No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Purification and characterisation of an extracellular exo-D-galacturonase of Aspergillus niger
Abstract
A D-galacturonanase (EC 3.2.1.67) catalyzing the degradation of D-galacturonans by terminal action pattern was purified from a culture filtrate of Aspergillus niger by a procedure including the salting-out with ammonium sulfate, precipitation by ethanol, chromatography on DEAE-cellulose, and gel chromatography on Sephadex G-100. The obtained preparation was slightly contaminated by an enzymically inactive protein fraction. Maximum activity and stability of the enzyme was observed at pH 5.2. The enzyme degrades digalacturonic acid, p-nitrophenyl-alpha-D-galactopyranuronide, as well as oligogalacturonides containing at the nonreducing end 4-deoxy-L-threo-hexa-4-enopyranosyluronate. It differs from all A. niger enzymes so far described which degrade D-galaturonans by the terminal action pattern, in not clearly preferring low-molecular substrates. It is therefore classified as an exo-D-galacturonanase.
... Exo-PGs [E.C. 3.2.1.67] are produced by many fungi [8][9][10][11][12][13]. Exo-PG is an enzyme that eventually hydrolyses glycosidic bonds in pectate or other galacturons, yielding the corresponding 1,4-α -D-galacturonide and galacturonic acid. ...
Polygalacturonases are important enzymes used in industrial applications that treated plant material like Juice extraction, Textile, Clarification etc. Fungi from genus Aspergillus are one of the most important sources of this enzyme. We production Polygalacturonase by submerged fermentation using seaweed waste. Five PG P1-P5 were differentiated on DEAE-Sepharose column. The homogeneity of PGP2a was identified on Sephacryl S-200 chromatography. PGP2a had a molecular weight of 20 kDa by Sephacryl S-200 and SDS-PAGE. The enzyme had an optimal pH of 6 also the temperature optimal of PGP2a was 40 °C. The thermal stability of PGP2a was detected up to 50 °C and the enzyme was highly stable till 60 °C after 30min incubation. The V max and K m values of PGP2a were 4.27 mg/ml and 1.16µmol min-1 mg-1 , respectively. The metal ions except only Co 2+ and Hg 2+ was found to enhance the PGP2a activity.
... The particular exoPGs differ from each other by the range and rate of the effects toward substrate in relation to the chain length. The exoPGs of microbial origin favour oligomers of lower degree of polymerization (DP) (trimer or tetramer) (Heinrichová & Rexová-Benková 1976;Heinrichová et al. 1993;Kester et al. 1996;Stratilová et al. 2006), digalacturonic acid (Hasegawa & Nagel 1968;van Rijssel et al. 1993) or the substrate chain length is not a factor determining the enzyme effect (Musel & Strouse 1972). It was supposed that the most suitable substrates for exoPGs of plant origin are polymeric d-galacturonan (Heinrichová 1977) or a partially degraded d-galacturonan of DP about 20 (Hatanaka & Ozawa 1964;Pressey & Avants 1973). ...
The life style of Aureobasidium pullulans on pectin medium and its production of extracellular polygalacturonases are closely related. Polygalacturonases with random
action pattern (EC 3.2.1.15) were formed in the first phases of cultivation, whereas exopolygalacturonases (EC 3.2.1.67) with
terminal action pattern on pectin were produced during the whole growth of this yeast-like fungus. The production and inactivation
of individual enzyme forms during cultivation were strongly dependent on the pH value of the pectin medium. Various kinds
of stress can support the prolongation of the phase of endo-acting enzyme production, as well as the increase of their activity.
... The particular exoPGs differ from each other by the range and rate of the effects toward substrate in relation to the chain length. The exoPGs of microbial origin favour oligomers of lower degree of polymerization (DP) (trimer or tetramer) (Heinrichová & Rexová-Benková 1976;Heinrichová et al. 1993;Kester et al. 1996;Stratilová et al. 2006), digalacturonic acid (Hasegawa & Nagel 1968;van Rijssel et al. 1993) or the substrate chain length is not a factor determining the enzyme effect (Musel & Strouse 1972). It was supposed that the most suitable substrates for exoPGs of plant origin are polymeric d-galacturonan (Heinrichová 1977) or a partially degraded d-galacturonan of DP about 20 (Hatanaka & Ozawa 1964;Pressey & Avants 1973). ...
The main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate preference of a plant exopolygalacturonase
because it clearly preferred the substrates with degree of polymerization about 10. This form was separated from the others,
purified and characterized. Enzyme exhibited sharp pH optimum corresponding to pH 4.7, molecular mass 53.5 kDa, and isoelectric
point 5.3. It was stable at 50°C in 2-h assay and had optimum of temperature at 60°C (activation energy being 37.0 kJ/mol).
The interaction with concanavalin A indicated the glycosylation of enzyme. Substrates were cleaved from the non-reducing end.
Pectinases are a group of at least seven different enzymatic activities that contribute to the breakdown of pectin from a variety of plants. Pectin is a structural polysaccharide found in primary cell wall and middle lamella of fruits and vegetables. Pectin has a complex structure; the predominant structure consists of homopolymeric partially methylated poly-α-(1,4)-galacturonic acid. Sections of α-(1,2)-L-rhamnosyl- α(1,4)-D-galacturonosyl containing branch-points with L-arabinose and D-galactose can be incorporated in the main polymeric chain. Pectin may also contain residues of D-glucuronic acid, D-apiose, D-xylose and L-fucose attached to poly- α-(1,4)-D-galacturonic acid sections (Pérez et al. 2003). As other enzymes, pectinases contained in natural ingredients have been used long time ago, e.g. the production of coffee and chocolate, where pectinases produced by wild microorganisms improved the fermentation step to remove grain mucilage. Pectinases are very useful to determine the structural characteristics of pectic substances and to prepare plant cell protoplasts for studies of genetic engineering Shubakov & El’kina 2002). Due to its ability to degrade cell wall, pectinases have been used in juice and wine processing for the last 70 years. They are extensively used in food industry to increase juice yields, to accelerate juice clarification and to produce juice concentrates from grapes, berries, pears, apples, carrots, beets, green peppers and citrus fruits. Pectinases are also used to increase the colour of juices, promoting antioxidants formation and favour the extraction of colour, flavour components and fermentable sugars when added to grapes of musts during wine production. Removal of the inner wall of lotus seed, garlic, almond and peanut is also carried out by pectinases. Pectinase world-wide consumption is above 7 x 106 tons per year.
Purified endopolygalacturonase (EC 3.2.1.15) from Aspergillus niger 71 was immobilized covalently by NH2 or COOH groups to the supports. The influence of Ca, Mg, Mn, Zn, Cu as well as of Pb, Hg and Cd ions in concentrations from 0.1 to 1.2 mM on the soluble and immobilized enzyme was shown. Endopolygalacturonase activity after immobilization is stabilized even in the presence of Pb, Hg and Cd ions. When this enzyme was immobilized by COOH groups with the support, the metal ions used decreased its activity more effectively than that of the soluble form of this enzyme. In contrast, when endopolygalacturonase was immobilized by NH2 groups, the activity of this enzyme was not only stabilized, but Cu, Mg and Cd ions also caused a 40 to 60% increase in the enzyme activity.
Three forms of polygalacturonase (PG), designated PG-I, PG-II, and PG-III, were purified to homogeneity from culture filtrate of Penicillium italicum. PG-I was characterized as an exoenzyme, whereas PG-II and PG-III wee characterized as endoenzymes. All three enzymes were present in orange peel infected by P. italicum. Exo-PG-I was the predominant enzyme in infected tissue and constituted 69% of the total PG activity as compared with 4% in the culture filtrate. Endo-PG-II and endo-PG-III constituted only 24 and 7%, respectively, of the activity in the infected tissues, as compared with 71 and 25% in culture filtrate (...)
Various methods have been used for the separation of pectic enzymes, such as ion-exchange chromatography on DEAE-cellulose, CM-cellulose, cellulose phosphate, gel chromatography, and affinity chromatography on pectic acid cross-linked by epichlorhydrin and its amino derivative, or on poly(hydroxyalkyl methacrylate) containing glycosidically bound oligo-D-galactosiduronic acids. Because of the variability in the composition of pectic enzyme complexes and the new trends in some industrial processes to apply preparations containing certain components of the pectolytic complex or mixtures of definite composition, rapid and efficient separation procedures on an analytical and preparative scale are required in research and industry. High-performance liquid chromatography (HPLC) of biopolymers, especially of enzymes has permitted the application of this rapid method to the separation of pectic enzymes of microbial and plant origin. Microbial pectic enzymes from three commercial preparations of pectinases were separated in 35–70 min on various types of ion-exchange derivatives of hydrophilic polymer Spheron using a combination of isocratic and linear gradient elution.
An exopolygalacturonase [exo-PG; poly (1,4-alpha-D-galacturonide) digalacturonohydrolase, EC 3.2.1.82] was found in a culture of Bacillus sp. strain KSM-P576. The purified exo-PG had a molecular weight of approximately 115,000 and an isoelectric point of pH 4.6. The N-terminal amino acid sequence was Thr-Glu-Val-Ser-Pro-Lys-Ser-Pro-Ala-Ser-Pro-Val. Maximum activity toward polygalacturonic acid (PGA) was observed at 55 degrees C and pH 8.0 in 100 mM Tris-HCl buffer. The exo-PG was quite stable in various pH buffers between pH 6 and 12 when incubated at 30 degrees C for 1 h. Mg(2+,) Mn(2+,) Pd(2+) and Ca(2+) ions stimulated the enzyme activity. The exo-PG released digalacturonic acid from PGA, tri-, tetra-, and penta-galacturonic acids. The apparent K(m) values for oligogalacturonic acids were almost identical, and k(cat) values increased with the chain length of the substrates.
The use of enzymes in juice industry has contributed in increasing the yield and production of various types of juices. The addition pectinases aims in particular to degrade the pectic substances, in the cell wall and middle lamella of the cells of plants, aiming to minimise the impacts of these compounds on the characteristics of the final product, such as colour, turbidity and viscosity. Enzymes able to remove bitterness of citrus juice, extract pigments, among other applications, have also had great interest in the juice industry.
The ß-galactosidase enzymes of Aspergillus oryzae and Scopulariopsis sp. are secreted during growth of the fungi on polygalacturonic acid, but not during growth on a wide range of other substrates, including lactose. The enzymes are thermotolerant with temperature optima in the range 55–65°C. The results indicate that fungal extracellular ß-galactosidases are involved in fungal growth on complex polysaccharides but not on lactose.
Background
The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation.
Results
Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan.
Conclusions
The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.
A strain of Aspergillus niger isolated from onion, when cultured in a medium containing onion, glucose, and salt, secreted large amounts of polygalacturonase (PG). A simple and rapid three-step procedure was adopted to separate the extracellular PG into endo- and exo- forms. The method gave an overall yield of 47%. Determination of the reducing groups at a 50% decrease in the relative viscosity of 1% sodium polypectate (NaPP) as substrate revealed the presence of two exo- forms (PGI and PGII) and one endo- form (PGIII). All three forms of the enzyme were optimally active at pH 5.0 and preferred NaPP as a substrate over pectin. Km values for Napp were 0.24, 0.12, and 0.063% for PGI, PGII, and PGIII, respectively
A selective affinity-adsorbent for the extracellular endo-D-galacturonanase (E.C. 3.2.1.15) of Aspergillus niger was prepared by covalent coupling of tri(D-galactosiduronic acid) to Separon, a poly(hydroxyalkyl methacrylate) gel. Complexing of the enzyme with the adsorbent is pH dependent; maximal interaction occurs at the optimum pH for enzyme activity. The enzyme was quantitatively displaced from the adsorbent either by changing the pH or by bioelution with soluble tri(D-galactosiduronic acid) or other substrate. Within the range of substitution of Separon examined [content of tri(D-galactosiduronic acid) 1.7-6.7%] the amount of endo-D-galacturonanase retained was proportional to the content of affinity ligand. Under the same conditions, unsubstituted carrier did not complex with endo-D-galacturonanase. The dissociation constant of the affinity complex, as determined by zonal analysis, kinetic measurements, and by means of the adsorption isotherm KL (0.54 mmol.L-1), is close to the value (KI 0.44 mmol.L-1) obtained by the two first methods with soluble tri(D-galactosiduronic acid). The results show that adsorption of endo-D-galacturonanase on tri(D-galactosiduronic acid)-Separon is due exclusively to active-site-directed interaction with bound affinity-ligand.
An exopolygalacturonase [exo-PGase; poly (1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67] was found to be extracellularly produced by Bacillus sp. strain KSM-P443. The exo-PGase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, through sequential column chromatographies. The enzyme had a molecular weight of approximately 45,000 and an isoelectric point of pH 5.8. The N-terminal sequence was Ser-Met-Gln-Lys-Ile-Lys-Asp-Glu-Ile-Leu-Lys-Thr-Leu-Lys-Val-Pro-Val-Phe and had no sequence similarity to those of other pectinolytic enzymes reported to date. Maximum activity toward polygalacturonic acid (PGA) was observed at 60 degrees C and at pH 7.0 in 100 mM Tris-HCl buffer without requiring any metal ions. When the chain length of oligogalacturonic acids increased, the apparent Km for them decreased, but the kcat values increased. This is the first bacterial exo-PGase that releases exclusively mono-galacturonic acid from PGA, di-, tri-, tetra-, and penta-galacturonic acids.
1An exopolygalacturonase [exo-PG; poly (1,4-alpha-D-galacturonide) digalacturonohydrolase, EC 3.2.1.82] was found in a culture of Bacillus sp. strain KSM-P576. The purified exo-PG had a molecular weight of approximately 115,000 and an isoelectric point of pH 4.6. The N-terminal amino acid sequence was Thr-Glu-Val-Ser-Pro-Lys-Ser-Pro-Ala-Ser-Pro-Val. Maximum activity toward polygalacturonic acid (PGA) was observed at 55 degrees C and pH 8.0 in 100 mM Tris-HCl buffer. The exo-PG was quite stable in various pH buffers between pH 6 and 12 when incubated at 30 degrees C for 1 h. Mg(2+,) Mn(2+,) Pd(2+) and Ca(2+) ions stimulated the enzyme activity. The exo-PG released digalacturonic acid from PGA, tri-, tetra-, and penta-galacturonic acids. The apparent K(m) values for oligogalacturonic acids were almost identical, and k(cat) values increased with the chain length of the substrates.
A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 degrees C, pH 6.0 and retains 90% of activity after heating at 90 degrees C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of 1H NMR spectroscopy revealed an inversion of configuration during hydrolysis of alpha-(1-->4)-galacturonic linkage.
An exopolygalacturonase was partially purified from mycelia_??_ extracts of a strain of Acrocylindrium. The enzyme was most active at pH 4.5 and showed a higher affinity for polygalacturonic acid than for oligogalacturonic acids. The enzyme was found to hydrolyze glycosidic linkages at the non-reducing end of polygalacturonic acid molecules, giving only monogalacturonic acid as the reaction product.
S ummary
The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
1. A second exopolygalacturonase was separated from a mycelial extract of Aspergillus niger with a 265-fold purification and a recovery of 1%. 2. Unlike the first exopolygalacturonase this enzyme showed no requirement for metal activators, nor was it inhibited by chelating agents. 3. The two exopolygalacturonases were also distinguished by their pH optima and stability. 4. The enzyme progressively removed the terminal galacturonic residues from alpha-(1-->4)-linked galacturonide chains, converting digalacturonic acid, trigalacturonic acid and tetragalacturonic acid into galacturonic acid. Galacturonic acid was also released from pectic acid but complete digestion was not achieved.
Incubation of a mixture of extracellular pectolytic enzymes of Aspergillus niger with insoluble pectic acid cross-linked by epichlorhydrine under optimum conditions for endopolygalacturonase (poly-α-1,4-galacturonide glycanohydrolase, EC 3.2.1.15) action results in a selective adsorption of endopolygalacturonase by the insoluble support. The quantitative liberation of the enzyme into solution is achieved either in the presence of soluble sodium pectate or at pH values over pH 6. On the basis of these observations a chromatographic method for endopolygalacturonase purification was developed.
An oligogalacturonate hydrolase, free of transeliminase and unsaturated oligogalacturonate hydrolase activities, has been isolated from the cell extract of a Bacillus specie. This enzyme was purified by ammonium sulfate fractionation and by chromatography on DEAE-cellulose columns. The pH optimum was 6–6.5. The hydrolase was highly specific for saturated oligogalacturonides and completely inactive toward unsaturated oligogalacturonides. In contrast to transeliminases and polygalacturonases, the hydrolase preferentially attacked the short-chain uronides and hydrolyzed the terminal glycosidic bond from the nonreducing end of the molecule. The hydrolase activity was greatest with trigalacturonic acid followed by tetramer, pentamer, dimer, and acid-soluble pectic acid.
1. An exopolygalacturonase was separated from a mycelial extract of Aspergillus niger with a 290-fold purification and a recovery of 8.6%. 2. The enzyme displayed its full activity only in the presence of Hg(2+) ions; K(A) for mercuric chloride was about 6x10(-8)m. 3. The mercury-activated enzyme progressively removed the terminal galacturonic acid residues from alpha-(1-->4)-linked galacturonide chains and converted digalacturonic acid, trigalacturonic acid, tetragalacturonic acid and pectic acid into galacturonic acid.
Purified citrus pectinesterase (PE) does not hydrolyze the ester groups of (a) the diester of digalacturonic acid, (b) the diester of methyldigalacturonic acid, (c) the triester of methyltrigalacturonic acid, or (d) the half esters of digalacturonic acid. It readily attacks polygalacturonic acid polymethyl esters with D.P.'s of 10 and above.Purified fungal polygalacturonase (PG) does not hydrolyze the glycosidic galacturonide bonds of the three completely esterified oligogalacturonic acids, dimethyl ester of methyldigalacturonic acid, dimethyl ester of digalacturonic acid, or trimethyl ester of methyltrigalacturonic acid. It does not attack the glycosidic linkage of the half esters of digalacturonic acid. It hydrolyzes digalacturonic acid, methyl-α-d-digalacturonic acid, and trigalacturonic acid, but only at about 5% of the rate at which it attacks polygalacturonic acid. It attacks α-d-galacturonopyranosyl-l-galactonic acid. Thus PG attacks all polygalacturonide substrates which possess a glycosidic uronide bond between two adjacent free carboxyl groups, but fails to attack those where one or both of the adjacent carboxyl groups is esterified.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.