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WP6, WP7. Technical University Braunschweig. German Research Centre for Biotechnology, Braunschweig. Screening isolates for enzymatic activities (WP6 and WP7). Focus on enzymes from metagenomic expression libraries (WP7). Work objectives:. to explore the diversity of DHABs:.

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Technical University Braunschweig

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Technical university braunschweig

WP6, WP7

Technical University

Braunschweig

German Research Centre for

Biotechnology, Braunschweig


Technical university braunschweig

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


Technical university braunschweig

DNA size-fractionated,

partially digested with Sau3A

Ligation

Metagenomic expression library in lambda phage

Cosmid arms treated with

phosphatese

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


Technical university braunschweig

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

Esterases

Cellulases and amylases


Technical university braunschweig

From phage library to enzyme

Excision

Screening of ca. 10000 phage clones

yields ca. 20 positives

Insert cloned into the ZAP Express

vector excised out of the phage in the form

of the Km-resistant pBK-CMV phagemid

vector

Purification

Expression

MALDI-TOF

Sequencing of

selected clones

Product,

enzymology

Selected clones clustered


Technical university braunschweig

BIODIVERSITY FEATURES

Thirteen unique clones (pBK Oil2, pBK Oil7, pBK Oil8, pBK O02, pBK O03, pBKO04, pBK O08, pBKO09, pBK O10, pBK O11,pBKO12, pBKO14, pBKO16, pBK O21, pBK O23) encoding esterases have been cloned and sequenced, and their protein products have been purified and characterized.

One gene (pBKOA3) encoding a amylase has been detected and cloned and the sequence and characterization is in progress

One gene (pBKOC1) encoding a endoglucanase has been retrieved from phage library


Technical university braunschweig

Plac

Subcloned DNA fragments from positives

Plac

oil2

<45% similarity,

<30% identity

44 520 kDa

pI 10,88

oil7

<40% similarity,

<30% identity

32516 kDa

pI 10,25

<45% similarity,

<30% identity

32627 kDa

pI 9,26

oil8

Plac

pp-kinase (65 %)

yafH (29 %)

1 kb


Technical university braunschweig

molGC 58 %

Plac

esterase

esterase/deacetylase

Oil2

Conserved hypothetical

protein


Technical university braunschweig

Rhodanese

domain protein

AraC

regulatory protein

October 03

putative para-nitrobenzyl/carboxyl esterase,

Ca. 500 aa, < 35 % seq. similarity,

Abo 80% similarity

Rhodanese

domain

AraC regulatory protein

Plac

bolA

O02

molGC 68 %


Technical university braunschweig

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

O08= Oil2

Put. esterase,

ca. 130 aa

<30% smlr.

Plac

molGC 57 %


Technical university braunschweig

Plac

O09

molGC 56 %

2731-3550

GtPase

2029-1067

Esterase (44% similar,

32 % ident)

487-960

Cons‘d hyp. Protein (68%to PAO)

Enoyl CoA

hydratase

COG0657, Aes, Esterase/lipase

[Lipid metabolism], <50 % similarity

Plac

O12

molGC 57 %


Technical university braunschweig

New:

O10

1072 to 104, 323 aa

Metallo-beta-lactamase

family protein, 65% sim

KT2440

1117-ca.280 aa (one read left)

enoyl-CoA hydratase/isomerase

family protein 89 % sim. Abo_141

70 % Caulobacter

molGC 56 %

Plac

October 03

204 to 1251-ca 350 aa (full length sequenced)

Putative acetyl esterase (lipase/hydrolase)

40 % sim. Bacillus


Technical university braunschweig

O11

New:

molGC 56 %

108 to 1526, 483 aa

Sulfate transporter/permease

family protein, Abo_1815, Abo_1817 80%sim

Arabidopsis-chloroplast- next (56%)

409-486 aa

Sulfate transporter/permease

family protein, Abo_1815, Abo_1817 80%sim

Arabidopsis-chloroplast- next (56%)

Plac

2414 to 296 aa (putative lipase/hydrolase

40 % sim. Bacillus


Technical university braunschweig

New:

O16

October 03

Low homology ORF ca 960 aa

molGC 44 %

Plac

Putative membrane protein

Low homology <30 %

Hydroxyacyl

dehydrogenase

Cons. hypothetical,

low homology

Cons. hypothetical

Plac

O21

molGC 33 %


Technical university braunschweig

O23

New:

molGC 54 %

Glucosylhydrolase

family protein (junk sequence!)

Plac

-30 bp to the end of the fragment

Carboxylesterase

Ca 500 aa (one read left)

54% sim. Bacillus


Technical university braunschweig

Enzyme reaction products

TG

1,3-DG

1(3), 2-DG

MG

oil2

oil7

oil8

O.4

O.2

O.16

O.21

O.8

O.12

O.9

O.14

Tributyrin

Esterase in H2O:Acetonitrile


Technical university braunschweig

Enzyme purification:

i.e. Oil2, Oil7 and Oil8

Native gel

electrophoresis,

development with

a-naphtylbutyrate

Purification:

Cationic exchange on MonoS

Hydrophobic interaction (Phenylsuperose)

Gel filtration (Superose 12)


Technical university braunschweig

Specific activities with p-nitrophenol derivatives


Technical university braunschweig

Specific activities with p-nitrophenol derivatives


Technical university braunschweig

Screening hydrolases using a pH indicator method:

Scheme:

CONDITIONS:

96 microtiter plate containing 5µ enzyme solution (20 mg/ml), and 100 µl of the following mixture:

420 µL of a 30 mM solution of the ester in acetonitrile,

470 µL acetonitrile

4-nitrophenol (6oooµL of a 0.9115 mM solution in 5.o mM BES, pH 7.2


Technical university braunschweig

E > 20 INDUSTRIAL POTENTIAL (Enantiomeric ratio)

e.e. > 70 INDUSTRIAL POTENTIAL (Enantiomeric excess)

ENANTIOMERIC RESOLUTION OF

1-PHENYLETHANOL

BY TRANSESTERIFICATION

WITH VINYL ACETATE

+

2 mL Iso-octane

20 mg E. coli esterase clones

1 M Vinylacetate

1 M 1-phenylethanol


Technical university braunschweig

Screening of hydrolytic activity: Conisma strains (the others to come)

Substrate:-naphtylacetate, butyrate, laurate, palmitate, phosphate, glucoside, galactoside and Fast Blue RR


Technical university braunschweig

ABSTRACT: enzyme properties

Here we describe a comparative analysis of ten novel esterases from a large library created from deep-sea hypersaline anoxic basins of the Eastern Mediterranean.

A genomic DNA library of deep-sea hypersaline anoxic basins of the Eastern Mediterranean was established into Escherichia coli, and screened for esterase activity by using -naphtyl butyrate and Fast Blue RR. Eleven genes (pBKOil2, pBKOil7, pBKOil8, pBKO.2, pBKO.4, pBKO.9, pBKO.12, pBKO.14, pBKO.16, pBKO.21) encoding eleven esterases has been cloned and sequenced, and their protein products have been purified and characterized.

Sequence analysis revealed less than 40% identity to sequences of known esterases and lipases.

They preferred short chain water-soluble esters as substrates. The optimal pH and temperature of the purified enzymes were in the range 8.0-9.5 and 40-60°C, respectively.

Purified esterases exhibited hydrolytic activity without addition of any metal ions. However they were optimally active in the presence of different concentrations of Na+ or K+. Esterases from pBKO.4, pBKO.8, pBKO.12, pBKO13 were 2 times more active at concentration in the range from 25 to 75 mM. In contrast, esterases from pBKO.9, pBKO.14 and pBKO.2 were inhibited above 25 mM. Some other esterases, i. e. from pBKO.16, pBKO.21, pBKOil2, pBKOil7 and pBKOil8 showed a high activation at concentrations of Na+ or K+1.5-2.0, 1.0-1.5, 2.0-4.0, 3.5, 3.5, 0.8 and 4.0 M, which is tipical for halophilic enzymes.

The positive clones, pBKO.2, pBKO.4, pBKO.21, were found to have 1(3) positional specificity against triacylglycerols. Interestingly, pBKOil8 and pBKO.4 showed 2 positional specificity against triacylglycerols. The hydrolytic and synthetic activities and estimated enantioselectivities towards chiral ester library was assayed. We found 4 lactone hydrolases (pBKOil8, pBKO.2, pBKO.9 andpBKO.21), 6 highly specific for aromatic chiral compounds (pBKOil7, pBKO.2, pBKO.14 and pBKO.16). Esterases derived from pBKO.14, pBKO.16, pBKO.4, pBKO.21 and pBKOil8, were potentially used for industrial resolution of chiral carboxylic acid with a stereocenter  to carbonyl.

pBKO.2, pBKO.14 pBKO.9 and pBKOil8 were the most stable against organic solvent and 1.5-fold times more active in the presence of detergents. Taking the catalytic efficiency and the enantiomeric excess and ratio, we concluded that the esterases produced by pBKOil8, pBKO.2, pBKO.4, pBKO.23 and pBKO.21 exhibited the best properties of all tested esterases for application in the biotechnological resolution of chiral esters. We found that most of the esterase-encoding genes discovered are entirely new and the targeted activity and enantioselectivities test for the esterase product revealed a unique phenotype for some of the new biocatalysts.


Technical university braunschweig

WHAT NEXT???

Check the esterase in unknown reactions.

Characterization of amylase: halophilic??? progress

Characterization of endoglucanase

Check for new enzymes: DNase, proteases, oxidoreductases, alcohol dehydrogenases and dehydratases, laccases, etc…

Crystallization

Surfactants – a big struggle…


Technical university braunschweig

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 - finished

Nothing incredibly new or interesting obtained

Expression libraries are the best tool to exploit the diversity:

Work with separate isolates is less productive and time-consuming

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


Technical university braunschweig

Publications

Ready to publish after finishing sequences (2-3 months)

Env Microbiol Special Issue is planned

1. Env-specificity: bio(geo)chemistry

2. gradient Daniele etc

3. Misha etc

4. Henk etc

5. Marseille etc

6. Tassos etc

7. biotech applications (Peter and I), Proteus


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