Technical University Braunschweig

1. WP7 Technical University Braunschweig German Research Centre for Biotechnology, Braunschweig

2. Oil - degradative capacities Objectives Evaluation of major microbial groups Background for applications Adjusting protocols Sample chosen: U3

3. Isolates from oil enrichment. Marinomonas Oceanospirillum Alcanivorax Neptunomonas Marinobacter Oleiphilus messinensis Microbulbifer Pseudomonas Xanthomonas- (sensu stricto) Stenotrophomonas Colwellia group group Thiomicrospira Aeromonas group group Vibrionaceae Chromatiaceae Legionellaceae Enterobacteriaceae Pasteurellaceae Cardiobacteriaceae Methylococcaceae 0.1 substitution/site Ps. stutzeri-Ps. balearica group Marinobacter hydrocarbonoclasticus-Mb.CAB a-Proteobacteria (Ruegeria group)

4. “Archives” SuperCos1 - a „common“cosmid for cloning, Fragments to clone 40-50 kbp pBAC (e.g. pBeloBac11) large fragments 50-300 kbp Expression libraries l-based systems, e.g. LambdaZap METAGENOMIC LIBRARIES pLAFR3 - a replicon in a variety of Gram-negatives (e.g. P. putida) fragments to clone - 20-30 kbp

5. 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

6. 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

7. Enzymes Detection system Esterases/proteases Indoxyl acetate Phosphatases Indoxyl phosphate Sulfatases Indoxyl sulfate Esterases/proteases a- naphtyl acetate/ butyrate+FastBlueRR Lipases a- naphtyl palmitate + ”” Phosphatases a- naphtyl phosphate + ”” b -D- Galactosidases a- b naphtyl -D- galactopyranoside + ”” a -D- Galactosidases a- a naphtyl -D- galactopyranoside + ”” a/b -D- glucosidases a- a/b naphtyl -D- glucopyranoside + ”” Enzymes and detection systems

8. 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

9. 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

10. Enzyme purification: Native gel electrophoresis, development with a-naphtylbutyrate Purification: Cationic exchange on MonoS Hydrophobic interacti(on Phenylsuperose) Gel filtration (Superose 12)

11. Enzyme reaction products TG 1,3-DG 1(3), 2-DG MG oil2 oil7 oil8

12. Specific activities with p-nitrophenol derivatives

13. Temperature optima Features of the enzymes Thermostability

14. Features of the enzymes (cont’d) pH - activity profiles

15. Features of the enzymes (cont’d) Influence of surfactants Solvent resistance

16. Effect of cations on activity: Substrate: p-NPhBu

17. Conclusions and outlook 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) 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)