Technical biochemistry

TUHHTechnical University Hamburg-Harburg

PSYCHROPHILIC DEGRADATION PSYCHROPHILIC DEGRADATION OF LONG CHAIN ALKANESOF LONG CHAIN ALKANES

OutOutlines of the researchlines of the research IntroductionIntroduction Objective of the researchObjective of the research Results and discussionResults and discussion

IntroductionIntroduction

Importance of the researchImportance of the research

DesertDesert

SeasSeas

SoilSoil

LakesLakes Problem of oil spillProblem of oil spill??

Petroleum Aromatics Resins Asphaltenes Alkanes Alkanes

What are alkanes?

Major components of crude oil (20 –Major components of crude oil (20 – 50 %)50 %)

AlkanesCnH2n+2

Biodegradation of alkanesBiodegradation of alkanes

Biodegradation in natureBiodegradation in nature

Microbial activityMicrobial activity

Lower Lower MWMW Higher Higher MWMW

0 - 20 °C Cold polar seasPsychrophiles

Extremophiles microorganisms and their habitats

Growth conditionsExtremophilesThermophiles 50 - 110 °C Hot springs

Acidophiles pH < 2 Acidic fields

Alkaliphiles pH > 9 Alkaline soils

Halophiles 3 – 20 % salt Highly saline lakes

Habitats

90 % of marine water masses are colder than 4°C. Two-third of sea water covering 70 % of the earth is cold.

Psychrophiles?!

CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH33

Intermediary metabolismIntermediary metabolism

ßß-Oxidation-Oxidation

CHCH33-(CH-(CH22))nn-CH-CH22CHOCHOAldehydeAldehyde

NAD+

NADHalcDHalcDH

NAD+

NADH H2O

CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH22COOHCOOHFatty acidFatty acid

aldDHaldDH

alkBalkB

CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH22OHOHPrimary alcoholPrimary alcohol

Alkanes degradation pathwayAlkanes degradation pathway

Mode of alkanes uptakeMode of alkanes uptake

AdaptationEmulsification Modulation

Diffusion &

active transport

Emulsification

Direct contact (Alkanedroplets)

Solubility

biosurfactantsalkanes

OutsideOutside MembraneMembrane InsideInside

Biosurfactants biosynthesisCnH2n+2

Intermediary metabolismEmulsification

Intermediary metabolism

Biosurfactants biosynthesis

Genes encoding those enzymes couldbe under the same repressor control

orA part of the same operon

Retledge, 1988

Mode of alkanes uptakeMode of alkanes uptake

biosurfactantsalkanes

OUTSIDEOUTSIDE MembraneMembrane INSIDEINSIDE

Biosurfactants biosynthesisCnH2n+2

Diffusion&active

transport

EmulsificationIntermediary metabolism

Direct contact (Alkanedroplets)

Solubility

Biosurfactants biosynthesis

EmulsificationIntermediary metabolism

Mode of contaminant uptakeMode of contaminant uptake

Diffusion ( cell wall)Diffusion (cell wall) Diffusion (Cytoplasmic membrane)

Multi-Enzymes Complexes Organic Contamination

CnH

2n+2CnH

2n+2

CnH

2n+2

Results and discussionResults and discussion

Source of the mixed cultureSource of the mixed culture

Morphological & biological characteristicsMorphological & biological characteristics

PPseudomonasseudomonas frederiksbergensis frederiksbergensis

RRhodococcushodococcus erythropolis erythropolis

2700 x

16S rDNA Fatty acids analysis

RR.. erythropolis erythropolis (+) (+)

PP.. frederiksbergensis frederiksbergensis (-)(-)

2700 x

Growth and biodegradation of eicosane by Growth and biodegradation of eicosane by P. P. frederiksbergensis, R. erythropolis frederiksbergensis, R. erythropolis and mixed and mixed

culture at 4°Cculture at 4°C

0.00E+00

1.50E+07

3.00E+07

4.50E+07

6.00E+07

7.50E+07

9.00E+07

0 4 8 12 16 20 240

200

400

600

800

1000

R. erythropolis P. frederiksbergensis Mixed culture eicosane conc. (µM) eicosane conc. (µM) eicosane conc. (µM)

Gro

wth

(cel

l/ml)

Eico

sane

con

c. (µ

M)

Time (day)

4°C

Gas Chromatography - Mass SpectrometryGas Chromatography - Mass Spectrometry(GC-MS)(GC-MS)

0

0.1

0.2

0.3

0.4

0 10 20 30 40Temperature (°C)

R. erythropolis P. frideriksbergensis

0

0.05

0.1

0.15

0.2

2.5 4.5 6.5 8.5 10.5pH

R. erythropolis P. frederiksbergensis

µ =ln2·TD*

*TD = Time Doubling

Optimal conditions for the growth of Optimal conditions for the growth of P. P. frederiksbergensisfrederiksbergensis and and R. erythropolis R. erythropolis

20°C15°C 7.47.0

Gro

wth

rate

(d)

Growth and biodegradation of eicosane by Growth and biodegradation of eicosane by P. P. frederiksbergensfrederiksbergensisis, , R. erythropolisR. erythropolis and mixed and mixed

culture at optimal conditionsculture at optimal conditionsoptimal conditions

0.00E+00

1.50E+07

3.00E+07

4.50E+07

6.00E+07

7.50E+07

9.00E+07

0 2 4 6 8 10 120

200

400

600

800

1000

P. frederikesbergensis R. erythropolis Mixed cultureeicosane conc. (µM) eicosane conc. (µM) eicosane conc. (µM)

Gro

wth

(cel

l/ml)

Eico

sane

con

c. (µ

M)

Time (day)

Growth ofGrowth of P. frederiksbergensis P. frederiksbergensis on eicosan on eicosan and docosane crystalsand docosane crystals

EicosaneEicosane crystalcrystal

Docosane Docosane crystalcrystal

C10H20, C16H34, C18H38, C20H42, C22H46

Biodegradative capabilities of Biodegradative capabilities of P. P. frederiksbergensisfrederiksbergensis

0

200

400

600

800

1000

0 2 4 6 8 10 12Time (day)

Con

c. (µ

M)

hexadecane eicosane decane decane

eicosanehexadecane

Metabolic pathway of eicosaneMetabolic pathway of eicosane and decane and decane degraded by degraded by P. frederP. frederiiksbergensisksbergensis

Eicosane

Decane

Metabolic pathway of eicosaneMetabolic pathway of eicosane and decane and decane degraded by degraded by P. frederP. frederiiksbergensisksbergensis

CH3-(CH2)n-CH2-CH3CH2 -CH2OHCHO-CH2COOH

Terminal oxidationTerminal oxidation

CHCH33-(CH-(CH22))nn-CH-CH22-CH-CH33

0,00E+00

1,25E+07

2,50E+07

3,75E+07

5,00E+07

6,25E+07

7,50E+07

0 2 4 6 8 10 12

Time (days)

Gro

wth

(cel

l/ml)

5,5

5,75

6

6,25

6,5

6,75

7

7,25

7,5

pH v

alue

P. frederiksbergensis (a) R. erythropolis (b) pH (a) pH (b)

Effect of pH on P. frederiksbergensisgrowth

NAD+ NADH + H+

alkanes Alkanoic acid

H+

H+

H+

H+

H+

H+

Medium acidification

ConclusionConclusion TheThe isolates in this study isolates in this study were able to degrade were able to degrade (C(C1010- C- C2222) ) n-n-alkanes alkanes

from 4°C to 20°C.from 4°C to 20°C.

P. fredP. fredeeriksbergensis riksbergensis waswas obligate psychrophile obligate psychrophile, while , while R. R. erythropolis erythropolis waswas facultative psychrophilefacultative psychrophile..

Alkane degradation by Alkane degradation by P. fredriksbergensis P. fredriksbergensis strated with terminal strated with terminal oxidation and detection of the metabolites was possible.oxidation and detection of the metabolites was possible.

The alkane degradation in The alkane degradation in P.P. frederiksbergensisfrederiksbergensis starts with the starts with the hydroxylation of the alkane and subsequently to fatty acidshydroxylation of the alkane and subsequently to fatty acids

Mode of alkanes uptake by Mode of alkanes uptake by P. frederiksbergensisP. frederiksbergensis ?!?!

Detection of the P. frederiksbergensisbiosurfactants by (MBAS)

P. frederiksbergensis colonies on blue agar

P. frederiksbergensis colonies on eicosane MSM

Biosurfactants activity produced Biosurfactants activity produced byby P. P. frederiksbergensisfrederiksbergensis

0

15

30

45

60

75

90

0 2 4 6 8 10Time (day)

Surf

ace

tens

ion

(mN

/m)

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

O.D

. 580

nm

surface tension (mN/m)surface tension (mN/m) with pellet suspensionGrowth on hexadecane

Cell surface hydrophobicity and Cell surface hydrophobicity and adhesion to hexadecaneadhesion to hexadecane

Growth

Emulsification stabilityAdhesion to hexadecane

0

30

60

90

120

0 40 80 120 160 200 240

Time (h)

O.D

. 58

0 nm

0,0

0,5

1,0

1,5

2,0

Adh

esio

n (%

)

(73%)

(10%)

(88%)

0

30

60

90

120

Em

ulsi

ficat

ion

stab

ility

(%)

% Adhesion = (1- (OD shaken with hexadecane //OD original) x 100%

GC-profile of the fatty acids involved biosurfactants of P. frederiksbergensis

?1

24

3

? ? ?

Time (min)

?

1

Abu

ndan

ce

1. Methyl palmitate2. Methyl decanoate3. Methyl tetradecanoate4. Methyl eicosanoate

Characterization of Characterization of the biosurfactantsthe biosurfactants

Rhamnolipids possess hemolytic properties.

Blood agar (5 % sheep blood)

Glycolipids produced by Glycolipids produced by P. fredriksbergensis P. fredriksbergensis had a potential effect had a potential effect on substrate on substrate utilizutilizationation by: by:

ConclusionConclusion

Developing hydrophobic cell surface and reducing Developing hydrophobic cell surface and reducing media surface tension to media surface tension to 25 mNm-125 mNm-1

The stability of the substrate emulsion reached the maximum value after 8 days and

73% of hexadecane was converted to hexadecanoic acid in water emulsion

Molecular BiologyMolecular BiologyHow can we solve the problem of environmental pollution by

Genetics engineering?

Detection ofDetection of alk alkBB in in P. frederiksbergensis P. frederiksbergensis withwith oligonucleotide primers specific foroligonucleotide primers specific for alk alkBB

PCR product M

DNA Purification

PCR products M

DNA Amplification

550 bp

DNA Isolation

P. frederiksbergensisP. frederiksbergensis

Amino acid and corresponding

proteins

Sequence analysis of the alkane Sequence analysis of the alkane hydroxylase genehydroxylase gene probeprobe

Detection and Detection and llocalization ofocalization of alkane alkane hydroxylase hydroxylase ggeneene

SouthernSouthern hybridizationhybridization

EcoR I

1 2 3 4 5 6 7

1. M (1kb) 2. DNA probe 3. EcoR1 4. NdeI 5. NcoI 6. AvaI 7. BamI

AmplificationAmplification

M

PCR

DigestionDigestion RestrictionRestriction

7 6 5 4 3 2 1

Cloning Cloning ofof alkane hydroxylase alkane hydroxylase ggeneene

PCR

Cloning and sequencing of alkane Cloning and sequencing of alkane hydroxylase of hydroxylase of P. frederiksbergensisP. frederiksbergensis

pUC19

2894 bp2894 bp

ORF

B1

FalkB primers

R

B2F

A2pUC19

primers

A1

R

pUC19 primeralkB primer1 bp 2 894 bp

Analysis of (ORFS) and genetic organization in P. frederiksbergensis

2 188 bp85 bpORF1

alkB

alcDH 2 316 bp1 303 bp

ORF2

The arrows indicate the direction and translation of the gene

ORF3

Unknown protein

alkT absence?? (downstream of the gene) P. frederikesbergensis DAN (one cluster) distribution of the gene (Scattered on the chromosome)

1 2 3 4

1313 bp

Subcloning of Subcloning of alcalcDH from the wild type DH from the wild type P. frederiksbergensisP. frederiksbergensis and the cloned fragment and the cloned fragment

pET-155708 bp

1. M (kb) 2. CF 3. WT 4. R

Alignment of P. fredriksbergensis alcohol dehydrogenase amino acids sequences with

different organisms

TGXXXGXG (cofactors binding site) TXXXL (active centre)

Member of SCR family Highly conserved regions

Phylogenetic tree of P. frederiksbergensis alcDH based on amino acids sequences

Accession number of AAR134804

Alignment of P. fredriksbergensis alkane hydroxylase amino acids sequences with

different organisms

Residues of eight histidine box hydrophobic amino acids

Member of alkB family Highly conserved regions

Phylogenetic tree of P. fredriksbergensis alkB based on amino acids sequences

sp.

Accession number of number of AY452488AY452488

20

Optimal temperature of native alkane hydroxylase of P. fredriksbergensis

0

100

200

300

400

500

0 5 10 15 20 25 30 35 40

Temperature (°C)

Los

s of d

ecan

e co

nc. (

µM)

Measurements were carried out at different temperatures (after 2 h of incubation)

20

Periplasm spacePeriplasm space

Genetics of alkane degradationGenetics of alkane degradation

CH3

H

uptake?? Cytoplasmic membraneCytoplasmic membrane

Outer membraneOuter membrane

alkSp2

O

alkB alkF alkG alkH alkK alkL alkJ

ß-Oxidation

RegulationRegulation

Chemotaxis?Chemotaxis?

CytoplasmCytoplasm

palkB

alkS

alkFalkT

alkHNAD

HalkK

alkGFe++

B

O ATPCoA

NADH

FAD

TCA cyclealkSTalkSp1

alkSCatabolic

repression

alkanes alkanes absenceabsencealkanes

presenceơS

R SCOA

alcDHFAD

CH3

HO

A alkL

alkNalkBFe++

alkS

alkFalkT

alkGFe++

alkBFe++

alkS

alkanes presence RegulationRegulation

0

0,2

0,4

0,6

0,8

1

0 8 16 24 32 40Temperature (°C)

0

0,2

0,4

0,6

0,8

1

2 4 6 8 10 12pH

Act

ivity

(U/m

l)Optimal reaction conditions of recombinant

alcohol dehydrogenase (alcDH)

U = (ε [cm2µmol-1] of NADH = 6.22)

pH = 9

T= 30°C

0

25

50

75

0 50 100 150 200 250 300Time (min)

Act

ivity

[µM

/min

]

10°C 30°C 40°C 50°C

0

20

40

60

80

0 2 4 6 8 10Time (day)

RT4°C

Thermostability of recombinant alcohol dehydrogenase (alcDH)

U = (ε [cm2µmol-1] of NADH = 6.22)

met

hano

l

etha

nol

prop

anol

1-bu

tano

l

isob

utan

ol

amyl

alco

hol

isoa

myl

alco

hol

1-oc

tano

l

tetra

deca

nol 10 mM

0

0,2

0,4

0,6

0,8

Specific activity [U/mg]

Substrate

10 mM 50 mM

Substrate specrtum recombinant alcDH activity

Measurements were carried out at pH = 9.0 and T = 30°C

Substrate specrtum of recombinant alcohol dehydrogenase (alcDH)

A broad substrate spectrum

Tendency towards primary alcohols

100 mM inhibited the enzyme activity

ConclusionConclusion The gene cluster of alkane hydroxylase of The gene cluster of alkane hydroxylase of P. fredriksbergensis P. fredriksbergensis waswas

different from known alkane hydroxylases found in other different from known alkane hydroxylases found in other alkanes alkanes degrading bacteria.degrading bacteria.

The gene encoding The gene encoding for for alcalcDH belonged the groups of NADDH belonged the groups of NAD+ +

dependent, shortdependent, short chain alcohol dehydrogenasechain alcohol dehydrogenase..

The enzyme of alcDH had a wide range of substrates The enzyme of alcDH had a wide range of substrates spectrumspectrum with with T T = = 3030°°CC

pH pH == 9.0. 9.0.

Alcohol dehydrogenase of Alcohol dehydrogenase of P. fredriksbergensisP. fredriksbergensis has the tendency towards primary alcohols

Technical biochemistry

TUHHTechnical University Hamburg-Harburg