Technical University Braunschweig

German Research Centre forBiotechnology, Braunschweig

WP6, WP7

Screening isolates for enzymatic activities (WP6 and WP7)

Work objectives:to explore the diversity of DHABs:

to isolate, hyperexpress and characterize novel enzymesand other products (DL27 – M34; DL29 - M34, DL30 – M26, DL 31 – M34)

Two approaches: analysis of expression libraries andmicrobial isolates

Focus on enzymes from metagenomic expression libraries (WP7)

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

EsterasesCellulases and amylases

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

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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

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

BIODIVERSITY FEATURES

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

Plac

molGC 58 %

Oil2

esterase/deacetylase esterase

Conserved hypothetical protein

AraC

regulatory protein

Rhodanese domain protein

O02bolAPlac

Rhodanese domain

putative para-nitrobenzyl/carboxyl esterase,Ca. 500 aa, < 35 % seq. similarity, Abo 80% similarity

molGC 68 %

October 03

AraC regulatory protein

O08= Oil2Plac

COG0657, Aes, Esterase/lipase Ca. 180 aa 29% similarity

Put. esterase, ca. 130 aa <30% smlr.

Conserved hypoth. protein, proteobact. ca 70 % a.a. sim

Consvd. membrprot, 65 %pfam02517, Abi, CAAX amino terminal protease family

molGC 57 %

O04

Plac

COG0247, GlpC, Fe-S oxidoreductasePart of 980 aa, 50% simil.

Cholesteroloxidase pecursor

bolAConserved HypotheticalProtein ca. 70 %

molGC 59 %

O12

PlacCOG0657, Aes, Esterase/lipase [Lipid metabolism], <50 % similarity

Enoyl CoAhydratase

molGC 57 %

O09Plac

molGC 56 %

487-960Cons‘d hyp. Protein (68%to PAO)

2731-3550GtPase

2029-1067Esterase (44% similar, 32 % ident)

O10

Plac

1072 to 104, 323 aa Metallo-beta-lactamase family protein, 65% simKT2440

1117-ca.280 aa (one read left)enoyl-CoA hydratase/isomerase family protein 89 % sim. Abo_14170 % Caulobacter

molGC 56 %

October 03

204 to 1251-ca 350 aa (full length sequenced)Putative acetyl esterase (lipase/hydrolase) 40 % sim. Bacillus

New:

O11

Plac

108 to 1526, 483 aa Sulfate transporter/permease family protein, Abo_1815, Abo_1817 80%simArabidopsis-chloroplast- next (56%)

molGC 56 %

2414 to 296 aa (putative lipase/hydrolase 40 % sim. Bacillus

New:

409-486 aa Sulfate transporter/permease family protein, Abo_1815, Abo_1817 80%simArabidopsis-chloroplast- next (56%)

O16

Plac

Low homology ORF ca 960 aamolGC 44 %

October 03New:

Plac

Putative membrane proteinLow homology <30 % O21

Hydroxyacyl dehydrogenase

molGC 33 %

Cons. hypotheticalCons. hypothetical, low homology

O23

Plac

Glucosylhydrolase family protein (junk sequence!)

molGC 54 %

-30 bp to the end of the fragment Carboxylesterase Ca 500 aa (one read left) 54% sim. Bacillus

New:

Enzyme reaction products

TG

1,3-DG1(3), 2-DG

MG

O

O

O

O

O

O

oil2 oil8oil7 O.8 O.14O.12O.9

O.4 O.21O.2O.16

TributyrinEsterase in H2O:Acetonitrile

Enzyme purification:i.e. Oil2, Oil7 and Oil8

Purification: Cationic exchange on MonoSHydrophobic interaction (Phenylsuperose)Gel filtration (Superose 12)

Native gel electrophoresis,development with -naphtylbutyrate

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OIL 2 OIL 7

OIL 8 O.4

O.5 O.8

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O.9 O.10

Specific activities with p-nitrophenol derivatives

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Carbon atoms

O.11

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O.16

O.23

O.12

O.14

O.21

O.2

Specific activities with p-nitrophenol derivatives

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

Screening hydrolases using a pH indicator method:

2 mL Iso-octane20 mg E. coli esterase clones

ENANTIOMERIC RESOLUTION OF 1-PHENYLETHANOL

BY TRANSESTERIFICATION WITH VINYL ACETATE

+

1 M 1-phenylethanol 1 M Vinylacetate

E > 20 INDUSTRIAL POTENTIAL (Enantiomeric ratio)

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

strain identification homology,% basin protease phosphatase esterase lipase glucosidase

3A Alteromonas marina 98 A 3 37A Alteromonas macleodii 99 A 3 38A Pseudoalteromonas sp. 92 A 3

14A no identification A 1 117A Alteromonas macleodii 99 A 318A Slope str., DIIII I c 97 A 2 3

1B Marinobacter hydrocarbonoclasticus 97 B 2 33B Marinobacter hydrocarbonoclasticus 100 B 3 2 3 34B Idiomarina baltica 99 B 3 3

5B bis Marinobacter hydrocarbonoclasticus 100 B 3 36B no identification B 212B Bacillus licheniformis 98 B 217B Rhodospirilaceae bact 97 B 319B no identification B 3

2D Alteromonas macleodii 99 D 34D Alteromonas macleodii 99 D 25D Alteromonas macleodii 99 D 2 3

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

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

ABSTRACT: enzyme propertiesHere 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.

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…

WHAT NEXT???

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

Conclusions and outlook

Screening of “biosurfactant” producing isolates - finishedNothing incredibly new or interesting obtained

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

Progress estimates:

Expression libraries are the best tool to exploit the diversity:Work with separate isolates is less productive and time-consuming

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

Env Microbiol Special Issue is planned

1. Env-specificity: bio(geo)chemistry2. gradient Daniele etc3. Misha etc4. Henk etc5. Marseille etc6. Tassos etc7. biotech applications (Peter and I), Proteus

Publications