Technical University

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WP6, WP7
Technical University Braunschweig
German Research Centre for Biotechnology,
Braunschweig
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Screening isolates for enzymatic activities (WP6
and WP7)
Focus on enzymes from metagenomic expression
libraries (WP7)
Work objectives
to explore the diversity of DHABs
to isolate, hyperexpress and characterize novel
enzymes and other products (DL27 M34 DL29 -
M34, DL30 M26, DL 31 M34)
Two approaches analysis of expression libraries
and microbial isolates
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Metagenomic expression library in lambda phage
Lambda arms treated with phosphatase
Package in vitro
Library of dozens of thousands phage particles
with 0-12 kbp inserts
Transduce, select for antibiotics resistans and
score for white phages n X-Gal
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Phage l expression system

The ZAP Express vector allows bouth
eukaryotic and 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 DNA probes or antibody probes
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Screening hydrolases using a pH indicator method
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From phage library to enzyme
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Subcloned DNA fragments from positives
Plac
oil2
lt45 similarity, lt30 identity
44 520 kDa pI 10,88
lt40 similarity, lt30 identity
oil7
32516 kDa pI 10,25
lt45 similarity, lt30 identity
32627 kDa pI 9,26
oil8
Plac
pp-kinase (65 )
yafH (29 )
1 kb
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New clones coding for hydrolytic enzymes
Rhodanese domain
putative para-nitrobenzyl/carboxyl esterase, Ca.
500 aa, lt 35 seq. similarity
Plac
bolA
O02
molGC 68
COG0247, GlpC, Fe-S oxidoreductase Part of 980
aa, 50 simil.
Conserved Hypothetical Protein ca. 70
Cholesterol oxidase pecursor
Plac
bolA
O04
molGC 59
COG0657, Aes, Esterase/lipase Ca. 180 aa 29
similarity
Conserved hypoth. protein, proteobact. ca 70
a.a. sim
Consvd. membr prot, 65 pfam02517, Abi, CAAX
amino terminal protease family
Put. esterase, ca. 130 aa lt30 smlr.
O08
Plac
molGC 57
Conserved hyp. Ca. 150 aa, 60 sim. Esterase
motif, Low similarity
Consvd. membr prot, 65 , pfam02517, Abi, CAAX
COG0657, Aes, Esterase/lipase Ca. 280 aa low
similarity
COG1514, LigT, 2'-5' RNA ligase lt 50
COG0523, Putative GTPases (G3E family)
Plac
O09
molGC 56
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New clones coding for hydrolytic enzymes (contd)
Enoyl CoA hydratase
COG0657, Aes, Esterase/lipase Lipid
metabolism, lt50 similarity
Plac
O12
molGC 57
COG0523, COG0523, Putative GTPases (G3E
family) pfam02492, cobW, Cobalamin synthesis
protein/P47K.
COG1514, LigT, 2'-5' RNA ligase 58
Plac
O14
VPS29-like phosphoesterase-related, 172aa long
hypothetical protein Pyrococcus horikoshii lt30

Hypothetical, Low homology
O16
Plac
molGC 50
No ORFs
Putative membrane protein Low homology lt30
Hydroxyacyl dehydrogenase
Cons. hypothetical, low homology
Cons. hypothetical
Plac
O21
molGC 33
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Enzyme purification
Native gel electrophoresis, development with
a-naphtylbutyrate
Purification Cationic exchange on
MonoS Hydrophobic interaction (Phenylsuperose) Gel
filtration (Superose 12)
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Enzyme reaction products
TG
1,3-DG
1(3), 2-DG
MG
oil2
oil8
oil7
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Features of the enzymes
Thermostability
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Specific activities with p-nitrophenol derivatives
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Specific activities with p-nitrophenol derivatives
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Screening of hydrolytic activity Conisma strains
(the others to come) Substrate
?-naphtylacetate, butyrate, laurate, palmitate,
phosphate, glucoside, galactoside and Fast Blue RR
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Conclusions and outlook
Collection of hydrolytic enzymes from expression
libraries obtained after oil enrichment, have
been characterised they exhibit novel structures
(low homology to the homologs), have
a good potential for industrial applications and
tell the stories about the
environment and organisms they were derived from

Screening of biosurfactant producing isolates
started for further characterization of novel
structures analysed
Screening/characterisation of enzymatic
activities from the isolates will be continued
Progress estimates
DL29- M34 20 Structures of novel surfactants
etc (screening stage) DL30-M26 100
Clones, hyperexpression clones etc. DL31-M34
100 Data sets of activities of obtained
compounds all selected items characterized
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Publications???
Best regards from Peter, Manolo and Ken