BioMatNet Item: AIR2-CT94-1345 - Exploitation of microbial pectinolytic enzyme specificity in pectin manufacturing and other agro-industrial processes

AIR2-CT94-1345
Exploitation of microbial pectinolytic enzyme specificity in pectin manufacturing and other agro-industrial processes

Contacts
Summary Information

	Proposal No:	AIR2-CT94-1345
	Date Prepared:	February 1996, April 1998, May 1999, September 1999
	Source:	First Project Progress Report Consolidated Report, Year 3 Final Consolidated Report

First Project Progress Report

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.

Consolidated Report, Year 3

Work and achievements
With respect to overproduction of fungal and bacterial pectinolytic enzymes and identification of new pectinolytic genes/activities output remained high. Overproduction and easy purification is realised by using strong promoters to direct expression of the pectinolytic genes of interest: T7-, Lac- and the Trc-promoters for bacterial genes and the pyruvate kinase promoter for fungal genes in combination with an appropriate Aspergillus niger expression strain. Most of the fungal enzymes, including the newly identified rhamnogalacturonan acetylesterase and pectate lyase can be delivered to other partners in 100 mg quantities. Of the desired bacterial enzymes, also including newly identified enzymes, amounts sufficient for characterisation have been purified from the expression clones except for the Erwinia oligogalacturonase. As an alternative for a bacterial protopectinase attention is now focused on enzymes active towards the ramified parts of the pectin that may serve as a protopectinase activity by the solubilisation of the protopectin.

The continued search for new pectinolytic genes has been successful: both in A. niger and E. chrysanthemi new genes have been identified. In addition to the previously reported genes plyA and rgaeA encoding a pectate lyase and a rhamnogalacturonan acetylesterase, respectively, two other genes have been obtained from A. niger; one being a rhamnogalacturonan lyase encoding gene, rgl. Promoter-gene fusions for these new genes will be prepared. For genes plyA and rgaeA sequence analyses were completed and promoter-gene fusions were prepared. The E. chrysanthemi pectinase gene spectrum has been expanded by four more genes: one endopolygalacturonase encoding gene, pehR, two exopolygalacturonase genes, pehV and pehW and an esterase encoding gene. Sequence analysis of these three polygalacturonase genes is in progress as is the construction of over-expression vectors while this is completed for the esterase gene. For all previously reported new genes, pelZ and pel1, encoding endopectate lyases PELZ and PELI, respectively, pelX, encoding exo-pectate lyase PELX, and pehX, encoding exo-polygalacturonase PEHX, sequence data are complete and expression vectors have been prepared.

The biochemical characterisation of the fungal and bacterial pectinolytic enzymes has been continued and extended to new enzymes as they became available. In addition to the fungal endopolygalacturonases PGI, II, C and E (partner 01) two other fungal endopolygalacturonases, PGA and PGB, have been characterised with respect to pH optima, performance on methylated pectins of various degree of esterification and mode of action on oligogalacturonates of defined length. Subsite map determination for these two enzymes is in progress. The performance of all polygalacturonases was also studied on a complex substrate, apple pectin. This revealed that the enzymes have different specificities although it is not known yet which substrate parts are preferentially recognised by the individual enzymes. The fungal pectate lyase, which serves as the basis of a pectin identification kit developed by industrial partner 06, was studied in great detail with respect to pH optimum, ionic strength dependence, calcium dependence, substrate specificity and mode of action on oligogalacturonates of defined length. A similar analysis of the biochemical properties as carried out previously for the bacterial enzymes PELA, B, C, D and E (partner 03) i.e. pH and temperature optima, kinetic constants Km and Vmax, the influence of calcium and other divalent ions and the influence of the degree of esterification of the substrate was performed for the pectate lyases PELL, PELI and PELZ. The mode of action of exo-pectate lyase PELX and exo-polygalacturonase PEHX was studied in collaboration with partner 01. This revealed that PELX cleaves off dimers from the reducing end of the substrate while PEHX hydrolyses dimers from the non-reducing end.

The 3-D structure analysis program (partner 05) which focuses on Aspergillus and Erwinia PME, on E. chrysanfhemi PELL and Bacillus subtilis PEL has progressed as follows. Fungal PME failed to crystallise. Therefore enzymatic deglycosylation was addressed but proved to be unsuccessful. Partner 01 will try to obtain fungal PME without N- glycosylation by expressing the enzyme in the presence of tunicamycin or in E. coli. Partner 01 also purified bacterial PME expressed in B. subtilis. Unfortunately this enzyme preparation failed to yield crystals. Revision of the purification protocol by partner 01 may solve this problem. For PEL L new crystals have been obtained which yielded a data set with resolution down to 0.18 nm. The solution of the structure is being addressed the analysis of heavy atom derivatives. A new pectate lyase, PELI, has been crystallised. Since for Bacillus subtilis PEL the 3-D structure is known the main objective for this system remains to study the interaction with the substrate and to understand the function of the bound calcium. We reported last year that several crystallisation conditions were found that might allow for substrate binding. It turned out that none of these conditions resulted in binding of the substrate. Alternative conditions are presently addressed. However, partner 05 was very successful in solving the first pectin lyase structure, PELA originating from A. niger which was not described as a task in this project.

Another approach to biochemical and physiologically characterise the different pectinolytic enzymes is by looking into induction and to study their performance with artificial substrates and inhibitors (partner 04). We already reported the discovery that the 2- keto function of 2-keto-3-deoxy-gluconate (KDG), one of the normal inducers of pectinase production, is essential for induction. In order to identify which of the two anomeric forms (alpha or beta) of KDG is recognised and what the function of the intracyclic oxygen atom is, the synthesis of the two epimeric carbocyclic analogs was addressed. Both compounds will be available for testing soon. With respect to artificial substrates and inhibitors very good progress has been made. Firstly, the chemistry has been developed to obtain gram quantities of intermediates necessary for the synthesis of these compounds and secondly, di-and trigalacturonates methyl-esterified at defined positions can be made and are available (dimers) or will be available soon (trimers) to be studied with PELs and PGs. With respect to inhibitors of OGL, two compounds were synthesised while the synthesis of two additional inhibitors/substrates will be finished shortly.

In order to assess the essential information of 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 four sources viz lemon, apple, sugar beet and orange (partner 06) and made available to all partners. In addition protopectin and 'hairy regions', the highly ramified part of the pectin, were prepared and delivered. The physical properties of all these pectins have been determined and these substrate batches are used by all partners as reference compounds in various assays.

The ultimate testing of the individual pectinolytic enzymes for their applicability in industrial processes has been continued for several enzymes. For upgrading feed stuffs and improving food processing (partner 02) it has been concluded that other enzymes besides the polygalacturonases are required. Most of the auxiliary enzymes can be provided by partner 01. Partners 01 and 02 will further work out which combination of enzymes is required Partner 6 has produced pectins using fungal pectinases and studies the physical properties of the modified pectins. An important result has been the development of a pectin identification kit which is based on the fungal pectate lyase developed by partner 01. The kit will be evaluated and distributed to all industries and researchers in the pectin field belonging to the International Pectin Producers Association (IPPA).

Conclusions and highlights
Highlights are:

the unexpected large number of new pectinolytic gene functions cloned
the marked differences in enzyme specificity observed within a specific class of enzymes (endo-polygalacturonases; pectate lyases)
the development of new chemistry to synthesise inhibitors and di- and trigalacturonate substrates substituted at specific positions
the remarkable topological similarity between the pectate and pectin lyase structure
the development of a pectin identification kit.