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
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