Abstract

The objective is to develop novel genetic screening methods for retrieval of valuable genes from unculturable microbes. A multi-sequence database on starch-modifying enzymes will be a basis to obtain new and improved enzymes for various starch modifications. A novel genetic screening method for a particular protein family has been designed. We will retrieve environmental sequences without creating large gene libraries and avoid high throughput expression cloning. A diversity of new sequences will be found and a selection of the most unique genes will be expressed and produced for characterisation The results will improve our understanding of the correlation between gene sequence, structure and function of this enzyme family.

Objectives

The main objective of the project is to develop methods to retrieve DNA sequences from environmental samples without creating large gene libraries and using high throughput expression screening. With a multi-sequence database on starch-modifying enzymes, we plan to develope novel genetic screening methods for retrieval of valuable genes from unculturable microbes in hot springs. In particular, the goal is to focus on obtaining new and improved enzymes for various starch modifications. We expect to find a large number of new sequences and to express and produce a selection of the most unique enzymes for characterisation. The results obtained should contribute to an improved understanding of the correlation between gene sequence, structure and function of this enzyme family.

Description of the work

The project involves development of methods to extract high quality DNA directly from biomass and enrichment cultures obtained from diverse geothermal habitats.

The DNA will be genetically screened for starch modifying enzyme genes directly from biomass. Gene specific primers will be used to obtain sequences that will be grouped on the basis of similarity. Candidates will be selected for complete gene retrieval, cloning and expression Primary screening for activity of the obtained and expressed starch modifying enzymes of the alpha amylase family includes enzymes of varied activities. Different assay system will be set up for fast and efficient activity screening for these activities. This also includes secondary screening using biochemical criteria, such as thermostability and pH tolerance. On the basis of these results enzymes will be selected for more thorough biochemical studies including specific activity and other kinetic parameters, temperature and pH optima, substrate specificity and end-product formation. Enzymes for potential industrial applications will be identified. Structure/function studies, correlating structural features and functional properties will be done. State-of-the-art software will be used for sequence analysis, and homology-modeling Selected enzymes will be produced in large- scale for application studies.

Deliverables

• Methods to get high quality DNA from hot spring biomass and enrichments for genetic PCR screening of starch-modifying enzymes.
• An efficient gene retrieval method and at least 100 novel genes coding for various starch-modifying enzymes partially sequenced.
• Up to 50 genes expressed coding for starch-modifying enzymes from biomass.
• Full biochemical characterisation of 5-10 starch-modifying enzymes.
• Results on function/structure relationships of thermostable starch-modifying enzymes.

Contacts

Coordinator

• Jakob K. KRISTJANSSON / Prokaria ehf ICELAND

Participant

• Peter SCHÖNHEIT / Christian-Albrechts-Universität zu Kiel Institute für Allgemeine Mikrobiologie / GERMANY
• Olle HOLST / Lunds Universitet Centre for Chemistry and Chemical Engineering / SWEDEN