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AIR2-CT94-1345
Exploitation of microbial pectinolytic enzyme specificity in pectin manufacturing and other agro-industrial processes - Final Report |
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Proposal No: | AIR2-CT94-1345 |
| Date Prepared: | September 1999 February 1996, April 1998, May 1999 | |
| Source: |
Final consolidated report summary 1997 Project Summary and First and Third Year Reports |
Final consolidated report summary 1997
Summary
In order to establish the molecular basis for the specificity of technologically-important pectinolytic enzymes, numerous genes encoding these enzymes, of both fungal and bacterial origin, were cloned, sequenced and individually over-expressed. Those enzymes comprised the depolymerising pectin- and pectate lyases and endo- and exo- polygalacturonases in addition to pectin, pectin methyl, and rhamnogalacturonan acetyl, esterase. The purified enzymes were characterized with respect to their biochemical properties and substrate specificity. The latter was assessed using pectins of various degrees of esterification and oligomeric substrates of defined length. Furthermore, di- and tri- mer substrates mono-methylesterified at defined positions, galactose-galacturonate dimers and digalacturonate analogs were synthesized and used to assess the substrate specicificity of individual pectinases.
2-Keto-3-deoxygluconate analogs were synthesized in order to identify which element of the KDG is responsible for induction of pectinase expression in Erwinia chrysanthemi. Knowledge of the latter is essential for the over-production of pectinases and also for the design of specific inhibitors of pectinase production for crop protection. Three-dimensional structure analysis of several pectinases to further increase the knowledge of substrate specificity has led to the elucidation of the 3-D structure of an endopolygalacturonase from Erwinia and the establishment of crystallisation conditions for the Erwinia pectin methylesterase and pectate lyase L, a representative of a new class of pectate lyases. Selected pectinases in combination with other polysaccharide hydrolases were studied for their applicability in food and feed processing. For this, specific methodologies were developed. This allowed for the identification of enzymes critical for solubilisation of an insoluble fraction from the important feedstuffs soy and lupin. Assessment of the potential of selected pectinases in the pectin manufacturing has led to the application of specific enzymes for the production of new pectin products and furthermore, has resulted in the application of a fungal pectate lyase in a world wide accepted unambiguous pectin identification kit.
Introduction
This project focused on the establishment of the molecular basis of the technologically-important pectinolytic enzymes and to analyze the pectin fine structure in detail. This knowledge was to be exploited for selecting enzymes to improve and innovate pectin manufacturing and to reduce environmental pollution. The selected enzymes were also to be exploited in improving animal feedstuffs and in dedicated (liquefaction, maceration) and traditional (extraction, clarification) processes in food industry.
Objectives
As a result of the objectives the project was divided into four areas.
Results
The search for new pectinolytic genes in Aspergillis niger and Erwinia chrysanthemi has been very successful and has resulted in the identification of fifteen genes more than anticipated. In all, including the genes known at the start of the project, for A. niger and E. chrysanthemi twenty and eighteen pectinolytic genes respectively have been cloned, sequenced and over-expressed.
For A. niger those genes are:
For E. chrysanthemi these genes are:
To facilitate over-production and simplify purification of a particular pectinase, a strategy was chosen to individually over-express the pectinase gene using a promoter-gene fusion under conditions where other pectinases are repressed. The Erwinia genes were therefore over-expressed in E. coli, which is devoid of pectinolytic activities, using the strong T7-, Lac- and the Trc-promoters. The fungal genes were expressed in an appropriate A. niger expression strain with reduced extracellular protease activity, using the pyruvate kinase promoter. This promoter drives transcription during growth on glucose or fructose. Under these conditions other pectinase genes are repressed. Most of the fungal enzymes were easily obtained in 100 mg quantities.
Of the desired bacterial enzymes, amounts sufficient for characterisation were purified from the expression clones, whereas larger amounts were purified for those enzymes to be crystallised or tested in applications With respect to the biochemical characterization of the pectinolytic enzymes, the emphasis was on the A. niger polygalacturonases and the E. chrysanthemi pectate lyases. The polygalacturonases cleave the substrate by hydrolysis whereas the pectate lyases cleave the substrate by beta-elimination and require Ca2+ ions for catalysis.
Due to the type of mechanism, the pH optima of the enzymes are more or less defined. Hydrolysis generally occurs at acidic to neutral pH, whereas beta-elimination requires slightly acidic to basic pH. The A. niger polygalacturonases have optimal activity between pH 3.5 and 4 5 with the exception of PGB, which has optimal activity between pH 4.5 and 5.5 and requires 100 mM NaCl. The pH optima for the E. chrysanthemi pectate lyases varies from pH 8 (PelE) to pH 9.3 (PelB). For the polygalacturonases, as well as the pectate lyases, the activity regions are quite small: 2-2.5 pH units.
For optimal characterisation of the substrate-specificity of the enzymes, a set of substrates was prepared and characterized with respect to degree of methylation and acetylation, molecular mass, galacturonate content and neutral sugar composition. The substrates comprised a series of pectins with various degrees of esterification (DE) (% DE of 7, 22, 45, 60 and 75%) prepared from one non-calcium-sensitive parent pectin, pectins from four sources viz lemon, apple, sugar beet and orange, protopectin and 'hairy regions', the highly rammified part of the pectin.
Using polygalacturonic acid as a substrate, the specific activities of the A. niger endo-polygalacturonases at the pH optimum appeared to differ by up to two orders of magnitude. The following specific activities were determined:
The low activity for PG C and PG E suggests that polygalacturonic acid is not the natural substrate for these enzymes and that they may require a substituted galacturonate moiety at one or more subsites. Studies involving all six PGs using pectins of various degrees of esterification revealed that PG B preferred partially methylated pectin (22-45 % DE) whereas the other five enzymes were most active on the unmethylated substrate. PG II appeared most sensitive for methylation with 30 % loss of activity at 7 % DE compared to approximately 0 to 5 % loss of activity for the other enzymes. These results also demonstrate that a methyl function on a galacturonate moiety is not the right substitution for PG C and PG E.
Clear demonstration of the differences in substrate specificity was obtained by analysis of the performance of the enzymes on apple pectin. Indeed, the enzymes generated pectic fragments of different molecular mass that were typical for each enzyme. However, the nature of the exact pectic structures recognized by the individual enzymes is not known. The E. chrysanthemi endo-pectate lyases showed an even stronger variation in activity, up to three orders of magnitude, with polygalacturonic acid as a substrate in the presence of 0.1 mM CaCl2. Vmax values obtained were as follows:
As for the endopolygalacturonases C and E, likewise for those pectate lyases with very low activity, PelA, PelL and PelZ, polygalacturonic acid may not be the natural substrate. In this respect it is interesting to note that PelL and PelZ, based on their amino acid sequence, belong to different families of pectate lyases: families 4 and 5 respectively. Furthermore, in addition to 0.1 mM CaCl2, PelZ also required 0.2 mM MnCl2 for optimal activity. Studies on the influence of degree of esterification of the substrate on the activity of the enzymes revealed that PelB, PelC, PelI and PelL preferred 31 % of esterification, PelZ preferred 7% of esterification, and the remaining enzymes preferred the unesterified substrate. The A. niger pectate lyase (PLYA) appeared to prefer 45%-esterified pectin (180 U/mg). Using polygalacturonic acid and 1 mM CaCl2, activity was stimulated five-fold by the addition of 100 mM NaCl. However, when using hexagalacturonate as a substrate the reverse was observed: increasing the NaCl concentration also increased Km app, which indicates that the enzyme-substrate interaction occurs via ionic interactions. This is compatible with the 3-D structure of other pectate lyases which shows that the substrate binding cleft contains many charged residues (see below).
To further extend the knowledge of the substrate specificity of the individual enzymes, the mode of action of substrate cleavage was studied using oligogalacturonates of defined chain length with degrees of polymerisation (DP) of 2 to 8 (G2 to G8). For the polygalacturonases, reduced oligogalacturonates with DP 3-8 were used as well. This was done in order to establish the position of the active site within the array of subsites - since the reduced end serves as a tag - and also to establish the stereochemistry of the hydrolysis reaction of the polygalacturonases. The latter studies, using endo-polygalacturonases 1, 11 and exo-polygalacturonase (PGX), revealed that the polygalacturonases act via the single displacement mechanism, as inversion of configuration at the anomeric position was observed.
Studies to locate the active site showed that endopolygalacturonases (endoPGs) hydrolyze the oligomeric substrates from the reducing end, in contrast to PGX from A. niger and PehX from E. chrysanthemi which hydrolyzed the substrate from the non-reducing end, generating monomers or dimers respectively. The following generalized observations were made:
Depending on the enzyme and Gn, for endoPGs major products observed were GI to G3, whereas for the endo-pectate lyases the major products were unsaturated G2 to unsaturated G4. The major products are not determined by the number of productive binding modes but rather by the likeliness of such a binding mode occuring. This likeliness is determined by the affinity of a particular neighbouring set of subsites for the substrate. Thus, by quantitation of the products formed in relation to the chain length, an estimate can be made of the binding energies of certain subsites. This has allowed the estimation of subsite maps for the A. niger endoPGs.
In addition to the endolytic mode of hydrolysis, three endoPGs showed a very strong accumulation of GI during polymer hydrolysis. Analysis of the oligomer hydrolysis by these enzymes showed that this phenomenon was caused by multiple attack, or processivity. This means that after hydrolysis the larger product is not released from the enzyme, but shifts one subsite to form again a productive complex.
Further insight into the substrate specificity was obtained from the study of the performance of the enzymes' performance on synthetic substrates. For those enzymes capable of cleaving digalacturonate (KdgC, OGL, PGX) and trigalacturonate (KdgC, OGL, PGX, PelX and the A. niger endoPGs), the following synthetic substrates were made:
The partly-methylated substrates were also used to test the pectin methylesterases.
The studies have revealed that some enzymes have an absolute requirement of non-esterified galacturonate at a certain subsite. The heterodimer substrate could not be used by any of the enzymes, demonstrating the necessity of the carboxylate function for either recognition or involvement in catalysis. Since OGL is involved in the generation of the inducer of pectinase gene expression, 2-keto-3-deoxygluconate (KDG) via cleavage of di- and trimer substrate, digalacturonate analogs were synthesized that might inhibit the enzyme and which might serve for protection of plants and crops from soft-rot by Erwinia. Unfortunately, none of the potential inhibitors appeared to inhibit the enzyme.
Additional synthetic compounds were made - five KDG analogs in total - to identify the structural elements of the inducer required to exert its biological function. This led to the identification of gratuitous inducers (5-deoxy-, 5-0-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.
Structural Analysis
A powerful tool to understand the substrate specificity, which is complementary to the biochemical studies, is the 3-D structure analysis of pectinolytic enzymes. One of the first pectinases for which the structure was solved was the Bacillus subtilis pectate lyase (Bs Pel). The structure revealed a cleft which might be the substrate binding cleft. At the bottom of this cleft a Ca2+ ion was bound, revealing the necessity of Ca2+ for pectate lyases. Much effort was spent to obtain enzyme-substrate complexes using Bs Plel and G4 or G5. Unfortunately no such complexes were obtained. Even using inactive enzyme by replacement of Ca2+ by Ba2+, or by site-directed mutagenesis of Arg276 - a residue very important for catalysis and located close to the Ca2+ when bound to the enzyme - did not result in enzyme substrate complexes. A plausible explanation might be that the crystallisation of Bs Pel, which requires rather high salt concentrations, might interfere with substrate binding, as was observed for the NaCl dependent activity for the A. niger PLYA.
Crystallisation conditions have been established for Erwinia PemA, PelI and PelL and the search for heavy atom derivatives is at hand. Both PelI and PelZ structures cannot be solved by molecular replacement using known Pel structures, since these enzymes belong to different families, families which most probably prefer other substrates to homogalacturonan.
Very recently, in this project the 3-D structure of an Erwinia endopolygalacturonase was solved. The enzyme appears to have the same right-handed beta-helical structure as found for several pectate lyases and for the A. niger pectin lyases, PelA and PelB, as well as for the rhamnogalacturonase. This conservation of the structural topology among de-olymerising pectinases is rather surprising, since these families of enzymes share at most 20% sequence identity. Furthermore, the hydrolases and lyases operate via different mechanisms at distinctive pH. The substrate-binding cleft of the endopolygalacturonase appears to be composed of charged residues, as are the pectate lyases. This finding is consistent with both enzymes preferentially using polygalacturonate as a substrate.
In A. niger PG II, by chemical modification a histidine was found to be involved in catalysis. Based on sequence homology, only one His, H223 in A. niger PG II, is strictly conserved among all the polygalacturonases. Site directed mutagenesis of this His223 to Ala223 in PG 11 indeed confirmed its role in catalysis by reducing the activity to 1 % of wild type. The equivalent of this His223 in the Erwinia endoPG structure is located at the bottom of the active site, in close contact to two Asp residues which are also strictly conserved among polygalacturonases. This confirms the substrate binding functionality of the cleft. Further inspection of the amino acid residues forming this cleft, and comparison with the A. niger endoPG primary sequences will give insight into the origin of the biochemical properties of the A. niger endoPGs.
Applications
The A. niger polygalacturonases, in combination with other enzymes, were tested for their applicability in upgrading feed stuffs and improving food processing. It was concluded that enzymes other than the polygalacturonases are necessary in these processes. Selected pectinolytic enzymes, mainly of fungal origin, were also tested in pectin manufacturing. Indeed, by action of some of these enzymes, new pectin products were obtained. Also, a pectin identification kit has been developed which is based on the fungal pectate lyase. The kit was evaluated by all pectin industries and other researchers in the pectin field through the International Pectin Producers Association (IPPA) and has been recommended to become the standard method for pectin identification.
Contacts
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EC Scientific Officer
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