity

or other kind of bonding which the methanolysis cannot degrade but the sulphuric hydrolysis can. If

studying the glucose results, it can be determined that there most likely is more crystalline cellulose

25

in AIR1 compared to AIR2. This, since the methanolysis produced 80 μg per mg of AIR1 and the

sulphuric produced 130 μg per mg of AIR1, which is a quite high increase compared to the values of

AIR2 with the methanolysis results of 160 μg per mg of AIR2 and the sulphuric hydrolysis results of

180 μg per mg of AIR2.

4.2 Bradford protein assay The protein contents of the AIRs of approximately 1 % does not agree with the literature of the

primary cell wall composition isolated from cambial tissues of Populus Tremuloides where it was

determined to be approximately 10 %. As previously mentioned, this could likely be due to the fact

of the difference between the starting materials.

4.3 DNS assay The results of the DNS assay reflect the high specificity of the enzymes, but if incubated and treated

in the right conditions, they do have the ability to digest substrates, which they in theory should not

be able to digest. Studying table 4 of the overnight incubations, it can be seen that the majority of the

enzymes have high activity for their corresponding substrate but also medium activity for other

substrates. As an example, the cellulase has the highest activity for the β-glucan with 113.4 μg per mg

of β-glucan, glucose equivalent, and secondly for the carboxymethyl cellulose with 55.4 μg per mg of

carboxymethyl cellulose, glucose equivalent. The activity for the β-glucan explained by the β-(1,4)

linkage which occurs within every other glucose unit. The medium activities are for the standard sugar

substrates gluco- and galactomannan, arabinoxylan and galactan. This could be explained by the fact

of the cellulase not being specific enough for the structure of glucose chains, but also for mannan,

arabinoxylan and galactan, which all has the same kind of linkage, β-(1,4), between the sugar units.

The mannanase incubation overnight had as expected mainly activity for the gluco- and

galactomannan with values of 590.1 μg per mg of glucomannan, glucose equivalent and 412.6 μg per

mg of galactomannan, glucose equivalent. The same can be seen for the xylanase with main activity

for the arabinoxylan and xylan. The results of the polygalacturonase and arabinanase did not turn out

as expected, which could be explained by the fact that the enzymes are either not very active, or that

the enzymes cannot access the substrates. The galactanase incubation did have high activity for its

corresponding substrate, 253.8 μg per mg of galactan, glucose equivalent, but the highest activity was

for β-glucan with 362.8 μg per mg of β-glucan, glucose equivalent. There was also high activity for

carboxymethyl cellulose, 159.3 μg per mg of carboxymethyl cellulose, glucose equivalent. From this,

it could be concluded that the galactanase has the ability to act on glucose-chains with the β-(1,4)-

linkage as well as the galactose-chains with the same kind of linkage.

The results of the xyloglucanase incubation showed the highest activity for its corresponding substrate

xyloglucan, but had also high activity for glucomannan. This could be interpreted as the xyloglucanase

being able to cleave glucose units from glucose units or from mannan units without the xylose

substitutions. The fact of the xyloglucanase not being able to cleave glucose units of the

carboxymethyl cellulose could be explained by the fact that the carboxymethyl groups of the cellulose

blocks the xyloglucanase and hence sterically hinders the cleavage. Incubations of the unsaturated

rhamnogalacturonyl hydrolase did not provide satisfying results. The highest activities, 78.3 and 43.4

μg per mg of citrus pectin and apple pectin, glucose equivalent, respectively, matches the expectations

since there is rhamnogalacturonan in pectin. However, there was medium and medium low activity

for a lot of other substrates as well, including arabinoxylan, rhamnogalacturonan I, β-glucan, xylan,

arabinogalactan, arabinan and galactan.

If studying table 5 presenting the incubations with standard sugar substrates for one hour, one can

observe that it in most cases reflects the green results in table 4, showing the results of the incubations

with standard sugar substrates overnight, but most values are generally lower. The cellulase now only

has activity for the carboxymethyl cellulose, which makes it a great candidate for the final assay. The

26

same applies for mannanase, xylanase and xyloglucanase. The polygalacturonase only shows activity

for rhamnogalacturonan I, which is not the corresponding substrate which it should act on, but it does

however only show activity for one substrate. The expected substrates would be the pectins where

homogalacturonan exists. It can hence be concluded that the polygalacturonase has the ability to

cleave rhamnose units from galacturonic acid units in rhamnogalacturonan I, or that the standard

sugar substrate contains homogalacturonan which the enzyme acts on.

The arabinanase incubation resulted in acting only on galactan during one-hour incubation time and

is hence not a very good candidate for the final assay being unreliable. The results of the galactanase

after one hour showed main activity for galactan but also activity for β-glucan. Since β-glucan does

not exist in poplar, the galactanase qualifies for the final assay. The incubation with the unsaturated

rhamnogalacturonyl hydrolase had the lowest activities of all incubations where the highest activities

was found for the pectins, 25.6 and 24.2 μg per mg of citrus pectin and apple pectin, glucose

equivalent, respectively. It would be expected to have activity for rhamnogalacturonan I, but this was

not the case.

The only difference between the overnight incubations and the one-hour incubations is the time. From

the achieved results, there seems like the enzymes starts to digest substrates which they normally

would not act on if they get enough time. However, it is clearly more suitable for the final assay to use

the one-hour incubation time, mainly for the specificity of the enzymes but also for the rapidity of the

method in general.

Results from the incubations with enzymes and AIRs for both overnight and one-hour incubations in

table 6 shows that there is activity on the polysaccharides in the AIR. Some of the one-hour incubations

reflect the overnight incubations in the manner of having smaller values and hence could be believed

to have not been able to continue until completion. As an example, the overnight incubation with

cellulase yielded 77 μg per mg of AIR1, glucose equivalent as compared to the one-hour incubation of

only 9.4 μg per mg of AIR1, glucose equivalent. These incubations for AIR1 had the enzymes cellulase,

mannanase, polygalacturonase, galactanase and xyloglucanase. For AIR2, the incubations where the

value was higher for the overnight incubations with cellulase, polygalacturonase, arabinanase and

rhamnogalacturonyl hydrolase. These differences could be due to the fact that the biological

duplicates turned out different overall, with as previously mentioned more crystalline cellulose found

in AIR1 or as previously mentioned, the enzymes start acting on substrates if there is enough time or

have the possibility to due to some kind of degradation. The incubations not mentioned above for the

AIRs yielded approximately the same or values very close to each other, leading to the belief that the

reactions reached completion during both the one hour incubation and the overnight incubation.

From the methanolysis the sugars found in the smallest amounts were fucose, glucuronic acid,

mannose, xylose and rhamnose for both AIRs. If comparing to the overnight incubation values, since

the reaction should reach as close to completion as possible, one can see that the results agree. The

AIR1 incubations with the smallest relative values include the mannanase and xylanase, which

matches the fact that there according to the sugar analyses are very small amounts of these sugars in

AIR1. It is more difficult to compare the cellulase activity to the glucose amount found in the AIRs since

the glucose units could originate from more than only cellulose, but the highest relative activities do

agree with the achieved results from sugar analyses as well. The cellulase is among the highest for the

overnight incubations, which is explained from the large amount of glucose found in the AIRs, in which

can be assumed to be a large part cellulose. The same theory can be applied for the arabinanase, since

approximately 10 % was found to be arabinose.

The theory of more crystalline cellulose in AIR1 is supported from the fact that the activity of the

cellulase is among the highest activities in AIR2 for both the overnight, 411.5 μg per mg, and the one

hour, 59.3 μg per mg, incubations, whilst it had the lowest value for the one-hour incubation, 9.4 μg

27

per mg, for AIR1. However, the results may as well be due to AIR2 containing more amorphous

cellulose as compared to AIR1 and hence the cellulose showing higher activity towards AIR2.

4.4 Mass spectrometry The achieved spectra of the standard sugar substrates with their corresponding enzyme were easily

interpreted due to the clear signal and patterns found in the spectra, figures 8, 9 and 10. The

mannanse in figure 8 has successfully cleaved hexoses of mannan, assumingly. The xylanase, figure

10, has cleaved of oligosaccharides ranging from three to ten, of pentoses, assuming to originate from

xylan and the same can be seen for the xyloglucanase incubated with the xyloglucan, figure 9. In this

figure, several kinds of combinations of hexoses and pentoses can be seen. The combinations XXXG

and XXLG commonly found in plants, have been identified.

Spectra of the AIR incubations were more difficult to analyse as compared to the standard sugar

substrates due to low signal and high background signal. The spectra presented in the results section,

figures 11 and 12, provided good and original results. In figure 11, showing the incubation of AIR2 and

xylanase, several peaks of various lengths of pentoses can be found. Combinations where acetylations

and methylations can be found for oligosaccharides of two, three, seven and eight pentoses as shown

in the figure, which could not be found in any other spectra of AIR and enzyme incubations. The

sequential incubation of AIR2 with cellulase and xyloglucanase in figure 12 also provides readable and

original results of two peaks only identifiable with xyloglucanase, the peaks representing XXXG and

XLLG-Ac. These peaks could only be seen in the sequential incubation and not in the normal incubation

with only xyloglucanase, see figure 30 in supplementary. Figures 25-30 presented in the

supplementary contains spectra of AIR incubations and enzymes which also had readable peaks

possible to interpret as certain oligosaccharides.

28

5. Conclusions Conclusions possible to draw from the project summarizes that there are more studies required to

complete the final assay. As of currently, the method is not feasible due to unreliable and inconsistent

results. During the project, there was an additional need of purification steps necessary to perform to

continue forward.

There is also a requirement of optimization regarding the purification used, as an example the solid

phase extraction which seemed unstable at certain occasions, seeing as an example, large amounts of

HEPES buffer originating from the enzyme buffer in some of the samples, whilst other samples run at

the same time were pure. As seen in table 11 in supplementary, one interpretation has two

alternatives of which one consists of a HEPES molecule attached to another interpretation of three

pentoses, the peak with a mass-to-charge ratio of 754. This peak could be found in two spectra, the

incubations of AIR2 with xyloglucanase and xylanase. Differences could also be seen when the

background samples were created where there seemed as new signals appeared after the solid phase

extraction, not existing before.

To improve purification further there could be attempts to fractionate the polysaccharides of the

complex AIR and hence making it able for enzymes to reach substrates which currently could be

sterically hindered, a possible explanation for low absorbance values from DNS assay measurements

as well as lack of oligosaccharides to be seen in spectra. Possible fractionation methods to use could

include fractionation of hot buffer-soluble solids, chelating agent-soluble solids, diluted alkali-soluble

solids and concentrated alkali-soluble solids.

Further studies to be made should include sequential and synergetic incubations due to the presented

results of the sequential incubation of cellulase and xyloglucanase of AIR2, which gave promising

results of the commonly found plant oligosaccharides XXXG and XXFG. Tries using other sequences

and synergetic combinations were attempted in the project but no results could be obtained from

these.

Suggestions of the final multiplex assay is to design it in a 96-well plate where the enzymes would

constitute one of the sides (as an example the long side) and different tissues or samples could be

tested for each enzyme, placed on the other side (the short side). The 96-well plate could be easily

incubated for enzyme deconstruction, put through the DNS assay where the heating could be possible

using a thermo-mixer or polymerase chain reaction (PCR) equipment, making it possible to set

individual temperature programs for both incubation and colour development. Absorbance

measurements could be carried out in a 96-well-platereader.

Regarding the assay overall, there is great potential for its usage, as seen how the standard sugar

substrates provided good results. However, when attempts were made to the complex cell wall

material, complications arose which means more studies are required for the assays completion.

29

6. Supplementary

6.1 Sugar analysis of standard sugar substrates In figures 13 and 14 below, the percentage of monosaccharides obtained from trifluoroacetic acid

(TFA) hydrolysis (121°C, 3h) can be seen for the standard sugar substrates. 50 μl of solutions of 5 g l-1

of the standard sugars were hydrolysed in 950 μl TFA (2M).

Figure 13: Percentage of sugars achieved from TFA hydrolysis for the sugar standard substrates Avicel (Avi), carboxymethyl cellulose (CMC), galactomannan (CGM), glucomannan (KGM), arabinoxylan (WAX), xyloglucan (XG) and β-glucan (BBG).

Figure 14: Percentage of sugars achieved from TFA hydrolysis for the sugar standard substrates xylan (BX), rhamnogalacturonan I (RG1), citrus pectin (CPe), apple pectin (Ape), galactan (LG), arabinan (SA) and arabinogalactan (LAG).

30

6.2 AIR sugar concentration The concentration of sugar achieved from the sulphuric hydrolysis and methanolysis is presented in

figures 15 and 16 below for AIR1 and AIR2, respectively.

Figure 15: Concentration of sugars in μg of fucose, arabinose, rhamnose, galactose, glucose, xylose mannose, galacturonic acid and glucuronic acid established by sulphuric acid hydrolysis (blue) and methanolysis (blue) per mg of AIR 1.

Figure 16: Concentration of sugars in μg of fucose, arabinose, rhamnose, galactose, glucose, xylose mannose, galacturonic acid and glucuronic acid established by sulphuric acid hydrolysis (blue) and methanolysis (blue) per mg of AIR 2.

31

6.3 Bradford standard curve The standard curve for the Bradford protein assay is shown in figure 17 below. The linear equation

shown in the figure was used for calculating the protein concentration of the AIRs.

Figure 17: Standard curve used for the Bradford protein assay. The concentration bovine serum albumin (BSA) plotted against the achieved absorbance values at 595 nm. The achieved linear equation was used for calculating the protein concentration of the AIRs.

6.4 Glucose standard curves In figures 18 and 19 below, the glucose standard curves for the DNS assay are presented. One was

made using the DNSA-10 working reagent and one was made using the DNSA-20 working reagent. The

achieved linear equations shown in the figures were used for calculations of the amount of reducing

ends created by the enzymes.

Figure 18: Standard curve for the incubations where the DNSA-10 working reagent was used. Glucose concentration, x-axis, plotted against the achieved absorbance at 575 nm, y-axis, resulted in the linear equation shown in the figure.

y = 1,267x + 0,0242R² = 0,9921

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 0,1 0,2 0,3 0,4 0,5 0,6

Ab

sorb

ance

595

nm

Concentration BSA [g/L]

BSA Standard curve

y = 2,1824x + 0,0628R² = 0,996

0

0,5

1

1,5

2

2,5

0 0,2 0,4 0,6 0,8 1 1,2

Ab

sorb

ance

57

5 n

m

Glucose concentration [g/L]

DNSA-10

32

Figure 19: Standard curve for the incubations where the DNSA-20 working reagent was used (xylanase). Glucose concentration, x-axis, plotted against the achieved absorbance at 575 nm, y-axis, resulted in the linear equation shown in the figure.

An example of how the equations were used for calculating the amount of reducing sugars is shown

below using the mannanse and the standard sugar substrate glucomannan. Volume of sample was 1

ml.

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 = 1,3776

𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑒𝑞𝑢𝑎𝑡𝑖𝑜𝑛 𝐷𝑁𝑆𝐴 − 10: 𝑦 = 2,1824𝑥 + 0,0628

[𝑟𝑒𝑑. 𝑒𝑛𝑑𝑠] =𝐴 − 0,0628

2,1824=

1,3776 − 0,0628

2,1824= 0,6025 𝑚𝑔 𝑝𝑒𝑟 𝑚𝑔, 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡

= 602,5 𝑚𝑔 𝑝𝑒𝑟 𝑔, 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡

The achieved value above was included in a mean value of several absorbance measurements.

y = 2,1435x + 0,0937R² = 0,9952

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

Ab

sorb

ance

575

nm

Glucose concentration [g/L]

DNSA-20

33

6.5 Additional spectra

6.5.1 Guidelines specta interpretation

Table 7 below summarizes the guidelines used for spectra interpretation. The mass-over-charge values

for hexoses, pentoses, combination of hexoses and pentoses, uronic acids and possible additions have

been used for interpretation of the spectra.

Table 7: Guidelines used for spectra analysis summarized as mass-over-charge values for hexoses, pentoses, combinations of them, uronic acids and possible additions. For easy interpretation, colours have been added to hexoses (yellow), pentoses (orange) and the combination of hexoses and pentoses (blue).

Hexoses Peak [m/z]

Pentoses Peak [m/z]

Combinations Peak [m/z]

Uronic acids

Peak [m/z]

1H 203 1P 173 1H 1P 335 1U 217

2H 365 2P 305 1H 2P 467 2U 393

3H 527 3P 437 1H 3P 599 3U 569

4H 689 4P 569 1H 4P 731

5H 851 5P 701 2H 1P 497 Possible additions

6H 1013 6P 833 2H 2P 629 Na+ +22

7H 1175 7P 965 2H 3P 761 Methyl +14

8H 1337 8P 1097 3H 1P 659 Acetyl +42

9H 1499 9P 1229 3H 2P 791 H2O +18

10H 1661 10P 1361 3H 3P 923 Fuc/Rha +147

To understand the origin of certain unknown peaks, background samples were made. The background

samples consisted of AIR1, both boiled and unboiled, AIR2 both boiled and unboiled, boiled

rhamnogalacturonan I, boiled galactomannan, boiled galactanase and boiled unsaturated

rhamnogalacturonyl hydrolase. The samples were treated the exact same way as the incubations,

made with the 20mM sodium acetate buffer with pH 6.5 and put through solid phase extraction. The

background peaks able to be explained from the background samples are summarized in table 8

below.

Table 8: Summarized background peaks [m/z] described, if possible, and stated origin based on which background samples they were found in. Green colour represents background mainly found in the AIRs and pink colour mainly background from the

enzymes.

Peak [m/z]

Description Origin

146 Acetonitrile Buffer in ESI-MS and elution buffer in SPE

207 Unknown AIR1, AIR2, enzyme

227 Unknown AIR1, AIR2, standard sugar substrates

239 HEPES Enzyme

283 Unknown Enzyme

324 Unknown Enzyme

469 Unknown Enzyme

475 Graphite Graphite-beds from SPE

543 Unknown Enzyme

601 Unknown Enzyme

34

6.5.2 Spectra standard sugar incubations In figures 20, 21 and 22 below are the complete spectra of the standard sugar incubations presented.

All marked peaks have been interpreted and described in table 9 below.

Figure 20: Complete spectra of the standard sugar substrate glucomannan incubation with mannanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 9.

Figure 21: Complete spectra of the standard sugar substrate xyloglucan incubation with xylogluanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 9.

146,1

207,2

283,2

324,3

365,3

527,4

569,5

689,6

731,6

851,7

893,71 013,8

1 055,9

1,0

10 001,0

20 001,0

30 001,0

40 001,0

50 001,0

60 001,0

70 001,0

80 001,0

90 001,0

100 001,0

100 200 300 400 500 600 700 800 900 1000 1100

Inte

nsi

ty

[m/z]

KGM: Mannanase

146,2

283,3

324,3

377,4

419,4 543,5

629,6716,7

791,9

954,0

1 086,2

1 248,31 410,5

1 432,5

1,0

5 001,0

10 001,0

15 001,0

20 001,0

25 001,0

30 001,0

100 300 500 700 900 1100 1300 1500

Inte

nsi

ty

[m/z]

XG: Xyloglucanase

35

Figure 22: Complete spectra of the standard sugar substrate arabinoxylan incubation with xylanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 9.

Table 9: Summarization of peaks found in figures 20, 21 and 22 and interpretation. Pink and green colour represents background previously described in table 8 above. Yellow colour represents hexoses, orange colour represents pentoses and blue colour represents combinations of hexoses and pentoses.

Peak [m/z]

Interpretation Peak [m/z]

Interpretation Peak [m/z]

Interpretation

KGM treated with mannanase

WAX treated with xylanase

XG treated with xyloglucanase

146 Acetonitrile 146 Acetonitrile 146 Acetonitrile

207 Unknown 283 (Enzyme) 283 (Enzyme)

283 (Enzyme) 324 (Enzyme) 324 (Enzyme)

324 (Enzyme) 437 3P 377 1H + 1P + Ac

365 2H 569 4P 419 1H + 1P + 2Ac

527 3H 701 5P 543 (Enzyme)

569 3U/4P 833 6P 629 2H + 2P

689 4H 965 7P 716 Unknown

731 4H + Ac 1098 8P 791 3H + 2P XXG

851 5H 1230 9P 953 4H + 2P XXGG

893 5H + Ac 1362 10P 1086 4H + 3P XXXG

1013 6H

1248 5H + 3P XXLG/XLXG

1055 6H + Ac

1410 6H + 3P XLLG 1432 6H + 3P + Na XXFG + Na

146,2

283,2

324,3

437,4

569,5 701,6

833,7

965,9

1 098,0

1 230,1 1 363,1

1,0

5 001,0

10 001,0

15 001,0

20 001,0

25 001,0

30 001,0

100 300 500 700 900 1100 1300 1500

Inte

nsi

ty

[m/z]

WAX: Xylanase

36

6.5.3 Sequential AIR incubations In figure 23 below, the only successful sequential incubation of AIR is shown using the cellulase and

the xyloglucanase. The figure presents the complete spectra and marked peaks are interpreted and

described in table 10 below.

Figure 23: Complete spectra of AIR2 sequential incubation with cellulase followed by xyloglucanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 10.

Table 10: Summarization of peaks found in figure 23 and interpretation. Blue colour represents combinations of hexoses and pentoses.

Peak [m/z]

Interpretation

AIR2: Cellulase -> Xyloglucanase

237 Unknown

475 Graphite

1086 4H + 3P XXXG

1436 6H + 3P + Ac XLLG-Ac

237,3

475,7

1 086,2

1 436,8

1,0

501,0

1 001,0

1 501,0

2 001,0

2 501,0

100 300 500 700 900 1100 1300 1500

Inte

nsi

ty

[m/z]

AIR2: Cellulase --> Xyloglucanase

37

6.5.4 AIR incubations In figures 24-30 below, the complete spectra of all AIR incubations with enzymes which had readable

results are presented. All peaks are interpreted and described in table 11 below.

Figure 24: Complete spectra of AIR2 incubation with xylanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 11.

Figure 25: Complete spectra of AIR1 incubation with xylanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 11.

239,4

413,7

477,8

499,8

515,8

754,3

1 018,3

1 060,8 1 100,6

1 150,61 283,2

1,0

5 001,0

10 001,0

15 001,0

20 001,0

25 001,0

30 001,0

35 001,0

40 001,0

45 001,0

50 001,0

100 300 500 700 900 1100 1300 1500

Inte

nsi

ty

[m/z]

AIR2: Xylanase

175,3

249,4

263,4 393,6

475,8

569,8 702,0

1,0

10 001,0

20 001,0

30 001,0

40 001,0

50 001,0

60 001,0

100 200 300 400 500 600 700 800

Inte

nsi

ty

[m/z]

AIR1: Xylanase

38

Figure 26: Complete spectra of AIR1 incubation with galactanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 11.

Figure 27: Complete spectra of AIR2 incubation with galactanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 11.

175,1

365,1

454,3

475,3527,2

601,2

1 014,3

1 175,4

1,0

1 001,0

2 001,0

3 001,0

4 001,0

5 001,0

6 001,0

7 001,0

100 300 500 700 900 1100 1300 1500

Inte

nsi

ty

[m/z]

AIR1: Galactanase

175,3432

365,6062439,6691

475,9412

527,8168

601,9315702,1916

1,0

2 001,0

4 001,0

6 001,0

8 001,0

10 001,0

12 001,0

14 001,0

16 001,0

18 001,0

100 200 300 400 500 600 700 800

Inte

nsi

ty

[m/z]

AIR2: Galactanase

39

Figure 28: Complete spectra of AIR2 incubation with cellulase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 11.

Figure 29: Complete spectra of AIR2 incubation with RGase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 11.

175,4

180,3

337,6

365,6

475,9

527,8 702,3

1,0

1 001,0

2 001,0

3 001,0

4 001,0

5 001,0

6 001,0

7 001,0

8 001,0

100 200 300 400 500 600 700 800

Inte

nsi

ty

[m/z]

AIR2: Cellulase

175,3

347,6

365,6

475,9

527,8

585,9

690,0

1,0

501,0

1 001,0

1 501,0

2 001,0

2 501,0

3 001,0

3 501,0

4 001,0

4 501,0

100 200 300 400 500 600 700 800

Inte

nsi

ty

[m/z]

AIR2: RGase

40

Figure 30: Complete spectra of AIR2 incubation with xyloglucanase. Mass-to-charge ratio, x-axis, against intensity, y-axis. Significant peaks are marked in the figure and explained in table 11.

Table 11: Summarization of peaks found in figures 24-30 and interpretation of peaks. Yellow colour represents hexoses, orange colour represents pentoses and blue colour represents combinations of hexoses and pentoses. Pink colour represents background originating from enzyme.

Peak [m/z]

Interpretation Peak [m/z]

Interpretation Peak [m/z]

Interpretation Peak [m/z]

Interpretation

AIR1: Xylanase AIR1: Galactanase AIR2: Cellulase AIR2: Xyloglucanase

175 Unknown 175 Unknown 175 Unknown 239 HEPES

249 Unknown 365 2H 180 Unknown 318 2P + Me

263 Unknown 454 Unknown 337 1H + 1P 477 Graphite

393 2U 475 Graphite 365 2H 499 Unknown

475 Graphite 527 3H 475 Graphite 515 3P + Ac + 3Me

569 4P 601 Unknown 527 3H 531 Unknown

702 5P 795 Unknown 702 5P 754 1H + 4P + Na /515 + HEPES

1014 6H

1175 7H

AIR2: Xylanase AIR2: Galactanase AIR2: RGase

239 HEPES 175 Unknown 175 Unknown

413 2P + 2Ac + 2Me 365 2H 347 2P + Ac

477 Graphite 439 Unknown 365 2H

499 Unknown 475 Graphite 475 Graphite

515 3P + Ac + 3Me 527 3H 527 3H

754 1H + 4P + Na /515 + HEPES

601 Unknown 585 Unknown

1018 7P + Ac + Me 702 5P 690 4H

1060 Unknown

1100 8P

1150 8P + Ac + Me

1283 Unknown

239,4

318,5

477,9

499,8

515,8

531,8

754,2

1,0

5 001,0

10 001,0

15 001,0

20 001,0

25 001,0

30 001,0

35 001,0

40 001,0

45 001,0

50 001,0

100 200 300 400 500 600 700 800

Inte

nsi

ty

[m/z]

AIR2: Xyloglucanase

41

7. References

1. Stettler, R. F., Bradshaw, Jr. H. D., Heilman, P. E., Hinckley, T. M., Biology of Populus and its

Implications for Management and Conservation, 1996, 4-7.

2. Rine, J., A future of the model organism model, Molecular Biology of the Cell, 25(5), 2014, 549-553.

3. Cronk, Q. C. B., Plant eco-devo: the potential of poplar as a model organism, New Phytologist, 166(1),

2005, 39-48.

4. Carpita, N. and McCann, M. In Biochemistry & Molecular Biology of Plants, Buchanan B., Gruissem,

W., Jones, R., Eds, American Society of Plant Physiologists, 2000, 52-108.

5. Keegstra, K., Plant Cell Walls, Plant Physiology, 2010, 483-486.

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