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Biodeep October 12-15, 2003

Biodeep October 12-15, 2003. “Nature has more imagination than our dreams”. Contents. Strains and clones transferred Microbial screening Proteases Esterases Xylanases Molecular screening Analysis of the cloned genomic fragments Construction/expression

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Biodeep October 12-15, 2003

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  1. Biodeep October 12-15, 2003 “Nature has more imagination than our dreams”

  2. Contents • Strains and clones transferred • Microbial screening • Proteases • Esterases • Xylanases • Molecular screening • Analysis of the cloned genomic fragments • Construction/expression • Biochemical characterization • Biodeep Database • Communications • Conclusion

  3. Strains transferred • Milan University (Dr. T. Brusa) 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefore stored in collection) and can be used for future work

  4. Strains transferred • Essex University (Dr. A. Sass) 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefore stored in collection) and can be used for future work 3. The 19 strains of the 1st batch which could not be cultured in our lab have been sent a second time. The 40 strains of batch 3 are anaerobes (stricts or facultatives). 4 enrichment cultures currently used

  5. Strains transferred • LLM (Dr. F. Morel) 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work

  6. Strains transferred • Messina University (Dr. G. D’auria) 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work 3. See next slide 4. All the strains excepted the ones growing in P10 medium (petrol based medium)

  7. Strains transferred • Cultures from Univ. Messina • Enrichment cultures and pure strains • No synthetic media defined (from the water point of view) • We tried to grow the stains in a defined medium • The growth conditions defined for the 22 strains cultured in our lab are available • about 70 pure strains growing in ONR medium will be transfer soon

  8. Strains transferred • Review 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab and can be used for future work

  9. Clones transferred • clones from TUB (Dr. P. Golyshin) • 11 genomic fragments encoding 11 different esterases • Corresponding (partial or FL) sequences have been provided with some genomic fragments • Subcloning of oil8 have been done in fusion with pelB leader for periplasmic expression. Expression successful

  10. P 1600 After 3 days at 25°C Microbial screening : Protease • Extracellular proteases using skim milk (0.2%) screening assay • Review (aerobes) (1) 1. Planned with the pure strains to be transferred

  11. Microbial screening : Protease • Taxonomic position according Biodeep partners

  12. Microbial screening : Esterase • Esterases • Principle of the CLIPS-O™ substrates R = C2H5 C9 H19

  13. Microbial screening : Esterase • Preliminary results : Non induced (aerobes) • Preliminary results : Induced (aerobes). 1. Measured only using C10 CLIPS-O™ substrates

  14. Microbial screening : Xylanase • Xylanase using “xylan blue” (0.05%) screening assay (xylan was used as carbon and energy sources) • Preliminary results (aerobes)

  15. Microbial screening : Xylanase • Taxonomic position according Biodeep partners

  16. Molecular screening • Proteases from strain P 1972 (Alteromonas sp.) and strain P 1600 (Bacillus sp.) • Genomic library construction : partial restriction using Sau 3A I • Inserts size ranging from 2 to 5 kb • Quality control : less than 10% of empty clones • Screening performed • Positive hits identified and sequenced

  17. Analysis of the genomic fragment (PP 1972) 2989 bp Identity/Similarity with Subtilisin (P00780) in amino acids : - FL gene : 29%-48% - Nterm region of the gene : 34%-54%

  18. Constructions Construction Activity OK + OK + promoter P1972 FL gene 1. 2. pelB

  19. Subcellular localization

  20. Kinetic of expression Induction at 28°C Induction

  21. Biochemical characterization Thermostability at 30°C Thermostability at 40°C Thermostability at 30°C

  22. Analysis of the genomic fragment (PP 1600) 4212 bp Identity/Similarity with Intracellular Alkaline Protease (P29140) in amino acids : - FL gene : 56%-75%

  23. Constructions Construction Activity OK + In progress In progress promoter P1600 FL gene Potential SP pelB

  24. Biochemical characterization • Optimum pH : 8-9 • Other characteristics : in progress

  25. Clones collection

  26. Biodeep database • Protéus • Design of the data sheet model • Data related to 347 strains transferred in July to Univ. of Milano • University of Milano • Design of the database

  27. Communications • Our industrial partners • Congress : • MINATEC 2003 (Grenoble, France) • BIO2003 (Washington, USA) • INDUSTRIAL APPLICATIONS OF BIOCATALYSIS (Boston, USA)

  28. Conclusions and Outlook • Current status of the Biodeep microbial collection :407 strains • Current status of the Biodeep clones collection : 13 cloned genomic fragments containing enzymatic activities (3 ORF subcloned)

  29. Timetable of future activities

  30. …Thanks for your attention. www.proteus.fr

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