QLK3-2001-00149 Combinatorial engineering of glycoside hydrolases from the alpha-amylase superfamily (CEGLYC)

Contacts




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Type of Project	Shared Cost Research
Contract No	QLK3-2001-00149
Total Cost
EC Contribution	1,991,300 EUR
Start Date	01-01-2002
Duration	36 Hours

Abstract

The aim of the CEGLYC project is to apply combinatorial engineering approaches to obtain improved enzymes with high catalytic efficiency and better specificity and stability. The selected targets for in vitro molecular evolution techniques are four glucoside hydrolases from the alpha-amylase superfamily: amylosucrase, amylomaltase, maltogenic alpha-amylase and barley alpha-amylase, which share an identical active site architecture. These improved enzymes will enable the efficient transformation of European renewable agro-resources, starch and sucrose, into products of interest for food and beverages, nutrition and health applications. The project thus cobines both basic scientific targets, an improved understanding of glucoside hydrolase mechanisms and developing new industrial environmentally-friendly bio-catalytic processes.

Objectives

The aim of the project is to apply protein combinatorial engineering approaches to obtain enzyme with improved catalytic efficiency and better specificity and stability properties. The selected targets for gene random mutagenesis and in vitro evolution techniques are those coding for four glucoside hydrolases from the alpha-amylase superfamily: amylosucrase, amylomaltase, maltogenic alpha-amylase and barley alpha-amylase, which share a common, beta-alpha-8 barrel catalytic domain structure and an identical active site architecture. These improved enzymes will allow the efficient use of European renewable agro-resources, starch and sucrose, into high added-value products of interest for food and beverages, nutrition and health applications.

Activities

A multidisciplinary and concerted approach will be followed within this project aimed at the design of improved variants of four structurally closely related enzymes of industrial interest:

• amylosucrase from Neisseria polysaccharea: random mutagenesis cycles will be carried out on the coding sequence. Screening tests will be designed for variants with improved properties. Key mutation sites for improvement of amylosucrase will be identified by structural analysis.
• amylomaltase from Thermus thermophilus: a functional analysis of the active site will be carried by construction of site-directed mutants. Random mutants will be made using error-prone PCR, coupled with high throughput screening, and characterised particularly for their ability to form thermo-reversible gels from starch. The structure of these mutants will be determined by crystallography to identify mutation sites and determine new target regions for random mutagenesis.
• maltogenic alpha-amylase with optimised substrate specificity: a screening test will be developed to distinguish between enzymes with altered specificity for high throughput screening of Bacillus subtilis variant libraries. Random and
• in vitro
evolution variant libraries will be constructed. Pilot-scale amounts of enzyme will be produced for application tests.
• plant alpha-amylases: genes encoding barley alpha-amylases will be selected for in vitro evolution and gene shuffling, screening and production of protein variants representing modified effect of calcium on activity and modified sensitivity to proteinaceous inhibitors of plant origin. Crystal structures will be determined for selected variants.

Deliverables

The following deliverables are anticipated:

• an array of new enzyme variants from glycoside hydrolase from Family 13 and the closely related Family 77 (both from the alpha-amylase superfamily) obtained through in vitro evolution with screens focusing on optimisation for industrial applications.
• several three-dimensional structures of new improved enzymes and the interpretation of these structures with regard to their functions.
• optimised and tuned shuffling methods adapted to the various specificities encountered in family alpha-amylase superfamily.

Contacts

Coordinator

• INSA Bioprocess Laboratory / FRANCE

Participant

• Carlsberg Laboratory Deptment of Chemistry / DENMARK
• Richard HASER / Institut de Biologie et Chimie des Protéines UCBL, UMR5086 / FRANCE
• Torben Vedel BORCHERT / Novozymes Protein design / DENMARK
• Michael GAJHEDE / University of Copenhagen Protein Structure group / DENMARK
• Lubbert DIJKHUIZEN / University of Groningen Microbial Physiology / NETHERLANDS