Toxic Compounds in Natural Water

- A Case for Laccase -

Water pollution through microcontaminants

2

Water pollution is one of the main environmental worries of our society

Test of water quality:

• Approx. 250 human and veterinarian pharmaceuticals found

• Sufficient information available for only 70 of them

• No defined limit value for most of them

Committee on the Environment,

Public Health and Food Safety

Ibuprofen

Estradiol Estrone

Ethinyl estradiol

Diclofenac

Naproxen

Anthracene Naphthalene

Acenaphthene

Phenanthrene

Water pollution through microcontaminants

3

Lindane

Estradiol Estrone

Ethinyl estradiol

• Most important female sexual hormones

• Ethinyl estradiol is much slower

metabolized and thus more effective

Estrogens

4

Ethinyl estradiol

Impact of Estrogens

5

• Sewage plants are not able to degrade

estrogen derivates

• Hormone active substances in the water body

Influence on the Environment:

• Feminization of fish

• Feminization of birds

Suspected Influence on Humans:

• Decreasing sperm count

• Testicular cancer

6

Laccase

7

• polyphenol-oxidase (EC 1.10.3.2)

• ubiquitous enzyme found in many

plants, fungi and microorganisms

• broad range of substrates,

mainly phenolic compounds

Laccase from T. versicolor, created with Jmol

with data from www.rcsb.org

Overview

8

Overview

9

The following laccases were produced successfully:

organism name of

protein

name given by

iGEM team used chassis

Bacillus pumilus DSM 27 CotA BPUL E. coli KRX

Escherichia coli BL21(DE3) CueO ECOL E. coli KRX

Thermus thermophilus HB27 TthL TTHL E. coli Rosetta-Gami 2

Bacillus halodurans C-125 Lbh1 BHAL E. coli Rosetta-Gami 2

Trametes versicolor Lcc1 TVEL0

Establishment of methods with TVEL0, a laccase from Trametes versicolor

Cultivation and Purification

10

Overview

11

Activity Assay

12

Oxidation of ABTS measured at 420 nm

H2O

O2

Laccase

Laccaseox

ABTS

ABTSox

Field et al., 1998 (modified)

Absorb

ance [

-]

Wavelength [nm]

1

0.5

0

1.5

200 300 400

ABTS ABTSox

Reaction:

ABTS - artificial substrate

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

Activity Assay

13

0 1 2 3 4 50

10

20

30

40

50

60

ECOL

BPUL

BHAL

TTHL

AB

TS

ox [

µM

]

Time [h] n = 4ECOL BPUL BHAL TTHL

0.0

0.2

0.4

0.6

Sp

ec.

en

zym

e a

ctivity [

U m

g-1]

n = 4

1 U = 1 µmol ABTSox formed per minute

Comparison of four different laccases

Activity Assay

14

Assay of pH and temperature effects

pH 1 pH 3 pH 5 pH 7 pH 90.0

0.1

0.2

0.3

0.4

Sp

ec.

en

zym

e a

ctivity [

U m

g-1] ECOL

BPUL

n = 4

1 U = 1 µmol ABTSox formed per minute

10 °C 25 °C0.0

0.2

0.4

0.6

Sp

ec.

en

zym

e a

ctivity [

U m

g-1] ECOL

BPUL

n = 4

Activity Assay - Summary

15

Four active laccases (~0.5 U mg-1)

Activity optimum at pH 5

Activity at low temperatures

ECOL BPUL BHAL TTHL0.0

0.2

0.4

0.6

Sp

ec.

en

zym

e a

ctivity [

U m

g-1]

n = 4

pH 1 pH 3 pH 5 pH 7 pH 90.0

0.1

0.2

0.3

0.4

Sp

ec.

en

zym

e a

ctivity [

U m

g-1] ECOL

BPUL

n = 4

10 °C 25 °C0.0

0.2

0.4

0.6

Sp

ec.

en

zym

e a

ctivity [

U m

g-1] ECOL

BPUL

n = 4

Overview

16

Substrate Analysis

17

Without laccase TVEL0 With laccase TVEL0

Can laccases degrade ethinyl estradiol?

Substrate Analysis

18

without

laccase

BPUL ECOL TVEL00

20

40

60

80

100

Estr

ad

iol d

eg

rad

atio

n [

%]

Degradation of estradiol after 3 hours

Substrate Analysis

19

without

laccase

BPUL ECOL TVEL00

20

40

60

80

100

Eth

inyl e

str

adio

l deg

radation [%

]

without

laccase

BPUL ECOL TVEL00

20

40

60

80

100

Eth

inyl e

str

adio

l deg

radation [%

]

Degradation of ethinyl estradiol,

after 3 hours

Degradation of ethinyl estradiol using

ABTS as mediator, after 10 minutes

Substrate Analysis - Summary

20

Produced bacterial laccases degrade estradiol without ABTS

TVEL0 laccase degrades ethinyl estradiol

Produced bacterial laccases degrade ethinyl estradiol

with ABTS

without

laccase

BPUL ECOL TVEL00

20

40

60

80

100

Eth

inyl e

str

ad

iol d

eg

rad

atio

n [

%]

without

laccase

BPUL ECOL TVEL00

20

40

60

80

100

Eth

inyl e

str

adio

l deg

radation [%

]

without

laccase

BPUL ECOL TVEL00

20

40

60

80

100

Estr

ad

iol d

eg

rad

atio

n [

%]

Overview

21

Immobilization

22

CPC = Controlled Pore Carrier

Immobilization

23

TVEL0 BPUL ECOL0

20

40

60

80

100

Perc

enta

ge

of b

oun

d p

rote

in [

%]

n = 3

Percentage of immobilized laccases

relative to initial concentration

BPUL ECOL0

20

40

60

80

100

Rela

tive s

pec.

activity [%

]

n = 3

Relative specific activity of immobilized

laccases

Immobilization - Summary

24

Immobilization of produced laccases with a

binding capacity > 98%

Immobilized BPUL active

TVEL0 BPUL ECOL0

20

40

60

80

100

Perc

enta

ge

of b

oun

d p

rote

in [

%]

n = 3

BPUL ECOL0

20

40

60

80

100

Rela

tive s

pec.

activity [%

]

n = 3

Overview

25

Human Practice - Overview

26

Human Practice - Overview

27

Interview with an expert of the

Institute of Fisheries Ecology

taken from wikipedia

First CeBiTec Student Academy

SynBioDay Germany,

Street Science in Bielefeld

Human Practice - Overview

28

Strategy process „Biotechnologie 2020+“

CAS conference for Synthetic Biology

Meeting with a member

of the german parliament

Feasibility studies with two

sewage treatment plants

Human Practice – Real world application

29

Goal: Real world application using existing structures within treatment plants

Cooperation with two sewage treatment plants to identify potential operation sites

Best suited location: Flocculation filtration

Human Practice – Real world application

30

Taken and modified from sewage treatmant plant Obere Lutter

Human Practice – Real world application

31

Taken and modified from sewage treatmant plant Obere Lutter

Model

32

Simulation of ABTS oxidation using laccase from B. pumilus (BPUL) according to

the conditions in the sewage treatment plant

Assumptions

• 80 % of substrate degraded

• Substrate concentration << KM

• Values of a typical german sewage plant

Overview

33

Achievements

34

We successfully…

generated four well characterized laccases for the community

produced four purified and active laccases

immobilized two of our produced laccases and measured their activities

improved a BioBrick by clarifying its true function

created a database for the community to organize their samples

conducted a feasibility study of our project with sewage plant experts

created a model which simulates the usability of our own laccases in an

actual treatment plant

did an extending outreach work

did collaborations with SDU-Denmark and UCL

constructed a shuttle vector for site directed integration in P. pastoris

Outlook

35

The expression of eukaryotic laccases in P. pastoris using our own

shuttle vector

Measurement of other substrates with HPLC and screening for

degradation products with LC-MS

Optimizing the immobilization conditions of ECOL and analyzing

those for BHAL and TTHL

Cellulose binding domain as an alternative immobilization

strategy for sewage treatment

Testing our degradation system in lab scale and sewage plants

Acknowledgements

36

Thank You!

WG Fermentation Engineering

WG Microbial Genomics and Biotechnology

Prof. Dr. Alfred Pühler

Prof. Dr. Erwin Flaschel

Dr. Jörn Kalinowski

Dr. Christian Rückert

Nils-Christian Lübke

Timo Wolf

Dr. Marcus Persicke

Dr. Thomas Hug

Dominik Cholewa