Engineering of substrate specificity in a plant cell-wall modifying enzyme through alterations of carboxyl-terminal amino acid residues.

Structural determinants of substrate recognition remain inadequately defined in broad specific cell-wall modifying enzymes, termed xyloglucan xyloglucosyl transferases (XETs). Here, we investigate the Tropaeolum majus seed TmXET6.3 isoform, a member of the GH16_20 subfamily of the GH16 network. This enzyme recognises xyloglucan (XG)-derived donors and acceptors, and a wide spectrum of other chiefly saccharide substrates, although it lacks the activity with homogalacturonan (pectin) fragments. We focus on defining the functionality of carboxyl-terminal residues in TmXET6.3, which extend acceptor binding regions in the GH16_20 subfamily but are absent in the related GH16_21 subfamily. Site-directed mutagenesis using double to quintuple mutants in the carboxyl-terminal region - substitutions emulated on barley XETs recognising the XG/penta-galacturonide acceptor substrate pair - demonstrated that this activity could be gained in TmXET6.3. We demonstrate the roles of semi-conserved Arg238 and Lys237 residues, introducing a net positive charge in the carboxyl-terminal region (which complements a negative charge of the acidic penta-galacturonide) for the transfer of xyloglucan fragments. Experimental data, supported by molecular modelling of TmXET6.3 with the XG oligosaccharide donor and penta-galacturonide acceptor substrates, indicated that they could be accommodated in the active site. Our findings support the conclusion on the significance of positively charged residues at the carboxyl terminus of TmXET6.3 and suggest that a broad specificity could be engineered via modifications of an acceptor binding site. The definition of substrate specificity in XETs should prove invaluable for defining the structure, dynamics, and function of plant cell walls, and their metabolism; these data could be applicable in various biotechnologies.

Definition of the Acceptor Substrate Binding Specificity in Plant Xyloglucan Endotransglycosylases Using Computational Chemistry.

Xyloglucan endotransglycosylases (XETs) play key roles in the remodelling and reconstruction of plant cell walls. These enzymes catalyse homo-transglycosylation reactions with xyloglucan-derived donor and acceptor substrates and hetero-transglycosylation reactions with a variety of structurally diverse polysaccharides. In this work, we describe the basis of acceptor substrate binding specificity in non-specific Tropaeolum majus (TmXET6.3) and specific Populus tremula x tremuloides (PttXET16A) XETs, using molecular docking and molecular dynamics (MD) simulations combined with binding free energy calculations. The data indicate that the enzyme-donor (xyloglucan heptaoligosaccharide or XG-OS7)/acceptor complexes with the linear acceptors, where a backbone consisted of glucose (Glc) moieties linked via (1,4)- or (1,3)-ß-glycosidic linkages, were bound stably in the active sites of TmXET6.3 and PttXET16A. Conversely, the acceptors with the (1,6)-ß-linked Glc moieties were bound stably in TmXET6.3 but not in PttXET16A. When in the (1,4)-ß-linked Glc containing acceptors, the saccharide moieties were replaced with mannose or xylose, they bound stably in TmXET6.3 but lacked stability in PttXET16A. MD simulations of the XET-donor/acceptor complexes with acceptors derived from (1,4;1,3)-ß-glucans highlighted the importance of (1,3)-ß-glycosidic linkages and side chain positions in the acceptor substrates. Our findings explain the differences in acceptor binding specificity between non-specific and specific XETs and associate theoretical to experimental data.

Broad Specific Xyloglucan:Xyloglucosyl Transferases Are Formidable Players in the Re-Modelling of Plant Cell Wall Structures.

Plant xyloglucan:xyloglucosyl transferases, known as xyloglucan endo-transglycosylases (XETs) are the key players that underlie plant cell wall dynamics and mechanics. These fundamental roles are central for the assembly and modifications of cell walls during embryogenesis, vegetative and reproductive growth, and adaptations to living environments under biotic and abiotic (environmental) stresses. XET enzymes (EC 2.4.1.207) have the ß-sandwich architecture and the ß-jelly-roll topology, and are classified in the glycoside hydrolase family 16 based on their evolutionary history. XET enzymes catalyse transglycosylation reactions with xyloglucan (XG)-derived and other than XG-derived donors and acceptors, and this poly-specificity originates from the structural plasticity and evolutionary diversification that has evolved through expansion and duplication. In phyletic groups, XETs form the gene families that are differentially expressed in organs and tissues in time- and space-dependent manners, and in response to environmental conditions. Here, we examine higher plant XET enzymes and dissect how their exclusively carbohydrate-linked transglycosylation catalytic function inter-connects complex plant cell wall components. Further, we discuss progress in technologies that advance the knowledge of plant cell walls and how this knowledge defines the roles of XETs. We construe that the broad specificity of the plant XETs underscores their roles in continuous cell wall restructuring and re-modelling.

Plant Xyloglucan Xyloglucosyl Transferases and the Cell Wall Structure: Subtle but Significant.

Plant xyloglucan xyloglucosyl transferases or xyloglucan endo-transglycosylases (XET; EC 2.4.1.207) catalogued in the glycoside hydrolase family 16 constitute cell wall-modifying enzymes that play a fundamental role in the cell wall expansion and re-modelling. Over the past thirty years, it has been established that XET enzymes catalyse homo-transglycosylation reactions with xyloglucan (XG)-derived substrates and hetero-transglycosylation reactions with neutral and charged donor and acceptor substrates other than XG-derived. This broad specificity in XET isoforms is credited to a high degree of structural and catalytic plasticity that has evolved ubiquitously in algal, moss, fern, basic Angiosperm, monocot, and eudicot enzymes. These XET isoforms constitute gene families that are differentially expressed in tissues in time- and space-dependent manners during plant growth and development, and in response to biotic and abiotic stresses. Here, we discuss the current state of knowledge of broad specific plant XET enzymes and how their inherently carbohydrate-based transglycosylation reactions tightly link with structural diversity that underlies the complexity of plant cell walls and their mechanics. Based on this knowledge, we conclude that multi- or poly-specific XET enzymes are widespread in plants to allow for modifications of the cell wall structure in muro, a feature that implements the multifaceted roles in plant cells.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Structural characterization of the Pet c 1.0201 PR-10 protein isolated from roots of Petroselinum crispum (Mill.) Fuss.

The native dimeric Petroselinum crispum (Mill.) Fuss protein Pet c 1.0201 and a monomeric xyloglucan endotransglycosylase enzyme (Garajova et al., 2008) isolated from the root cells co-purify and share similar molecular masses and acidic isoelectric points. In this work, we determined the complete primary structure of the parsley Pet c 1.0201 protein, based on tryptic and chymotryptic peptides followed by the manual micro-gradient chromatographic separation coupled with offline MALDI-TOF/TOF mass spectrometry. The bioinformatics approach enabled us to include the parsley protein into the PR-10 family, as it exhibited the highest protein sequence identity with the Apium graveolens Api g 1.0201 allergen and the major Daucus carota allergen Dau c 1.0201. Hence, we designated the Petroselinum crispum protein as Pet c 1.0201 and deposited it in the UniProt Knowledgebase under the accession C0HKF5. 3D protein homology modelling and molecular dynamics simulations of the Pet c 1.0201 dimer confirmed the typical structure of the Bet v 1 family allergens, and the potential of the Pet c 1.0201 protein to dimerize in water. However, the behavioural properties of Pet c 1.0201 and the celery allergen Api g 1.0101 differed in the presence of salts due to transiently and stably formed dimeric forms of Pet c 1.0201 and Api g 1.0101, respectively.

Diversity of yeasts in the soil adjacent to fruit trees of the Rosaceae family.

Yeasts are common constituents of different types of soil. Their diversity depends on the season, the type and depth of the soil, the plant species, and the locality. In this study, diversity of yeasts isolated from the soil adjacent to five fruit trees (apple, appricot, peach, pear, and plum) in two localities (in Slovakia) in four sampling periods was examined. Our results demonstrated differences in the species richness and evenness among the yeast populations, which inhabited the soil beneath individual fruit tree species in both localities. Altogether, 32 ascomycetous and 27 basidiomycetous yeast species were discovered. The highest species richness was found in the soil adjacent to the apricot trees. Galactomyces candidum, Metschnikowia pulcherrima, Hanseniaspora uvarum, Schwanniomyces capriottii, and Tausonia pullulans, as well as the genus Apiotrichum, were present in soil samples in all samplings. Two species of the genus Holtermanniella (H. festucosa and H. takashimae) were exclusively isolated during Sampling IV in April. Cyberlindnera spp., Clavispora reshetovae, S. capriottii, and Trichosporon asahii were found only in one of two localities. Ascomycetous yeasts were present more frequently than their basidiomycetous counterparts in the three samplings (one in June and two in October); they formed from 65.6% to 70.8% of the total yeast population, whereas basidiomycetous yeasts prevailed in the April sampling (61.2%).

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.

Characterization of a long-chain α-galactosidase from Papiliotrema flavescens.

α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and ß-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.

Catalytic properties of Phr family members of cell wall glucan remodeling enzymes: implications for the adaptation of Candida albicans to ambient pH.

Fungal wall formation is a dynamic process involving several categories of enzymes. The GH72 family of ß(1,3)-glucanosyltransferases is essential for the determination of cell shape, for cell integrity and for virulence in pathogenic fungi. Candida albicans has five GH72 genes: PHR1 and PHR2 are pH dependent, the first being expressed at pH ≥ 6 and repressed at lower pH and the second regulated in the opposite manner, PGA4 is transcribed independently of pH whereas PHR3 and PGA5 have low expression levels. To characterize the catalytic properties of Phr1p-2p and probe the activity of Pga4p, we heterologously expressed these proteins and used a fluorescent assay based on the transfer of oligosaccharyl units from a donor to a sulforhodamine-labeled acceptor. Phr1p-2p used exclusively ß-1,3-glucan or cell wall glucan as donor and laminarin-derived oligosaccharides as acceptor. The acceptor efficiency increased with the length of the oligosaccharide. The temperature optimum was 30°C. The pH optimum was 5.8 for Phr1p and 3 for Phr2p. Overall, adaptation to pH of C. albicans appears to involve a fine interplay among the pH-dependent activity of Phr1p and Phr2p, the pH-regulated expression of their genes and protein stability. Unexpectedly, Pga4p was inactive suggesting that it turned into a structural mannoprotein.

Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, ß-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited ß-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET).

Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc(4)Xyl(3)Gal(2) (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium (Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K(m) for individual acceptors increased in the order [1-(3)H]XLLGXLLG-SR>XLLG-ANTS>[1-(3)H]XLLGol>[1-(3)H]XLLG>XLLG-AA. Catalytic efficiency (expressed as k(cat)/K(m)) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1-(3)H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET.

Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: isolation and characterization of the major form.

Five forms of xyloglucan endotransglycosylase/hydrolase (XTH) differing in their isoelectric points (pI) were detected in crude extracts from germinating nasturtium seeds. Without further fractionation, all five forms behaved as typical endotransglycosylases since they exhibited only transglycosylating (XET) activity and no xyloglucan-hydrolysing (XEH) activity. They all were glycoproteins with identical molecular mass, and deglycosylation led to a decrease in molecular mass from approximately 29 to 26.5 kDa. The major enzyme form having pI 6.3, temporarily designated as TmXET(6.3), was isolated and characterized. Molecular and biochemical properties of TmXET(6.3) confirmed its distinction from the XTHs described previously from nasturtium. The enzyme exhibited broad substrate specificity by transferring xyloglucan or hydroxyethylcellulose fragments not only to oligoxyloglucosides and cello-oligosaccharides but also to oligosaccharides derived from beta-(1,4)-d-glucuronoxylan, beta-(1,6)-d-glucan, mixed-linkage beta-(1,3; 1,4)-d-glucan and at a relatively low rate also to beta-(1,3)-gluco-oligosaccharides. The transglycosylating activity with xyloglucan as donor and cello-oligosaccharides as acceptors represented 4.6%, with laminarioligosaccharides 0.23%, with mixed-linkage beta-(1,3; 1,4)-d-gluco-oligosaccharides 2.06%, with beta-(1,4)-d-glucuronoxylo-oligosaccharides 0.31% and with beta-(1,6)-d-gluco-oligosaccharides 0.69% of that determined with xyloglucan oligosaccharides as acceptors. Based on the sequence homology of tryptic fragments with the sequences of known XTHs, the TmXET(6.3) was classified into group II of the XTH phylogeny of glycoside hydrolase family GH16.

Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts.

Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated glass slides, was incubated with crude plant extracts, along with a series of fluorophore-labelled acceptor oligosaccharides. After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities in crude extracts from nasturtium (Tropaeolum majus) and mouse-ear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the formation of homo- and hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence of diverse transglycosylase activities in crude plant extracts.

Pectate hydrolases of parsley (Petroselinum crispum) roots.

The presence of various enzyme forms with terminal action pattern on pectate was evaluated in a protein mixture obtained from parsley roots. Enzymes found in the soluble fraction of roots (juice) were purified to homogeneity according to SDS-PAGE, partially separated by preparative isoelectric focusing and characterized. Three forms with pH optima 3.6, 4.2 and 4.6 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates) while the form with pH optimum 5.2 was a typical exopolygalacturonase [EC 3. 2.1.67] with relatively fast cleavage of polymeric substrate. The forms with pH optima 3.6, 4.2 and 5.2 were released from the pulp, too. The form from the pulp with pH optimum 4.6 preferred higher oligogalacturonates and was not described in plants previously. The production of individual forms in roots was compared with that produced by root cells cultivated on solid medium and in liquid one.

Effect of salt stress on the production and properties of extracellular polysaccharides produced by Cryptococcus laurentii.

The composition, main structural features and molecular properties of exopolysaccharides (EP) produced by Cryptococcus laurentii var. laurentii CCY 17-3-16 under optimal (EPo) and NaCI-stress conditions (EPs) as well as their subfractions isolated by gel chromatography were studied using chemical, FT-IR and NMR spectroscopy methods. The results showed that under stress conditions the yeast produced EP with a lower content of protein and phosphorus. In comparison to EPo, the EPs exhibited a substantially larger proportion of high molecular mass populations. NMR analysis of EPs revealed a higher degree of branching with single xylose side chains of the heteromannan components. The increase of the molecular mass and degree of branching of the macromolecular chains of the heteromannan components might in part be related to the function of EPs to protect the yeast cells from water loss and maintain growth conditions under the salt stress.

Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylamide gels.

The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M(r) polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure.

Purification and biochemical characterization of polygalacturonases produced by Aureobasidium pullulans.

The extracellular polygalacturonases produced by Aureobasidium pullulans isolated from waters of the Danube river were partially purified and characterized. The pH optima of polygalacturonases produced in the first phases of cultivation (48 h) and after 10 d as well as their optima of temperature, thermal stabilities, molecular masses, isoelectric points, action pattern and ability to cleave polymeric and oligomeric substrates were compared. Polygalacturonases with a random action pattern (random cleavage of pectate forming a mixture of galactosiduronides with a lower degree of polymerization) [EC 3.2.1.15] were produced only in the first phases of growth, while exopolygalacturonases [EC 3.2.1.67] with a terminal action pattern (cleavage of pectate from the nonreducing end forming D-galactopyranuronic acid as a product) were found during the whole growth. The main enzyme form with a random action pattern was glycosylated and its active site had the arrangement described previously for the active site of polygalacturonase of phytopathogenic fungi.

Oligogalacturonate hydrolase from carrot roots.

The presence of multiple forms of enzyme with terminal action pattern on pectate was evaluated in the protein mixture obtained from carrot roots. The form with pH optimum 3.8 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates). Its molecular mass, isoelectric point, glycosylation as well as cleavage of pectate from nonreducing end corresponded to an exopolygalacturonase [EC 3.2.2.67]. The affinity of this enzyme to the substrates increased with the increasing degree of polymerization, and the difference was observed only in the maximal ratio of catalysis of oligomeric and polymeric substrates. Sterical hindrance for substrates with more than six D-galactopyranuronic acid units is supposed and an oligogalacturonate hydrolase rather than exopolygalacturonase is considered.

Biosorption of cadmium ions by different yeast species.

Toxicity and accumulation of Cd2+ in yeasts were studied in eight different yeast species. The adaptation to toxic concentration of this metal was dependent on the production of extracellular yeast glycoproteins. The highest concentration of Cd2+ ions in the growth medium was tolerated by a Hansenula anomala, strain while the lowest tolerance was found by the strain of species Saccharomyces cerevisiae. Extracellular glycoproteins of Hansenula anomala absorbed nearly 90% of the total content of Cd2+ ions bound by yeast cells, while extracellular glycoproteins of Saccharomyces cerevisiae bound only 6% of the total amount of cadmium. This difference is caused by the variable composition of the saccharide moiety in the extracellular glycoproteins. The composition of extracellular glycoproteins changed during the adaptation of the yeast cells to the presence of Cd2+ ions.