Skip to main content
European Commission logo print header

EXPLOITATION OF MICROBIAL PECTINOLYTIC ENZYME SPECIFICITY IN PECTIN MANUFACTURING AND OTHER AGRO-INDUSTRIAL PROCESSES

Obiettivo

The aim of this project is to investigate the nature and function of pectin degrading enzymes at the molecular level as well as with respect of industrial applications.

Pectinolytic enzymes are widely applied in the agro-food industry. However, this is the first time that these important enzymes and substrates are investigated in combination at the molecular level. The skills of the partners involved complement each other in a unique way, and combine expertise in the biochemistry and molecular genetics of pectin modifying and degrading enzymes (partners 01, 02), in x-ray crystallography (partner 03), in carbohydrate chemistry (partner 04), in the structural analysis of pectin and enzymatically obtained pectin degradation products (partner 05), and in various applications (partner 05, 06).

The project will focus on:

1. The overproduction of individual fungal and bacterial pectinolytic enzymes both by genetic manipulation and by proper management of microbial physiology.

2. Characterization of enzyme specificity, by thorough kinetic analysis (pH-, T-profiles; oligomeric, polymeric substrates; subsites; mode of action; enzyme inhibition using inhibitors designed from oligogalacturonates); and supported by enzyme structural data.

3. Analysis of the fine-structure of different pectins in relation to enzyme specificity.

4. Analysis of pectin degradation in situ.

Application studies involve two different industries and are directed:

1. To improve product quality, to develop new products (novel pectins, oligosaccharides) and to reduce environmental problems.

2. To improve protopectin extraction.

3.To improve fruit and vegetable processing (extraction yield, clarification, liquefaction, maceration).

4. To increase the nutritional efficiency of raw material and to incorporate previously unsuitable materials into animal feed.

A further spin-off comes from the synthesis of specific pectinase inhibitors as potential plant-protecting agents and to enzymatically convert and upgrade agricultural biomass and waste preparing carbohydrate mixtures as feed stocks for fermentation and chemical industries.

Pectin is one of the components of plant cell walls, occurring in some abundance in citrus fruit, for example, from which it may be extracted for use as a food ingredient. Conversely, pectin degrading enzymes may be used to remove particulate matter in clarification of juices and beverages as well as finding many other applications in the food industry. The purpose of this project is to investigate the nature and function of these enzymes at the molecular level as well as with respect of industrial applications. ACTIVITIES

Genetic manipulation: So far, progress with respect to overproduction of pectinolytic enzymes and identification of new pectinolytic genes/activities of both fungal and bacterial origin has been good. Many genes encoding pectinolytic enzymes (pectin and pectate Iyases [PL and PAL, respectively], oligo-/poly-galacturonases [OGL and PG, respectively], pectin methylesterases [PME]) have been engineered under the control of strong promoters. In the case of the fungal genes, the strong constitutive pyruvate kinase promoter was used; while in the case of bacterial genes, several strong inducible promoters were used. As a result of this work, many of the fungal enzymes can now be distributed to other partners in 100 mg quantities. Through use of these promoter-gene fusions, the choice of an appropriate Aspergillus niger expression strain and easy purification protocols have been made. One of the fungal genes (pectin methylesterase) has not yet been successfully fused. Of the desired bacterial enzymes, deliverable amounts have been purified from the expression clones with the exception of Erwinia protopectinase and polygalacturonase from Erwinia carotovora. It is expected that by cloning this gene into a new vector, sufficient expression will be obtained. A search for enzymes with protopectinase activity has started. New pectinolytic genes have been identified in A. niger and E. chrysanthemi. Four additional PG genes have been cloned from A niger and one of these has been completely sequenced and fused to the pyruvate kinase-promoter; while two genes have been partly sequenced. Two new genes were cloned and sequenced from E. chrysanthemi, (encoding a PME and a PAL); while two more pectinolytic clones were identified, but not yet characterised.

Biochemistry: Considerable progress has been made in biochemical characterisation of pectinolytic enzymes from fungi. Temperature and pH optima have been determined for all three enzymes. Kinetic analysis has been completed for some enzymes and continues for other. This work includes the determination of physico-chemical and kinetic properties for the recently detected bacterial PME B and PAL L.

Induction: Another approach adopted has been the study of inducers of enzyme synthesis. It is known that 2-keto-3-deoxy-gluconate (KDG) is one of the normal inducers of pectinase production. A set of 5 analogs was synthesised and tested for inducing properties. This led to the identification of gratuitous inducers (5-deoxy-, 5-O-methyl- and 5 epi-KDG) which can be used for expression purposes and to the discovery that the 2-keto function is essential for induction. Two routes have been explored for the synthesis of artificial substrates and inhibitors. These routes proved not to be successful. Other routes are now being explored.

Structure: Characterisation of several pectinolytic enzymes, based on 3-D structure analysis, is in progress. This work focuses on a number of enzymes from Aspergillus, Erwinia chrysanthemi and Bacillus subtilis. Efforts are directed at optimising crystallisation conditions in order to obtain crystals that allow high resolution data, as well as at obtaining heavy atom derivatives and simultaneously using replacement techniques (using the known pectate Iyase structure as search model). For Bacillus subtilis PAL the 3-D structure is known and has been used to study the interaction with the substrate and to understand the function of the bound calcium. Experiments are aimed at obtaining crystals with substrate bound and crystals with other divalent ions such as barium.

Mode of action: In order to assess the mode of action and activity of the pectinolytic enzymes on pectins of different degrees of esterification and amounts and nature of side chains, pectins were isolated from lemon with various degrees of esterification (70%, 60%, 45%, 20% and 7%), as well as three other sources (apple, sugar beet and orange). These have been characterised and will be used as reference compounds in various assays. The testing of the individual pectinolytic enzymes for their applicability in industrial processes like upgrading feed stuffs and improving food processing has not started yet.

Invito a presentare proposte

Data not available

Meccanismo di finanziamento

CSC - Cost-sharing contracts

Coordinatore

WAGENINGEN AGRICULTURAL UNIVERSITY
Contributo UE
Nessun dato
Indirizzo
Dreijenlaan 2
HA Wageningen
Paesi Bassi

Mostra sulla mappa

Costo totale
Nessun dato

Partecipanti (5)