Klonierung, Expression und Produktion einer marinen Chitindeacetylase. Hayssam Zakaria, Christian Schmalz, Arno Cordes & Bernd Otto

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1 Klonierung, Expression und Produktion einer marinen Chitindeacetylase Hayssam Zakaria, Christian Schmalz, Arno Cordes & Bernd Otto

2 CHITOSAN- what it is Deacetylated derivative of chitin Deacetylation grade > 50% Chain length variable Secondary structure still remaining Proteins and Calcium carbonat present or not

3 Chitosan Chemical Properties Polymer of D-glucosamine Reactive amino groups Reactive hydroxyl groups available Chelates many metal ions Forms films

4 Chitosan Biological Properties Biocompatible Natural polymer Biodegradable Non-toxic Binds to mammalian cells Regenerative effect on conective tissue Accelarates the formation of osteoblasts Fungistatic Spermicidal

5 CHITOSAN- chemical production Decalcification in dilute HCl solution Deproteination in dilute NaOH solution Decolorization in 0.5% KMnO4 and Oxalic acid Chitin Deacetylation in hot (80 C) concentrated (40-50%) NaOH solution Neutralization Chitosan

6 Chitinabbau

7 Chitin-Deacetylase

8 CHITOSAN- Anwendungen Membranes and paper Permeability control Reverse osmosis Photographic paper Surface treatment Cosmetics Moisturizer Antimicrobial agent Fungistatic Wastewater Treatment Removal of metal ions Flocculant/ Coagulant Biotechnology Wound healing Slow release of drugs Burn treatment Enzyme immobilization Contac lens Agriculture Seed coating Fertilizer Animal feed additive

9 Chitin - sources Crustacean shells Insect exosceleton Fungal cell walls Plankton

10 Basics High tech Chitosan für medizinische Anwendungen Neue Chitindeacetylase (CDa) zur enzymatischen Konvertierung von Chitin zu Chitosan Enzymatische Prozesse sind leichter zu kontrollieren / zu reproduzieren (uniforme Qualität) Bisherige Enzyme sind dafür unzureichend

11 Der Plan Alignment bekannter Enzyme mit ähnlicher Aktivität Design degenerierter Oligonukleotide Degenerierte PCR von DNA aus isolierten marinen Mikroorganismen Direkte genomische Sequenzierung für eine full length Sequenz Heterologe Expression des klonierten Gens in E. coli Aufreinigung (Affinitäts-Chromatographie)

12 Alignment

13 Design of the degenerated primers Protein-level: DNA-level:

PCR-products lane 1: Marker λ-dna-bst EIIDigest lane 2 + 20: Marker φx174

14 PCR-products lane 1: Marker λ-dna-bst EIIDigest lane : Marker φx174 DNA-HaeIII-Digest lane 3-19: PCR-products with several primer-pairs 2% TBE agarose-gel

15 Die neue Chitindeacetylase

16 Alignment of the new Chitindeacetylase

17 Characteristics Original-Stamm grampositive bacteria open reading frame gene length [bp] molecular weight [kd] theoretical IP startcodon promicromonospora/ actinobacteria ,65 11,86 CTG ,05 10,64 CTG Biologische Daten keine Sekretions-Sequenz möglicherweise Teil eines Chitindegradations-Operons ,69 8,52 GTG ,50 6,84 ATC ,01 8,52 GTG ,09 6,83 CTG spezifische Aktivität (ORF 5): 5 nkat/mg

18 Klonierung der neuen Chitindeacetylase expression in E. coli strain BL21 (D3) vector: pask-iba 7 induction with 200 µg/l Anhydro- Tetracycline expression for 4 h at 25 C expression levels: - soluble: 100 µg/g wet-weight - Inclusion bodies: > 10 mg/g wetweight

19 Klonierung der neuen Chitindeacetylase expression in E. coli strain BL21 (D3) vector: pask-iba 7 induction with 200 µg/l Anhydro- Tetracycline expression for 4 h at 25 C expression levels: - soluble: 100 µg/g wet-weight - Inclusion bodies: > 10 mg/g wetweight

20 Aufreinigung - lösliche Produktion (ORF 5) coomassie stained sds-gel westernblot lane1: total lysat; lane 2-4 and 6-8: fractions of the Strep-tag II purification; lane 5: prestained low-range marker, Biorad

Purification - inclusion bodies (ORF 5) coomassie stained sds-gel lane1: prestained low-range marker, Biorad; lane 2: total lysat; lane 3: purification from

21 Purification - inclusion bodies (ORF 5) coomassie stained sds-gel lane1: prestained low-range marker, Biorad; lane 2: total lysat; lane 3: purification from inclusion bodies pro: high total yield high purity contra: protein has to be denaturated and refolded specific activity is lower compared to soluble preparation

22 Temperatur Optimum 20 Temperature Optimum 15 acetic acid [mm] C 15 C 25 C 30 C 37 C 42 C

23 ph-optimum 20 Deacetylation of 45 mm N-Acetyl-Chitosan-Oligomers by 1 µm enzyme at 25 C for 48h in 100 mm PBS buffer 15 acetic acid [mm] ph 6.0 ph 7.0 ph 8.0

24 Kinetik Deacetylation of 45 mm N-Acetyl-Chitosan-Oligomers by 1 µm enzyme at 25 C in PBS buffer, ph acetic acid [mm] deacetylation [%] h 24 h 48 h 72 h 96 h 120 h time

25 Substrat Spezifität acetic acid [mm] Crab-Chitin N-Acetyl-Chitosan-Oligomers

26 Ausblick Austesten weiterer Chitin-Substrate Expression in patentfreiem System Erhöhung der Proteinausbeute Upscaling zum industriellen Maßstab Optimierung des enzymatischen Prozesses

27 Die Hände & Köpfe Tierärztliche Hochschule Hannover (ehemals FhG-IGB) Prof. Bernd Otto Dr. Hayssam Zakaria Dr. Christian Schmalz (Hugo-Geiger-Preis) FH Oldenburg/ Ostfriesland/ Wilhelmshaven Prof. Eike Siefert Uta Bünger Bernd Schmietenknop Universität Hannover Dr. Jochen Meens Dipl. Biochem. Christine Schreiber Dr. Irene Wagner-Döbler ASA Spezialenzyme GmbH Dr. Arno Cordes

28 Das Portemonaie Niedersächsischer Forschungsschwerpunkt Meeresbiotechnologie Themenbereich 1: Chitosan und Chitosanoligomere

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