Klonierung, Expression und Produktion einer

marinen Chitindeacetylase

Hayssam Zakaria, Christian Schmalz, Arno Cordes & Bernd Otto

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

Chitosan Chemical Properties

Polymer of D-glucosamine

Reactive amino groups

Reactive hydroxyl groups available

Chelates many metal ions

Forms films

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

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

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Chitin-Deacetylase

CHITOSAN- Anwendungen

Biotechnology

Wound healingSlow release of drugs

Burn treatmentEnzyme immobilization

Contac lens

Agriculture

Seed coatingFertilizer

Animal feed additive

Membranes and paper

Permeability controlReverse osmosis

Photographic paperSurface treatment

Cosmetics

MoisturizerAntimicrobial agent

Fungistatic

Wastewater Treatment

Removal of metal ionsFlocculant/ Coagulant

Chitin - sources

• Crustacean shells

• Insect exosceleton

• Fungal cell walls

• Plankton

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

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)

Alignment

Design of the degenerated primers

Protein-level:

DNA-level:

PCR-products

lane 1: Marker λ-DNA-Bst EII-Digest

lane 2 + 20: Marker φX174 DNA-HaeIII-Digest

lane 3-19: PCR-products with several primer-pairs

2% TBE agarose-gel

Die neue Chitindeacetylase

Alignment of the new Chitindeacetylase

Characteristics

Original-Stamm

• grampositive bacteria

• promicromonospora/ actinobacteria

Biologische Daten

• keine Sekretions-Sequenz

• möglicherweise Teil eines

Chitindegradations-Operons

open reading frame

gene length [bp]

molecular weight [kD]

theoretical IP startcodon

1 1068 38,65 11,86 CTG

2 915 33,05 10,64 CTG

3 789 28,69 8,52 GTG

4 759 27,50 6,84 ATC

5 747 27,01 8,52 GTG

6 633 23,09 6,83 CTG

• spezifische Aktivität (ORF 5): 5 nkat/mg

Klonierung der neuen Chitindeacetylase!"#$%&'(($) ) ) **))

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• 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 wet- weight

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 wet- weight

Aufreinigung - lösliche Produktion (ORF 5)

lane1: total lysat; lane 2-4 and 6-8: fractions of the Strep-tag II purification; lane 5: prestained low-range marker, Biorad

westernblotcoomassie stained sds-gel

Purification - inclusion bodies (ORF 5)

coomassie stainedsds-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

Temperatur Optimum

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4°C 15°C 25°C 30°C 37°C 42°C

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Temperature Optimum

pH-Optimum

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pH 6.0 pH 7.0 pH 8.0

Deacetylation of 45 mM N-Acetyl-Chitosan-Oligomers by 1 µM enzyme at 25°C for 48h in 100 mM PBS buffer

acet

ic a

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[mM

]

Kinetik

Deacetylation of 45 mM N-Acetyl-Chitosan-Oligomers by 1 µM enzyme at 25°C in PBS buffer, pH 7.0

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0 h 24 h 48 h 72 h 96 h 120 h

time

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Substrat Spezifität

0

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Crab-Chitin N-Acetyl-Chitosan-Oligomers

acet

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[mM

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Ausblick

Austesten weiterer Chitin-Substrate

Expression in patentfreiem System

Erhöhung der Proteinausbeute

Upscaling zum industriellen Maßstab

Optimierung des enzymatischen Prozesses

Die Hände & Köpfe

Tierärztliche Hochschule Hannover Prof. Bernd Otto

(ehemals FhG-IGB) 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

Das Portemonaie

Niedersächsisches Hochschulgesetz (NHG)

Die wichtigsten Änderungen

Niedersächsischer Forschungsschwerpunkt Meeresbiotechnologie

Themenbereich 1: Chitosan und Chitosanoligomere

1998 - 2002