Technical University Braunschweig
17
Technical University Braunschweig German Research Centre for Biotechnology, Braunschweig WP7
description
WP7. Technical University Braunschweig. German Research Centre for Biotechnology, Braunschweig. Oil - degradative capacities. Objectives. Evaluation of major microbial groups. Background for applications. Adjusting protocols. Sample chosen: U3. Isolates from oil enrichment. - PowerPoint PPT Presentation
Transcript of Technical University Braunschweig
Technical University Braunschweig
German Research Centre forBiotechnology, Braunschweig
WP7
Oil - degradative capacities
Sample chosen: U3
Objectives
Evaluation of major microbial groups
Background for applications
Adjusting protocols
0.1 substitution/site
Thiomicrospiragroup
Chromatiaceae
Legionellaceae
CardiobacteriaceaeMethylococcaceaeEnterobacteriaceaePasteurellaceae
Vibrionaceae
Aeromonasgroup
Colwelliagroup
Xanthomonas- Stenotrophomonas group
Microbulbifer
Neptunomonas
Marinomonas
Alcanivorax
Oleiphilus messinensis
Oceanospirillum
Marinobacter
Pseudomonas(sensu stricto)
Isolates from oil enrichment.
Ps. stutzeri-Ps. balearica groupMarinobacter hydrocarbonoclasticus-Mb.CABProteobacteria (Ruegeria group)
METAGENOMIC LIBRARIES
pBAC (e.g. pBeloBac11) large fragments 50-300 kbp
SuperCos1 - a „common“cosmid for cloning, Fragments to clone 40-50 kbp
“Archives”
based systems, e.g. LambdaZap
Expression libraries
pLAFR3 - a replicon in a variety of Gram-negatives(e.g. P. putida)fragments to clone - 20-30 kbp
Metagenomic expression library in lambda phage
Cosmid arms treated with phosphatese
Transduce, select for antibiotics resistans and score for white phages n X-Gal
DNA size-fractionated,partially digested with Sau3A
Ligation
Package in vitro
Library of dozens of thousands phage particles with 0-12 kbp inserts
The ZAP Express vector allows bouth eukaryoticand prokaryotic expression and accomodates DNA insert from 0 to 12 kb in length.
„Oil“ library = 1,8 x 106 phage particles. Average insert size - 7.5 kbp
Clones in the ZAP Express vector can be screend with either DNAprobes or antibody probes
Phage expression system
Enzymes Detection systemEsterases/proteases
Phosphatases
Sulfatases
Esterases/proteases
Lipases
Phosphatases
-D-Galactosidases
-D- Galactosidases
-D-glucosidases
Indoxyl acetate
Indoxyl phosphate
Indoxyl sulfate
naphtyl acetate/butyrate+FastBlueRR
naphtyl palmitate + ””
naphtyl phosphate + ””
naphtyl -D-galactopyranoside + ””
naphtyl -D- galactopyranoside + ””
naphtyl -D-glucopyranoside + ””
Enzymes and detection systems
Insert cloned into the ZAP Express vector excised out of the phage in the form of the Km-resistant pBK-CMV phagemid vector
Screening of ca. 10000 phage clones yields ca. 20 positives
Excision
Selected clones clustered
500 600 700 800 900 1000 1100
20
40
60
80
100 518.3
569.2598.1
637.2
673.4711.3
775.3
826.0
867.0925.5
994.71035.4
Expression
MALDI-TOF
Purification
Sequencing of selected clones
Product, enzymology
From phage library to enzyme
Subcloned DNA fragments from positives
oil2<45% similarity,<30% identity
Plac
oil8Plac
<40% similarity,<30% identityoil7Plac
yafH (29 %)pp-kinase (65 %)
<45% similarity,<30% identity
1 kb
44 520 kDapI 10,88
32516 kDapI 10,25
32627 kDapI 9,26
Enzyme purification:
Purification: Cationic exchange on MonoSHydrophobic interacti(on Phenylsuperose)Gel filtration (Superose 12)
Native gel electrophoresis,development with -naphtylbutyrate
Enzyme reaction products
TG
1,3-DG1(3), 2-DG
MG
O
O
O
O
O
O
oil2 oil8oil7
Specific activities with p-nitrophenol derivatives
Carbon atoms
2 4 6 8 10 12 14 16
Spe
cific
act
ivity
(µm
ol/m
in/m
g)
0
1
2
3
4
5
6
Features of the enzymes
Temperature (°C)
20 40 60 80
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120
Temperature (°C)
20 40 60 80
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120
Temperature (°C)
20 40 60 80
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120
Temperature-dependent activity
Oil 2 Oil 7Oil 8
Temperature optima
Time (min)
0 30 60 90 120 150 180
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120
Time (min)
0 30 60 90 120 150 180
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120
Time (min)
0 30 60 90 120 150 180 210
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120
OIL 2 OIL 7 OIL 8
30 °C
40 °C
50 °C
THERMOSTABILITY Thermostability
Features of the enzymes (cont’d)
pH
5 6 7 8 9 10 11
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120
pH
5 6 7 8 9 10 11R
elat
ive
activ
ity (
%)
0
20
40
60
80
100
120
pH
5 6 7 8 9 10 11
Rel
ativ
e ac
tivity
(%
)
0
20
40
60
80
100
120Oil 2 Oil 7 Oil 8
pH activity profile
Buffers used were as follows:
pH 5.5-7.0 MESpH 7.0-8.0 HEPESpH 8.0-10 Tris-HCl / Sodium carbonate / Bicine / CHES
pH - activity profiles
Features of the enzymes (cont’d)
Addition ConcentrationmM
Activity
% % (OIL 7) % (OIL 2) % (OIL 8)None 100 100 100Tween 20 0.05 47 70 58
0.4 52 55 742 27 36 29
Tween 80 0.05 75 56 720.4 62 38 68
2 29 0.1 40Titon X-100 0.05 80 57 151
0.4 76 33 1492 42 0.1 55
SDSa 0.05 27 14 350.250 0.1 0 0
1 0 0 0
Influence of surfactants
Addition Percentage Activity% % (OIL 7) % (OIL 2) % (OIL 8)
None 100 100 100n-propanol 1 80 0.1 87
5 70 75 72Ethanol 1 84 131 101
5 73 83 133DMSO 1 76 39 86
5 67 54 9925 30 44 59
t-amylalcohol 1 68 66 655 36 26 21
25 25 25 23Acetonitrile 1 89 98 92
5 99 168 12425 15 0.1 67
Solvent resistance
Effect of cations on activity:
Substrate: p-NPhBu
Addition Concentration ActivitymM % (OIL 7) % (OIL 2) % (OIL 8)
None 100 100 100Na+ 1 103 120 114
10 102 108 145125 103 55 117
Ca2+ 1 104 44 12210 57 70 100
125 58 0.1 45K+ 1 99 83 93
10 108 76 84125 88 65 108
Mg2+ 1 92 55 9010 60 43 114
125 40 17 27Li+ 1 102 61 102
10 78 57 126125 82 68 110
Zn2+ 1 86 0 14410 59 0 62
125 0 0 0Sr2+ 1 89 63 110
10 75 31 143125 44 0.1 38
Co2+ 1 92 0 13310 45 0 73
125 26 0 0Fe2+ 1 81 0.1 124
10 13 0 0125 0 0 0
EDTA 1 84 102 11010 72 76 103
125 50 42 63EDTA + Mg2+ 10 + 10 69 45 87EDTA + Ca2+ 10 + 10 64 40 111Mercaptoethanol 1 95 0 52PMSF 1 80 32 54
5 43 11 27
Few hydrolytic enzymes from expression libraries obtained after oil enrichment, have been characterised
Isolation will be continued with other samples(e.g. Bannock interface has a number of positives, characterization in progress)
Conclusions and outlook
The natural microbial communities from interface capable of petroleum oil degradation
Isolation of a variety of enzymes will be continued
Screening/characterisation of enzymatic and antimicrobial activities from the isolates
Interface libraries from the last RV”Urania” sampling cruise to be analysed(e.g. Bannock interface has a number of positives, characterization in progress)
