Placement Thesis: Cover Letter

During my work placement I produced recombinant class II CDH in Trichoderma reesei,

purified and preliminary characterized the enzyme. I worked on the transformation of the

construct into the expression host, established the shaking flask cultivation of T. reesei

transformants and production of CDH by continuous cultivation in a laboratory fermenter.

Finally I chromatographically purified the enzyme and performed initial characterisation by

SDS-PAGE and activity measurements. My supervisor was a post-doctoral researcher Su Ma.

Su is originally from China and her area of expertise included gene cloning, protein

production, protein purification and electrochemistry. She has an in-depth knowledge of

Cellobiose dehydrogenase and Lytic polysaccharide mono-oxygenases which are enzymes

that she has been currently working with during her research. During the work placement I

performed my own individual experiment under her supervision which involved recombinant

production of an engineered cellobiose dehydrogenase from Corynascus thermophilus in

Trichoderma reesei QM9414.

Class II cellobiose dehydrogenase purification and classification

[Document subtitle]

Traineeship Title:

Class II cellobiose dehydrogenase purification and characterisation using Trichoderma reesei

as an expression system.

Supervisor: Su Ma Post-Doctoral Researcher

Organisation: University of Natural Resources and Life Sciences, Vienna.

Department of Food Science and Technology

Address of Organisation: Muthgasse 18, 1190 Vienna, Austria

Name: Stephen Nagle

I.D: 12318996

1

Table of Contents

Cover Letter 0

Traineeship Title 1

Table of Contents 2

Abstract 3

Abbreviations 3

1.Introduction 4

Acknowledgements 4

2. Materials and Methods 8

2.1 Strains and culture conditions 9

2.2 Protein Purification 9

2.3 Enzyme Assays……………………………………………………………………......10

2.4 SDS-PAGE and Protein Concentration…………………………………………..........10

2.5 T. reesei DNA Isolation………………………………………….................................11

2.6 Genetic Transformation of T. Reesei……………………………………………….....12

2.7 Selection, Purification and Genotyping of Transformants…………………………….14

2.8 Preparation of MA-Medium for cultivation…………………………………………...15

2.9 Preparing spore solution for MA Medium and Culture Collection…………………...16

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

3.1 Flask Cultivation and Enzyme Activity 17

3.2 Purification of CDH 20

3.3 Comparison of T. reesei and P. pastoris……………………………………………….22

Discussion 23

Conclusion 25

References 26

2

Abstract

Due to recent demands for the production of biofuels there has been significant interest in

producing engineered cellulases. One enzyme that is attractive for enzymatic cellulose

conversion is Cellobiose dehydrogenases (CDH) which is produced by Trichoderma reesei

and can be expressed at levels exceeding 100g per litre. Trichoderma reesei has the ability to

secrete large amounts of cellulolytic enzymes such as cellulases and hemicellulases. These

enzymes have the ability to convert cellulose which is a major component of plant biomass

into monomeric sugars. These sugars can then be converted to bio-ethanol through

fermentation. By engineering CDH it could be possible to increase its efficiency in degrading

cellulose, hemicellulose and lignin. Cellobiose dehydrogenase (CDH) is also an emerging

enzyme in the field of bioelectrocatalysis. CDH has been studied for applications in glucose

powered biofuel cell anodes. CDH has a unique structure with two redox active centres (FAD

and Haem b) in two separate domains which allows CDH to direct electron transfer (DET)

with modified graphite electrodes. This feature of CDH provides the possibility of 3rd

generation blood glucose biosensors as well as biofuel cell anodes. The aim of the

experiment was to produce an engineered CDH with improved enzyme activity which could

be expressed in Trichoderma reesei in large quantities. In this study we recombinantly

produced an engineered cellobiose dehydrogenase from Corynascus thermophilus (rCtCDH)

in Trichoderma reesei QM9414 (93.6 U/ml, 12.4 U/mg). Cellobiose dehydrogenase produced

a mass of 75kDa which was isolated and partially characterised, generating a 42% yield in

small scale flask cultivation.

Abbreviations

CDH, cellobiose dehydrogenase; CtCDH, CDH from Corynascus thermopilus; cyt c,

cytochrome c; DCIP, 2,6-di chloroindophenol; DET, direct electron transfer; FAD, flavin-

adenin-dinucleotid; GDH, glucose dehydrogenase; GOx, glucose oxidase; rCtCDH,

recombinantly expressed CDH from Corynascus thermophilus; HmB, Hygromicin B, KOH,

Potassium hydroxide

3

Acknowledgments

I thank Roland Ludwig and Su Ma (Universität für Bodenkultur) for their performance in

many aspects of the practical work, including plasmid isolation, screening, enzyme

production/purification, SDS-PAGE and enzyme assays. I thank Lisa Kappler (Vienna

University of Technology) for her involvement in the genetic transformation of T. reesei and

offering equipment needed for the transformation. I thank Nenad Mardetko for his help with

SDS PAGE and DNA Isolation. I thank Diarmaid de Barra for his help with enzyme assays. I

once again thank my supervisor Su Ma for her encouragement and interest in my work and

her valuable discussions on Cellobiose dehydrogenase.

1. Introduction

Trichoderma reesei is a filamentous fungus with 7 chromosomes and a 33Mb genome size. It

has the ability to secrete large amounts of cellulolytic enzymes such as cellulases and

hemicellulases. These enzymes can convert cellulose which is a major component of plant

cell biomass into glucose. They are used in various industrial applications, such as pulp,

paper production in the food and feed industries and in the textile industry. (Ander P et al,

1996). Engineered cellulases from Trichoderma reesei or other fungal sources are in demand

for the production of biofuels from plant dry matter. Fungal cellulases can be expressed at

levels exceeding 100g per liter in fungal hosts such as Trichoderma reesei and therefore are

attractive for enzymatic cellulose conversion (Cherry and Fidantsef., 2003; Kubicek et al.,

2009). The different types of cellulases secreted by T. reesei are cellobiohydrolases,

endoglucanases and different B-glucosidases which synergistically degrade cellulose.

(Henrissat et al. 1985) Trichoderma reesei is an extremely efficient producer of cellulases.

Trichoderma reesei can be used for investigating the regulation of (hemi)-cellulase gene

expression. The transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are

the main regulators of their expression. Fungi are essential decomposers in nature and they

4

are heavily involved in the carbon and nitrogen cycle. Cellulose, Hemicellulose, and lignin

are main organic polymers that are produced through carbon fixation by plants. These strains

are also being used for the conversion of plant biomass materials into second generation

biofuels. (Chundawat et al, 2011: Viikari et al, 2012). The main industrial source of cellulases

and hemicellulases is the mesophilic soft-rot fungus T. reesei (teleomorph Hypocrea

jecorina), valued for the high protein secretion capacity of its mutant strains obtained by

random mutagenesis. It is established that CDHs are produced in cellulolytic conditions and

are involved in cellulose and lignin degradation. However CDH is not an essential component

of the lignocellulose-degrading enzyme complex but can enhance both cellulose and lignin

degradation (Bao W et al, 1992).

By cultivating better industrial strains of this fungus it is possible to yield larger quantities of

bioethanol and a range of biochemical building blocks such as succinate, malate, fumarate

and glycerol. Certain techniques such as genetic engineering, gene knockout protocols and

DNA mediated transformation systems are being used to improve T. reesei strains.

Mutagenesis of T. reesei enzymes can produce mutant proteins with improved properties

which can be expressed in micro-organisms such as E.coli S.cerevisae and P.pastoris.

However this can prove difficult because of the inability of these expression systems to

provide post translational modifications such as disluphide bridges and N or O glycosylation.

Post translational modifications are needed for protein stability and protein function. The

production of T.reesei cellobiohydrolase CEL7A in P. pastoris and S. cervisia resulted in

hyper over glycosylated enzymes with compromised activities. (Boer et al., 2000; Godbole et

al., 1999; Jeoh et al., 2008; Penttilä et al., 1988; Arsdell et al., 1987). Cellulase induction in

T.reesei is reliant on the function of the Zn2Cys6 transcriptional regulator XYR1 and

knockout mutants in xyr1 are cellulase negative (Stricker et al., 2006). Most cellulases and

hemicellulases are glycosylated proteins. Glycosyl hydrolases break down the glycosidic

bonds in carbohydrates. The B (1-4) bonds on cellulose are specifically cleaved by cellulases

which results in producing cellulosic ethanol from lignocellulosic material. However to

achieve full hydrolysis of structured cellulose other enzymes such as exoglycanase,

endoglucanase and cellobiase are needed.

The extracellular fungal flavocytochrome cellobiose dehydrogenase is secreted by several

wood degrading fungi within the phyla of Basidiomycota and Ascomycota. According to

their origin, CDHs show different catalytic properties. The schematic of (Fig 1) shows that

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CDH has two distinct domains (heme domain and flavin domain) the flavin domain allows

for oligosaccharides oxidation followed by electron transfer to the heme domain. Due to these

catalytic properties CDH from T. reesei is a promising competitor for mediator less glucose

biosensors and biofuel cell anodes to be used on body tissues and fluids (Coman V et al.,

2010, Coman V et al., 2008).

The present glucose biosensors are based on glucose oxidase (GOx) or glucose

dehydrogenase (GDH). These enzymes exert a high specific activity and selectivity for

glucose. However, they promote some major defects. Due to their design, direct electrode

communication is not feasible. As a result, they depend on electron shuttling species to

contact the electrode. Electrons can be transferred by migrating reaction products (e.g.

hydrogen peroxide, “first generation” biosensors) or the help of redox mediators (“second

generation” biosensors). First generation glucose biosensors based on glucose oxidase are

dependent on a stable oxygen concentration for accurate measurements. Second generation

glucose biosensors can be affected by varying oxygen concentrations or hydrogen peroxide

production. CDH, such as GDH, cannot use oxygen as alternative electron acceptor and

prevent this negative effect (Heller A et al., 2008). Glucose oxidase and glucose

dehydrogenase based biosensors leads to oxidation of interfering compounds such as

acetaminophen, an active agent of common painkillers. CDH-based biosensors avoid these

negative effects by working at a low operational potential (Heller A et al, 2008).

The applicability of a heterologously expressed CDH would allow customization of CDH for

bioelectronics applications by genetic engineering. CDH has many attractive properties, e.g.,

its specificity for β-1, 4-linked disaccharides, its redox potentials and the increased

availability of the T. reesei enzyme could enable a range of technological applications, such

as biosensors, bioremediation, or biocatalysis. CDH has been used in colorimetric assays

(Canevascini G et al, 1982) and in amperometric biosensors (Elmgren M et al, 1992) for the

detection of lactose. CDH-based biosensors also have been used for the sensitive and

selective detection of diphenols, widely distributed toxic pollutants. In addition, CDH has a

potential role in bioremediation, since it can directly reduce munitions such as 2, 4, 6-

trinitrotoluene and indirectly degrade many more chemicals, including polyacrylate polymers

(Cameron M D et al, 2000). Finally, CDH can be used in biocatalysis for the preparation of

organic acids such as lactobionic acid (Baminger U et al., 2001). Therefore, high reported

expression levels and an easy genetic manipulation were the reason to select Trichoderma

6

reesei as expression host in this work. We report the efficient recombinant expression of

rCtCDH in T. reesei, its purification and a comparative characterization with fungal CtCDH

in respect to physical, catalytic and bioelectrochemical properties.

7

2. Materials & Methods

The chemicals for enzyme assays and buffers and reagents were purchased from Sigma

Aldrich (Steinheim, Germany) and were the highest of purity. Table 1, Table 2 and Table 3

clearly show the common solutions and mediums prepared to carry out this experiment.

Trichoderma reesei strain QM9414 (ATCC 26921) and the expression vector

pLH1_hph_PcDNA1 was used as the transformation host. CDH from Corynascus

thermophilus CBS 405.69 (CtCDH) was produced according to a published procedure

(Coudray, M. R et al, 1982)

8

2.1 Strains and culture conditionsT. reesei QM9414 (ATCC 26921) and the Δxyr1 strain (Stricker et al. 2006) derived from it

were used throughout this study. They were maintained on potato dextrose agar (PDA) plates

at 28°C. For flask cultivation strains were grown for the indicated time in 100 mL medium in

500ml flasks at 28°C on a rotary shaker at 180 rpm (Multitron 2, Infors AG). A modified

medium (Vaheri et al. 1979) containing 10 g/L carbon source, 1.4 g/L (NH4)2SO4, 2.0 g/L

KH2PO4, 0.3 g/L MgSO4·7H2O, 0.4 g/L CaCl2·2H2O, 1 g/L peptone, 0.02% (w/v)

Tween80, and 1/50 (v/v) of the trace element solution (0.25 g/L FeSO4·7H2O, 0.08 g/L

MnSO4·H2O, 0.07 g/L ZnSO4·7H2O, 0.1 g/L CoCl2·2H2O) was adjusted to pH 5.5 by

KOH and not further controlled. The culture was regularly sampled, samples were clarified

by centrifugation, and CDH activity, Cyt c activity, and protein concentration were assayed.

2.2 Protein PurificationThe enzyme was purified by hydrophobic interaction chromatography using phenyl sepharose

followed by anion exchange chromatography using Q source (AKTA explorer). The purity of

the enzyme preparation was verified by SDS-PAGE. CtCDH was prepared as published

previously (Harreither, W et al, 2010). Homogeneous CDH solutions were sterile filtered,

aliquoted and stored at –80°C.

9

2.3 Enzyme assays

CDH activity was specifically determined by following the reduction of 1 mM 20uL

cytochrome c in 860uL sodium acetate buffer, 100mM pH 5.5, containing 300mM 100uL

lactose and 20uL clear supernatant. The DCIP assay was performed by measuring the time-

dependent reduction of 3 mM DCIP at 520 nm in 100 mM 780uL sodium acetate buffer at pH

5.5, 300mM 100uL lactose solution and 20uL clear supernatant. Initial rates for the

determination of activity with DCIP and cyt c were determined at 30°C. The CDH activity

assay using DCIP measured CDH in crude extracts using lactose as electron donor and DCIP

as electron acceptor. In contrast to the DCIP method, the cytochrome c method determines

the activity of the holoenzyme CDH and not the flavin fragment.

2.4 SDS PAGE and Protein ConcentrationThe supernatants were loaded to SDS gel after EtOH precipitation as follows: 500uL of

culture supernatant was mixed with 1ml 96% ethanol and stored at -20C. After the

centrifugation at 13,000rpm for 20min at 4C, the protein pellets were suspended with 40uL

ddH2O. Samples were run with current 15 mA/gel on 12% denaturing SDS gel. The total

protein concentrations in the culture supernatants were measured by the Bradford assay

(Bradford 1979).

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2.5 T. Reesei DNA Isolation

Mycelium was removed from the agar plate with a small sterile spatula and added to 500µl

lysis buffer. The Eppendorf tube (Eppi) was left at room temperature for 10mins. 150µl of K-

acetate buffer was added to Eppi and left on ice for 5mins. The Eppi was then vortexed and

centrifuged at 15,000xg\rcf for 5mins. The supernatant was transferred to a fresh Eppi and

centrifuged again. 500µl of isopropanol was added to a fresh Eppi and the supernatant was

added. This solution was mixed by inverting. The Eppi was left at -20C for 20mins. The Eppi

was centrifuged at 4C at 12,000rpm for 10mins and the supernatant was discarded. The DNA

pellet was washed with 300µl 70% ethanol and centrifuged again. The supernatant was

discarded and the pellet was left to dry at room temperature for 5mins. The DNA was

suspended in 50µl of ddH2O.

11

2.6 Genetic Transformation of Trichoderma reesei The method for transformation was based on producing protoplasts by removal of fungal cell

wall with lytic enzymes. Purified protoplasts were able to take up DNA in the presence of

CaCl2 and polyethylenglycol(PEG). The DNA fragment was integrated into the genome.

Protoplasts are widely used for DNA mediated transformation of fungi. They are sensitive to

osmotic pressure and were kept in 1M sorbitol solutions to stabilize them. For this

experiment expression vectors were used for the transformation of T. reesei strains. Materials

and Solutions needed for genetic transformation can be seen on Table 1 and Table 3. Freshly

sporulated plate were used for preparation of T.reesei spore solution. Spores were harvested

by pouring about 1.5ml of a freshly sterilized physiological salt solution(NaCl,Tween) on a

plate. The spores were removed from the medium by a Drigalski spatula and filtered through

glass wool Eppendorf tubes into a 15ml plastic tube. One cellophane disc was added per

prepared PDA plate and the cellophane disc was flattened with a Drigalski spatula. 50-100µl

of spore solution was streaked out onto every cellophane covered plate. 6 plates were

prepared and incubated at 28C for 16-20hrs.

The plates were examined, the cellophane was covered with freshly grown mycelium. The

overlay medium was then autoclaved and placed in a water bath at 48C. The hygromicin B

was added to a final concentration of 100µg/ml. The protoplasting solution was prepared by

adding 0.15g of lysing enzymes(Trichoderma harzianum) to 30ml solution A. The enzymes

were dissolved by stirring the solution and filtered into a sterile petri dish. The cellophane

disc was put into the protoplasting solution and the mycelium was carefully scratched off of

the cellophane. This step was repeated for all cellophane discs and when all the mycelium

was transferred to the protoplast solution, the mycelium was torn into little pieces with a

sterile tweezers. The cellophane discs were incubated in the petri dish for 90 min at 28C. The

centrifuge was then cooled to 4C.

After the 90mins the mycellium was further disintegrated by gently up and down pipetting of

the mycelial debris with a cut blue 1ml tip. The mycelial suspension is then filtered through

the glass wool in the glass funnel into a sterile 50ml centrifuge tube. The glass wool was

washed with a few ml of solution A. For the rest of the procedure an ice bath was needed.

The 50ml centrifuge tube was centrifuged for 10mins at 2000rpm at 4C. The supernatent was

decanted and the protoplasts were resuspended in 10ml precooled solution B. This was again

centrifuged for 10min, 2000rpm at 4C. The supernatant was discarded and the protopßlasts

were resuspended in 200µl solution B(4C). The protoplasts were stored on ice. For the low

12

copy plasmid content 3µl of purified DNA fragment was transformed. For high copy plasmid

content 10µl of purified DNA fragment was transformed. 50µl of PEG was added to both

soluitons to enable uptake of DNA. The plates were sealed and stored in the 28C incubator

for 3 days.

13

2.7 Selection, Purification and Genotyping of TransformantsTransformants were cut out with a sterile needle from the transformation plates and

transferred to selection plates. The selection plates were stored at 28C for 3-5days to allow

sporulation of colonies. These spores were then transferred from these plates to plates

containing triton X-100(decreases growth colonies) and selection marker to separate them

(Table 4). After 2-3days colonies from the single spores from Triton X-100 plates were

transferred to small 3cm Petri dishes with selection medium for another 1-2days of growth at

28C(Table 5). Three generations of screening were performed to ensure correct genetic

transformation and to achieve optimal colony growth/spore production. Genomic DNA was

extracted from colonies of single spores and tested for correct integration of the DNA

Fragment by PCR.

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2.8 Preparation of MA-Medium for cultivation2.8g of Ammonium sulfate, 4g of monopotassium phosphate, 0.6g of Magnesium sulfate

heptahydrate, 0.6g of calcium chloride dihydrate was added to 2.5L bottle and filled to 1.76

mark and mixed. The trace element solution was prepared- 0.025g of iron sulfate

heptahydrate, 0.008g Manganese sulfate monohydrate, 0.007 zinc sulfate and 0.01g cobalt

chloride were weighed out and added to bottle. 80ml of dH2O was added and mixed and then

filled up to the 100ml mark. 2% glucose solution was prepared by adding 40g of glucose to

200ml dH2O

The trace element solution was then filtered into an autoclaved bottle. 40ml of the trace

element solution was added to the medium and mixed. Then 200ml of the 2% glucose

solution was added and mixed. The final volume in the MA medium flask was 2L. 100ml of

medium was added to 18 autoclaved flasks. The autoclaved plugs were put on top of the

flasks. The pH of the medium was changed to pH5.5 using KOH to allow appropriate pH for

fungal cell growth.

15

2.9 Preparing spore solution for MA Medium and Culture Collection

In the fume hood 1ml of NaCl/Tween solution was added to each of the 18 small PDA plates.

The solution was pipetted up and down a few times to collect all the spores. This solution was

filtered through glass wool Eppis and collected in an Eppi underneath which was then closed

and labelled. 300µl of the spore solutions was added to the 100ml MA medium flasks. These

flasks were labelled as seen below and put into the shaking incubator at 160rpm on 28

degrees. 16-1-2, 25-2-1, 12-2-1, 19-1-1, 19-3-1, 21-2-1, 12-6-1, 16-2-1, 12-2-1, 20-5-2,

19-3-2, 25-2-2, 16-1-1, 20-4-2, 33-6-1, 20-4-1, 12-4-1, 20-5-1,

For the culture collection 1ml of glycerol solution was added to 18 labelled culture collection

tubes, in the fume hood mycelium from labelled small PDA plates were transferred to these

sterile culture collection tubes with a sterile tweezers. These tubes were shaked to ensure that

the mycelium was fully submerged in the solution and they were stored in the -30 degrees

freezer. The culture collection tubes were labelled as above.

16

3.0 Results

3.1 Flask Cultivation and Enzyme Activity

CDH activity was carried out using DCIP. Enzyme activity was examined after 4 days

incubation. The results can be seen on (Fig 2). The optimal enzyme activity was recorded on

the 6th day of incubation. The flasks that showed best enzyme activity were 19-3-2, 20-5-2,

21-2-1, and 19-1-1. It was apparent that the flask 20-5-2 showed the highest enzyme activity

throughout day 4 to day 7 of incubation. The peak on the sixth day was (0.262 U/ml). A

Bradford Assay for protein concentration was also carried out (data not shown). The colour

of the 13 culture flasks were noted as seen in table 6. For the second cultivation 6 flasks were

screened altogether; the strains used were 21-2-1, 20-5-2 and 19-1-1. The results of the

enzyme activity can be seen on (Fig 3). The enzyme activity was highest on the 8th day

0.2246 U/ml (19-1-1). The standard deviation was also calculated which can be seen on the

graph (Fig 3S). The Bradford assay for protein concentration was carried out (data not

shown).

17

18

19

3.2 Purification of CDHNo CDH activity was lost during harvest, removal of the mycelia by centrifugation, and

initial concentration and desalting of the medium prior to separation on DEAE Sepharose.

The purification steps, i.e., hydrophobic interaction chromatography and anion exchange

chromatography, gave single activity peaks in Fig 4 and three activity peaks in Fig 5. This

two-step procedure (Fig 6) yielded a reddish-brown protein that was apparently homogenous

as judged by SDS-PAGE (Fig.4). The purified CDH had a specific activity of 12 U · mg−1

for DCIP reduction and 5 mg total protein content when using the Bradford protein assay).

The molecular mass was 75 kDa as estimated by SDS-PAGE (Fig 4) and (Fig 5). The DCIP

activity after Q source measured 93 U/ml.

20

21

3.3 Comparison of T. reesei and P. pastorisFrom examining the results in (Fig 7) it’s possible to compare the two different expression

systems used to express CDH. The enzyme activity for T. reesei using DCIP and Cyt c was

93.6 U/ml and 35.3 U/ml respectively. Compared to 87.2 U/ml and 23 U/ml for P.pastoris.

Examining the specific activity for T. reesei and P.pastoris using DCIP and Cyt c shows 12.4

U/mg and 4.7 U/mg for T.reesei respectively and 6.5 U/mg, 4.7 U/mg for P.pastoris.

22

Discussion

T. reesei is a model system for the degradation of plant biomass into monomeric sugars

applied in biofuel production and in glucose powered biofuel cell anodes. The aim of the

study was to recombinantly produce an engineered cellobiose dehydrogenase from

Corynascus thermophilus (rCtCDH) in Trichoderma reesei QM9414. The Class II cellobiose

dehydrogenase was purified and partially characterised. The protein concentration of rCtCDH

reached by flask cultivation was 7.5 mg/ml. This is, when compared to the other

heterologously expressed CDHs such as P.pastoris (13.2mg/ml) not extraordinary high, but

satisfactory. CtCDH has an optimum pH with Cyt c at 7.5 and with DCIP at 5.5. The DCIP

activity of 93 U/ml is considerably higher than the Cyt c activity of 35 U/ml which is as

expected due to the fact that DCIP measures both Cellobiose Quinone oxidoreductase and

Cellobiose dehydrogenase, whereas Cytochrome c only measures CDH activity, indicating

that the FAD domain efficiently reacts with cellobiose and Quinone’s regardless of the

presence of the heme domain.

The CDH enzyme reported from T. reesei QM 9414 was described as a monomeric protein of

75 kDa. The enzyme oxidized cellobiose, cellooligosaccharides, and lactose, and could be

reoxidized by DCIP and also by Cyt c, although the catalytic efficiency with the latter

electron acceptor was several orders of magnitude lower (93 U/ml compared to 35 U/ml). P

pastoris as seen in (Fig 6) has a lower specific activity compared to T. reesei which is a

promising result for the expression of CDH in T. reesei. However the low percentage yield

(42%) CDH expression in T. reesei compared to 70% yield in P. pastoris means that

upscaling the production of CDH in T.reesei is needed for it to be used in industrial

processes. Nevertheless the 2 fold increase in activity in T. reesei means it has a greater

possibility to be used in glucose powered biofuel cell anodes or in the production of biofuels.

CDH was also produced by continuous cultivation in a laboratory fermenter, the solutions

and mediums in table 3 were prepared and the fermenter was set up according to detailed

protocol. However the fermentation proved to be unsuccessful. Enzyme assays using DCIP

and cyt c were performed along with Bradford assay, nevertheless no enzyme activity or

protein concentration were recorded.

23

Purification results as seen in Fig 3, Fig 4 and Fig 5 indicate the necessity to carry out a two-

step purification procedure. The first purification step was hydrophobic interaction

chromatography using phenyl sepharose and the second purification step was anion exchange

chromatography using Q source. The SDS PAGE results show that the purity of the proteins

samples increase with each further purification step. However it is possible to see in (Fig 4)

some unwanted bands in lanes 1, 2, 3 but these bands are identical to the molecular ladder,

therefore some crossover of dyes must have occurred when running of the gel. Although it

could be said that after the hydrophobic interaction chromatography the enzyme purity was

sufficient due to the single absorbance peaks seen in (Fig 3). The result shows that the

absorbance peaks in (Fig 4) are not as defined as in (Fig 3). However according to the

purification scheme in Fig 5 an increase in DCIP activity, specific activity and purification

factor indicate the need for anion exchange chromatography.

Several interesting applications have been detailed promoting the unique catalytic properties

of CDH. Its unique structure with two redox active centres (FAD and haem b) in two separate

domains allows for direct electron transfer with an electrode. CDH has many applications for

use in analytical biosensors, enzymatic assays for the detection and quantification of

substrates and analogous molecules. The CtCDH was successfully used as biocatalyst for

glucose biosensors and glucose fuelled biofuel cell anodes (Safina, G et al, 2010), and can

efficiently communicate via DET with either thiol-modified gold electrodes or spectrographic

graphite electrodes at physiological pH. It can be concluded that the successful recombinant

expression of CtCDH and its proven applicability are important steps towards its use in

bioelectronics, but there is need for further optimization. Further research will focus on

genetic engineering of the substrate specificity of rCtCDH.

24

Conclusion

We can conclude from the experiment that T. reesei is a suitable expression system for the

production of engineered CDHs. The aim of the experiment was to purify and characterize a

Class II cellobiose dehydrogenase. From my findings it can be said CtCDH expressed in T.

reesei produced significantly higher enzymatic activity compared to other expression systems

such as P. Pastoris. A two fold increase in DCIP activity and a threefold increase in cyt c

activity shows that further research should be taken to enable the production of CDH with

optimal enzyme activity and increased percentage yields. A 43% yield of CDH in T. reesei is

quite low compared to other expression systems, therefore it should be possible to design an

experiment that would enable higher production yields of pure CDH. We can also conclude

that DCIP in the activity assay acted as an electron acceptor. The natural substrate cellulose

inhibited the substrate therefore it was replaced with lactose which acted as the electron

donor. This was similar for the cyt c activity assay, however cyt c acted as the electron

acceptor and it determined the activity of the holoenzyme CDH and not the Flavin fragment.

It can also be concluded that the genetic transformation of DNA into T. reesei genome was

successful upon examination of the PDA selection plates with selection marker. The selection

marker Hygromicin B inhibited polypeptide synthesis and translocation, therefore it was

possible to efficiently select the best transformants by liquid cultivation. We also concluded

that the selection plates that had a larger quantity of spore growth, seemed to produce higher

enzyme activity during cultivation. This can also be said for the liquid culture flasks, the

flasks which produced a yellow/green colour had higher enzyme activity than the flasks that

had a cloudy colour.

25

References

Ander P, G Daniel, B Pettersson, and U Westermark, Possible applications of

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Plagiarism of Declaration

In respect of the report above, I, Stephen Nagle, do declare that I understand fully that plagiarism is the act of copying, including or directly quoting from the work of another without adequate acknowledgement, in order to gain benefit. I do declare that the work undertaken is my own. I have not plagiarized in the past and never intend on plagiarizing in the future. The reference used in the report were taken from published documents and were used to enhance the findings in the report.

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