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Technical University Braunschweig

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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  1. WP6, WP7 Technical University Braunschweig German Research Centre for Biotechnology, Braunschweig

  2. 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

  3. DNA size-fractionated, partially digested with Sau3A Ligation 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

  4. 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

  5. Screening hydrolases using a pH indicator method:

  6. 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

  7. 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

  8. New clones coding for hydrolytic enzymes Rhodanese domain putative para-nitrobenzyl/carboxyl esterase, Ca. 500 aa, < 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 <30% smlr. O08 Plac molGC 57 % Consvd. membr prot, 65 %, pfam02517, Abi, CAAX Conserved hyp. Ca. 150 aa, 60 % sim. Esterase motif, Low similarity COG0657, Aes, Esterase/lipase Ca. 280 aa low similarity COG1514, LigT, 2'-5' RNA ligase < 50 % COG0523, Putative GTPases (G3E family) Plac O09 molGC 56 %

  9. New clones coding for hydrolytic enzymes (cont‘d) Enoyl CoA hydratase COG0657, Aes, Esterase/lipase [Lipid metabolism], <50 % 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] <30 % Hypothetical, Low homology O16 Plac molGC 50 % No ORFs Putative membrane protein Low homology <30 % Hydroxyacyl dehydrogenase Cons. hypothetical, low homology Cons. hypothetical Plac O21 molGC 33 %

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

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

  12. Temperature optima Features of the enzymes Thermostability

  13. Specific activities with p-nitrophenol derivatives

  14. Specific activities with p-nitrophenol derivatives

  15. 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

  16. Screening of hydrolytic activity: Conisma strains (the others to come) Substrate:-naphtylacetate, butyrate, laurate, palmitate, phosphate, glucoside, galactoside and Fast Blue RR

  17. 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

  18. Publications??? Best regards from Peter, Manolo and Ken

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