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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 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

  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 Esterases Cellulases and amylases

  5. 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

  6. 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

  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. molGC 58 % Plac esterase esterase/deacetylase Oil2 Conserved hypothetical protein

  9. 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 %

  10. 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 %

  11. 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 %

  12. 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

  13. 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

  14. 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 %

  15. 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

  16. 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

  17. 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)

  18. Specific activities with p-nitrophenol derivatives

  19. Specific activities with p-nitrophenol derivatives

  20. 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

  21. 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

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

  23. 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.

  24. 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…

  25. 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

  26. 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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