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Purification and partial characterization of an endo-polygalacturonase from Aspergillus niger
Journal of Food Biochemistry
Abstract
ABSTRACTA pectinase was identified and isolated from a commercial Aspergillus niger pectinase preparation. The crude enzyme preparation, which was prepared by precipitation of the water extract of the culture of A. niger with ammonium sulfate, was further fractionated by three steps of chromatography, i. e., cation exchange, hydrophobic interaction and onion exchange, to obtain an electrophoretically homogeneous pectinase. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be about 40.4 kDa under both nonreducing and reducing conditions, with the optimum pH at 5.0 and the optimum temperature at 36C. The enzyme was stable at temperatures below 35C. The partial N-terminal ammo acid sequence data analysis of the first 19 amina acids of the obtained pectinase revealed 94.7% and 89.5% homology with two reported pectinases from A. niger.
... The chromatography of DEAE- Sepharose PGase pooled fractions on Sephacryl S-200 column give a purified PGase with specific activity of 1125 units/mg protein and 40% recovery. This specific activity higher than that reported for polygalacturonases from T. harzianum (276 units/mg protein) (Mohamed et al. 2006), T. reesei (916 and 708 units/mg protein) (Mohamed et al., 2003) and Aspergillus niger (418 units/mg protein) (Fahmy et al., 2008). The homogeneity of PGase was detected by SDS-PAGE (Fig. 2). ...
... This molecular weight was confirmed by SDS-PAGE (Fig. 2) and estimated to be 29 kDa as single subunit. The majority of purified fungal polygalacturonases have molecular weight in the range from 25 kDa to 82 kDa (Barense et al., 2001; Sakamoto et al., 2002; Fahmy et al. 2008), whereas the polygalacturonase of Neurodpora sitophila had a low molecular mass of 13 kDa (Fogarty and Kelly, 1983). A number of citrus pectins were tested as substrates for T. harzianum PGase (Table 2). ...
... Each point represents the average of two experiments. optimum (pH 4 -5) was reported for polygalacturonases from Fusarium moniliforme (Niture and Pant 2004), T. reseei (Mohamed et al., 2003) and A. niger (Fahmy et al. 2008). The remarkable property of A. kawachii PGI was its high activity in the pH range 2-3 towards soluble and insoluble substrates, while being inactive at pH 5.0 (Contreras-Esquivel and Voget, 2004). ...
A polygalacturonase (PGase) was purified from Trichoderma harzianum, grown on citrus peel, by anion exchange and size exclusion chromatographies. The enzyme had molecular mass of 29 kDa. The extent of hydrolysis by PGase was decreased with increasing of pectin esterification degree. m max K and V , using polygalacturonic acid as substrate, were 1.42 mg and 0.66 ìmol reducing sugar, respectively. PGase had a broad pH optimum ranged from 4.5 to 7.0 and temperature optimum at 55<C. PGase was stable up to 50°C after incubation for 30 min. All the examined metal cations showed partial inhibitory effects, except for Hg which was completely inhibited the enzyme activity. 2+ The effect of PGase and wheat á-amylase on apple juice was studied, where the mixture of the two enzymes increased the quantity (135% fold) and clarity degree of apple juice.
... In recent years, vigorous researches on biosynthesis and purification of endo-PG have been carried out (Naidu et al., 1998;Jayani et al., 2005;Tewari et al., 2005). Significant progress has been made in purifying endo-PG to homogeneity by using different purification methods (Manachini et al., 1987;Nagai et al., 2000;Guo et al., 2001;Gummadi, 2003). In Vietnam, however, due to high cost and lack of pectinase production, the utilization of this enzyme in the food industry has still been limited (Pham, 2007a). ...
... Ammonium sulfate has a great advantage to precipitate protein because of its low cost and rarely causing irreversible denaturation of protein molecules (Scopes, 1994). According to previous research by Afifi et al. (2002); Guo et al. (2001); Jaffar et al. (1993);Miyazaki (1991), the saturation degree of ammonium sulfate appropriate for endo-PG purification varied from 60 to 80% and that depended on the enzyme sources. In this study, the suitable concentration of ammonium sulfate, which is 47.2% (w/v), is equivalent to 70% saturation degree of this salt in the enzyme solution. ...
The purification of extracellular endopolygalacturonase (endo-PG) from the filtrate of Aspergillus awamori L1 submerged culture was carried out using a two-step procedure: enzyme precipitation by organic solvents, polymers or salts; and gel filtration using Sephadex G-75 column. The results show that ammonium sulfate was the suitable precipitant in the two-step procedure for endo-PG purification. Firstly, ammonium sulfate was added to the crude enzyme solution with the concentration of 47.2% (w/v) for endo-PG precipitation. The precipitates were then isolated and redissolved in a buffer solution to prepare for the gel filtration step. Consequently, the purification factor achieved 30.4-fold and the endo-PG recovery yield obtained 68.60% in comparison to the crude enzyme solution.
... No activity was detected after heating the enzyme at 65 ºC for 15 min. Data obtained are comparable to those of TengGuo et al. (2002), where they reported that PG from Aspergillus niger was stable at 35 ºC, and its incubation at 40 ºC, and 50 ºC for 30 min resulted in activity losses by 45 and 74 %, respectively .Also, they reported that PG was almost inactivated at a temperature above 60 ºC for 5 min. The endo-PG from Aspergillus japonicus lost more than 50 % of its activity during 5 min at 50 ºC (Semenova et al., 2003). ...
Polygalacturonase (PG) produced by Aspergillus niger U-86 was purified by ammonium sulfate
fractionation followed by gel filtration on sephadex G-75. Purification fold of 6.1 and enzyme yield of
64% were achieved. SDS-PAGE of the purified polygalacturonase showed the presence of two bands
with the molecular weight of 36 and 38 kDA. The purified polygalacturonase was stable at acidic pH
(3.0-6.0) and at 30ºC. The apparent Km value was calculated to be 1.42 mg. ml-1.
The clarification of pomegranate and grape juices was greatly affected by both PG concentration
(units) and reaction time. Maximum reduction in turbidity and viscosity with the retention of the original
color was achieved by applying PG at the concentration of 58.5 units for 60 min and 39 units for 90 min
for pomegranate and grape juice, respectively. The clarified juices were stable upon storage at both 4 and
25 ºC.
An endo-polygalacturonase (endo-PGase) that exhibits excellent performance during acidic fruit juice production would be highly attractive to the fruit juice industry. However, candidate endo-PGases for this purpose have rarely been reported. In this study, we expressed a gene from Penicillium oxalicum in Pichia pastoris. The recombinant enzyme PoxaEnPG28C had an optimal enzyme activity at pH 4.5 and 45°C and was stable at pH 3.0-6.5 and < 45°C. The enzyme had a specific activity of 4,377.65±55.37 U/mg towards polygalacturonic acid, and the Km and Vmax values of PoxaEnPG28C were calculated as 1.64 g/L and 6127.45 U/mg, respectively. PoxaEnPG28C increased the light transmittance of orange, lemon, strawberry and hawthorn juice by 13.9±0.3%, 29.4±3.8%, 95.7±10.2% and 79.8±1.7%, respectively; it reduced the viscosity of the same juices by 25.7±1.6%, 52.0±4.5%, 48.2±0.7% and 80.5±2.3%, respectively; and it increased the yield of the juices by 24.5±0.7%, 12.7±2.2%, 48.5±4.2% and 104.5±6.4%, respectively. Thus, PoxaEnPG28C could be considered an excellent candidate enzyme for acidic fruit juice production. Remarkably, fruit juice production using hawthorn as an ingredient was reported for the first time.
The production of a notable, safe and highly active pectinase by the local fungal strain Trichoderma viride EF-8 utilizing the abundant pigmented Egyptian onion (Allium cepa L.) skins (6.5%, w/v) was achieved in 4 days submerged fermentation (SMF) cultures, at temperature and pH of 30 °C and 4.0, respectively. The indigenously produced pectinase was partially purified by 50% batch ethanol precipitation and its general properties were studied following the standard procedures. The lyophilized enzyme preparation was free of any ochra or aflatoxins. The optimum conditions for the partially purified enzyme form were 2 mg/mL and 1% (w/v) enzyme protein and substrate (citrus pectin) concentrations, reaction pH and temperature of 7.0 and 40 °C, respectively. The results presented the low cost onion skins waste as the major substrate for the fungal pectinase production and its subsequent use in perfect fruit (apple, lemon and orange) juices clarification with remarkable stability during and after this process, which certainly enhance fruit juices processing in the tropics.
Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts and wines. However, most of reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endo-polygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively; and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner releasing oligo-galacturonates from PGA as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.
Two inducible polygalacturonases (PG-1 and PG-2) from culture filtrates of Trichoderma koningii were purified to homogeneity by CM-cellulose chromatography and isoelectric focusing in a narrow pH range (pH 6 to 8). They were both hydrolytic enzymes classifiable as endopolygalacturonases [poly(1,4-α-D-galacturonide) glycanohydrolase; EC 3.2.1.15]. PG-1 and PG-2, focusing at pH 6-41 and 6-57 respectively, each consisted of a single polypeptide chain having an apparent molecular weight of 32000 as determined by gel filtration on Sephadex G-100; they were both glycoproteins and had carbohydrate contents of 0-033 and 0-062 mg sugar (mg protein)-1 respectively. When the isoenzymes were incubated with different plant tissues, they were not absorbed by any of them.
The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].
A polygalacturonase from culture filtrates of a strain ofRhizopus
stolonifer was purified about 80 fold by ethanol precipitation, followed by ion exchange chromatography (CM-Sepharose 6B) and gel filtration (Sephadex G-100). The purified preparation was homogeneous when examined by PAGE. The enzyme is an endopolygalacturonase with an optimum catalytic activity at pH 5.0 and 45°C, and a molecular weight of 57,000±500 daltons. The activity was stimulated by Fe+++, Mg++, Co++, and inhibited by Mn++ and Zn++. The enzyme was stable in the pH range of 3.0 to 5.0. The purified enzyme was specific for nonmethoxylate polygalacturonic acid, with Km and Vmax values respectively of 0.19 mg/ml and 1.3 μmol/μg/min. In addition, this enzymatic preparation degraded pectic substances in organge peel.
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Thin-layer chromatography, with its inherent simplicity of operation and sufficiently high degree of sensitivity and resolution, can, as a preliminary technique in the analysis of sugars and related compounds, replace other more sophisticated procedures such as gas-liquid and ion-exchange chromatography. Furthermore, as far as the identification of the sugar compounds is concerned, TLC is superior because it permits the use of rapid differential reactions on the layer and the utilization of other criteria such as RF values and colour variations.
The polygalacturonase (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) activity of Pectinol is resolved into two fractions (E1 and E2) of about equal total activity on DEAE-cellulose. These fractions are purified from other pectinolytic enzyme activity by Sephadex G-75 chromatography. Both E1 and E2 reduce the viscosity of polygalacturonate by 50% after 7% of the glycosidic bonds are hydrolysed. Their activities are not affected by iodoacetate (1 mM) or EDTA (10 mM). E1 and E2 have different molecular weights (35 000 and 85 000, respectively) and different electrophoretic mobilities on sodium dodecyl sulphate polyacrylamide gels. Their pH (4.1 and 3.8 respectively) and ionic strength optima and specific activities also differ. Both enzymes are inhibited at similar rates by diethyl pyrocarbonate at pH 6 but only E2 is protected from this inhibition by 2% (w/v) polygalacturonate. The rate of change of protein absorbance at 250 nm accompanying this inhibition, and the residues are essential for the activities of both E1 and E2. About 2 molecules of carbethoxyhistidine per subunit of E2 and 0.6 molecules per subunit of E1 are present in the completely inhibited enzymes.20
Polygalacturonase (EC 3.2.1.15) produced by Fursarium oxysporum f. sp. lycopersici was purified by chromatography on DEAE-cellulose, CM-cellulose, and hydroxyapatite. The purified enzyme consisted of two electrophoretically distinct "isozymes", that behaved as charge isomers during electrophoresis in several different concentrations of polyacrylamide gel. The two isozymes had similar "endo" modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction, reducing group release, and thin-layer chromatography of oligomeric hydrolysis products. Both isozymes hydrolzyed 5% of the substrate bonds in reaching 50% viscosity reduction. The amino acid compositions of the isozymes were similar and their molecular weights were about 37000 as determined by sedimentation equilibrium. Removal of large amounts of carbohydrate during purification did not affect heat stability of the enzymes. A large proportion of the remaining carbohydrate appeared to be covalently linked to the enzyme protein.
The endopolygalacturonase (EC 3.2.1.15) enzyme produced in vitro by Sclerotinia sclerotiorum were found to consist of numerous isoforms covering a broad pI range. Two of the isoforms, labelled PG2 and PG3, were purified using gel-filtration chromatography, isoelectric focusing and anion-exchange chromatography. The pIs of PG2 and PG3 were, respectively, 4.8 and 4.9. Their molecular weights were similar. Both enzymes hydrolysed 0.9% of the bonds in reaching a 50% reduction in viscosity. However, their enzymic parameters were different. Their amino acid compositions differed only in the aspartic acid-asparagine content. The N-terminal sequences differed at the fourth amino acid only. PG2 and PG3 exhibited a high level of glycosylation compared to a similar enzyme isolated from Aspergillus niger. Antibody raised against PG3 was shown to crossreact with PG2, but not with the enzyme purified from Aspergillus niger.
PolygalacturonaseII of Aspergillus niger was fragmented using CNBr and the NH2-terminal fragment and another fragment were partially sequenced. The polygalacturonaseII (pgaII) gene was then isolated by using an oligonucleotide mixture based on the internal amino acid sequence as a probe. The nucleotide sequence of the pgaII structural gene was determined. It was found that polygalacturonaseII is synthesized as a precursor having an NH2-terminal prepro-sequence of 27 amino acids. The cloned gene was used to construct polygalacturonaseII over-producing A. niger strains. PolygalacturonaseII was isolated from one such strain and was determined to be correctly processed and to be fully active.
Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.
A 1319 bp long cDNA encoding for a polygalacturonase (EC 3.2.1.15) from Aspergillus niger RH5344 comprises a single open reading frame of 1089 bp which includes the mature protein of 362 amino acids and an NH2-terminal signal peptide of 27 amino acids. The directly determined peptides of the mature polygalacturonase confirmed the sequence information deduced from the cDNA.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
An endopolygalacturonase (poly,α-1,4-galacturonide glycanohydrolase, EC 3.2.1.15) was purified 60-fold from the culture fluids of Verticillium albo-atrum by chromatography on columns of carboxymethyl cellulose and hydroxylapatite, gel filtration, and preparative electrophoresis. Final preparations were homogeneous on the basis of disc-gel electrophoresis, ion-exchange and gel-filtration chromatography, and sedimentation in the ultracentrifuge. The purified endopolygalacturonase had a molecular weight of about 30,000, was rich in basic amino acids, and contained 1.2% carbohydrate. The enzyme catalyzed the random hydrolysis of sodium polypectate to short-chain oligouronides and exhibited macerating activity on cucumber pericarp tissues. The endopolygalacturonase had a Michaelis constant and maximum velocity on sodium polypectate of 0.15% and 2150 μmoles galacturonate equivalents liberated/ min/mg protein, respectively, at 35 ° and at the optimum pH of 6.5. The enzyme was inhibited by heavy metals but not by classical sulfhydryl or serine antagonists.
Nine forms of Aspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with free alpha-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from other Aspergillus species (72-75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66-71% residue differences toward the Erwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties.
In a culture filtrate of Trichosporan penicillatum B2, which is a gamma-ray irradiation mutant induced from T. penicillatum SNO3, we found three kinds of pectin-releasing enzymes, protopectinases SE1, SE2, and SE3, that have endo-polygalacturonase activity. These enzymes were purified to homogeneity with cation-exchange and size exclusion chromatographies. The major PPase in the culture filtrate was PPase SE1, which accounted for 75% of total activities in the culture filtrate, and the two others were 0.15% (PPase SE2) and 0.007% (PPase SE3). Their molecular masses were approximately 41, 41, and 42 kDa on SDS-PAGE, respectively. They had similar enzymatic properties but different PPase activity and pH- and thermo-stability. Antibody against PPase S, which is produced by strain SNO3, inhibited the activities of PPase SE1, SE2, and SE3. However PPase SE1 was completely inhibited by treatment with the anti-PPase S antibody, but the activities of PPases SE2 and SE3 remained at 20 and 50% of the original activity, respectively.
An exo-polygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with citrus pectin, using preparative isoelectric focusing. The enzyme, denominated PG2, had an apparent M(r) of 74000 Da upon SDS-PAGE. The pI of the main PG2 isoform was 4.5, and pH and temperature optima were 5.0 and 55 degrees C, respectively. PG2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by anaysis of degradation products. The enzyme was N-glycosylated. The N-terminal amino acid sequence, L-A-F-N-V-P-S-K-P-P, has no identify to other known polygalacturonases.
An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus.
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.
Thin-layer chromatography of carbohydrates Purification and characterization of anendo-polygalacturonase froma mutant of Sacchuromyces cerevisiae
- M Ghebregzabher
- S Rufini
- B Monaldi
- M Lato
- N Hirose
- M Kishida
- H Kawasaki
- T Sakai
GHEBREGZABHER, M., RUFINI, S., MONALDI, B. and LATO, M. 1976. Thin-layer chromatography of carbohydrates. J. Chromatogr. 127(2), HIROSE, N., KISHIDA, M., KAWASAKI, H. and SAKAI, T. 1999. Purification and characterization of anendo-polygalacturonase froma mutant of Sacchuromyces cerevisiae. Biosci. Biotechnol. Biochem. 63(6), 1 100-1103.
Purification and character-ization of three extracellular protopectinases with polygalacturonase activities from Trichosporon penicillutum Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger
- K Iguchi
- M Kishida
- T Sakai
- H C Kester
- J Visser
IGUCHI, K., KISHIDA, M. and SAKAI, T. 1996. Purification and character-ization of three extracellular protopectinases with polygalacturonase activities from Trichosporon penicillutum. Biosci. Biotechnol. Biochem. 60(4), KESTER, H.C. and VISSER, J. 1990. Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger. Biotechnol. Appl. Biochem. 12(2), 150-160.
Economic Microbiology
- F. W. ROMBOUTS
- W. PILNIK
- NELSON N.