Pullulan 4-glucanohydrolase from Aspergillus niger

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 6³-α-glucosylmaltotriose, 6²-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.     

Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[¹⁴C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)-α- and (1 → 6)-α-d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.     

Effect of two-stage temperature on pullulan production by Aureobasidium pullulans

The effect of a two-stage cultivation temperature on the production of pullulan synthesized by Aureobasidium pullulans CGMCC1234 was investigated. Pullulan production was affected by temperature; although the optimum temperature for pullulan production was 26°C, the optimal temperature for cell growth was 32°C. Maximum pullulan production was achieved by growing A. pullulans in a first stage of 32°C for 2 days, and then in a second stage of 26°C for 2 days. Pullulan production using these two-stage temperatures significantly increased: about 27.80% (w/w) compared to constant-temperature fermentation (26°C for 4 days). The morphology of the A. pullulans (CGMCC 1234) was also affected by temperature; the lower temperature (26°C) supported unicellular biomass growth. Results of this study indicate that fermentation using two temperature stages is a promising method for pullulan production.     

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1.

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp.

A cell-bound cyclodextrin-degrading enzyme with a relative molecular mass (M_r) of around 62 000 and an isoelectric point (pI) near 8.0 was isolated and purified to 94% homogeneity from Flavobacterium sp. The enzyme hydrolysed maltooligosaccharides and cyclodextrins to glucose, maltose, and maltotriose. Less glucose, but larger amounts of the line of maltooligosaccharides from maltose to (in case of cyclodextrins) the linearized substrates were found in short-term digests. Digestion of maltotriose yielded glucose, maltose, and some maltotetraose to maltohexaose, i.e. the enzyme catalysed both hydrolysis and transglycosylation. Starch was a poorer substrate, and was hydrolysed to mainly glucose and maltose, presumably by a kind of exo-attack. Pullulan was slightly digested, the products being glucose, panose/isopanose, and larger saccharides containing -1,6-glucosidic bonds. Since maltohexaose to maltooctaose were hydrolysed at higher rates than the cyclodextrins of corresponding lengths, the enzyme of Flavobacterium sp. was proposed to be classified as a decycling maltodextrinase. Correspondence to: H. Bender     

Development of aqueous two‐phase systems comprising poly ethylene glycol and dextran for purification of pullulan: Phase diagrams and fiscal analysis

Pullulan is a commercially important Exopolysaccharide (EPS) with wide‐spread applications which is produced by Aureobasidium pullulans. The alternative α (1 4) & α (1 6) configuration in pullulan provides it the specific structural and conformational properties. Pullulan is currently being exploited in food, health care, pharmacy, lithography, cosmetics. The fermented broth is processed by organic solvent precipitation for isolation and purification of pullulan. In this study, we have tried to analyze the potential of aqueous two phase system as an alternate technique to extract pullulan from fermented broth. Including this viability of ATPS was also compared with conventional organic solvent precipitation system in terms of cost and time. It was found that ATPS process produced a higher yield of pullulan (80.56%) than organic solvent precipitation method (71.6%). ATPS was also found more economical and less time consuming method.     

Catalytic properties of the cloned amylase from Bacillus licheniformis.

A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformis was double-digested with EcoRI and BamHI and ligated the pBR322. The transformed E. coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B. licheniformis DNA. The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch. It was active over a pH range of 6-8 and its optimum temperature was 50 degrees C. The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4. It degraded soluble starch by cleaving maltose units preferentially but did not attack alpha-1,6-linkage. The enzyme also hydrolyzed pullulan to panose units exclusively. In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of alpha-1,6-linkage. The specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose. The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.     

Discovering the role of the apolipoprotein gene and the genes in the putative pullulan biosynthesis pathway on the synthesis of pullulan,heavy oil and melanin in <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Pullulan produced by Aureobasidium pullulans presents various applications in food manufacturing and pharmaceutical industry. However, the pullulan biosynthesis mechanism remains unclear. This work proposed a pathway suggesting that heavy oil and melanin may correlate with pullulan production. The effects of overexpression or deletion of genes encoding apolipoprotein, UDPG-pyrophosphorylase, glucosyltransferase, and α-phosphoglucose mutase on the production of pullulan, heavy oil, and melanin were examined. Pullulan production increased by 16.93 and 8.52% with the overexpression of UDPG-pyrophosphorylase and apolipoprotein genes, respectively. Nevertheless, the overexpression or deletion of other genes exerted little effect on pullulan biosynthesis. Heavy oil production increased by 146.30, 64.81, and 33.33% with the overexpression of UDPG-pyrophosphorylase, α-phosphoglucose mutase, and apolipoprotein genes, respectively. Furthermore, the syntheses of pullulan, heavy oil, and melanin can compete with one another. This work may provide new guidance to improve the production of pullulan, heavy oil, and melanin through genetic approach.     

Pullulan production from synthetic medium by a new mutant of Aureobasidium pullulans

Pullulan with different molecular-weight could be applied in various fields. A UV-induced mutagenesis Aureobasidium pullulans UVMU6-1 was obtained from the strain A. pullulans CGMCC3.933 for the production of low-molecular-weight pullulan. First, the obtained polysaccharide from A. pullulans UVMU6-1 was purified and identified to be pullulan with thin-layer chromatography, Fourier transform infrared, and nuclear magnetic resonance. Then, culture medium and conditions for this strain were optimized by flask fermentation. Based on the optimized medium and culture conditions (pH 4, addition of 4?g/L Tween 80 for 96?hr of cultivation), continuously fermentation was performed. The highest pullulan production and dry biomass was 109 and 125?g/L after fermentation for 114?hr, respectively. The average productivity was about 1?g/L/hr, which was intensively higher than the previous reported. This study would lay foundations for the industrial production of pullulan.     

Hydrolysis of alpha-D-glucans and alpha-D-gluco-oligosaccharides by Cladosporium resinae glucoamylases

Culture filtrates of Cladosporium resinae ATCC 20495 contain a mixture of enzymes able to convert starch and pullulan efficiently into D-glucose. Culture conditions for optimal production of the pullulan-degrading activity have been established. The amylolytic enzyme preparation was fractionated by ion-exchange and molecular-sieve chromatography, and shown to contain alpha-D-glucosidase, alpha-amylase, and two glucoamylases. The glucoamylases have been purified to homogeneity and their substrate specificities investigated. One of the glucoamylases (termed P) readily hydrolyses the (1 leads to 6)-alpha-D linkages in pullulan, amylopectin, isomaltose, panose, and 6(3)-alpha-D-glucosylmaltotriose. Each of the glucoamylases cleaves the (1 leads to 6)-alpha-D linkage in panose much more readily than that in isomaltose.     

Optimization of pH for high molecular weight pullulan

Summary Pullulan is a polysaccharide produced by Aureobasidium pullulans. In this study, the effect of pH on the molecular weight of pullulan was investigated. High concentration of pullulan was obtained when initial pH was 6. Pullulan having molecular weight of 500,000–600,000 was produced at initial pH of 3.0, while pullulan with molecular weight of 200,000–300,000 was produced at pH above 4.5. To obtain high molecular weight pullulan with high concentration, pH was initially controlled at pH 6, followed by pH shift from pH 6 to pH 3. Transition of pH at 2 days of fermentation was observed to be optimum. Higher molecular weight pullulan was also obtained when sucrose concentration was 50 g/l compared to the result obtained at initial sucrose concentration of 20 g/l. Sucrose concentration and pH of the fermentation broth seem to be important parameters in obtaining high molecular weight of pullulan.     

Production of Pentameric Hybrid Immunoglobulins Consisting of IgA and IgM

The amylomaltase from Escherichia coli IFO 3806 was purified to homogeneity seen by SDS- polyacrylamide gel electrophoresis after DEAE-Sephadex, Ultrogel AcA 44, hydroxylapatite, and 1,6- hexane-diamine-Sepharose 4B column chromatographies. The molecular weight of the purified enzyme was 93,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5 and at 35°C, and stable up to 45°C at pH 7.0 and from pH 6.0 —7.3 at 40°C on 30min incubation. The enzyme acted on maltotetraitol, maltopentaitol, and maltosylsucrose besides maltooligosaccharides, but did not act on maltitol, maltotriitol, glucosylsucrose, isomaltose, panose, isopanose, or isomaltosyl- maltose. This enzyme did not catalyze hydrolytic action on maltotetraitol, maltopentaitol, or maltosylsucrose.     

Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1

Exopolysaccharide produced by a new novel colour variant strain of Aureobasidium pullulans FB-1 was purified by cell harvesting and precipitation of the polymer. Various organic solvents were screened for pullulan precipitation. Isolation and purification of pullulan from fermentation broth was carried out using single-step purification strategy by isopropyl alcohol precipitation. Ratio of culture supernatant to isopropyl alcohol and time of precipitation were optimized for pullulan precipitation. Maximum yield (4.47%, w/v) of polysaccharide was obtained when two volumes of ice-cold isopropyl alcohol were added to one volume of supernatant with precipitation time of 12 h. IR spectra as well as carbon-13 and proton NMR spectra in aqueous solution of intact polysaccharide obtained from A. pullulans FB-1 and commercially available pullulan (Sigma, USA) revealed solely α-(1 → 6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Maximum hydrolysis (94.25%) of purified pullulan at 50 °C by pullulanase was achieved under agitation (150 rpm) after 360 min.     

Purification of ferredoxin-NADP+ oxidoreductase from cyanobacteria by affinity chromatography on 2′,5′-ADP-Sepharose 4B

A simple and rapid method for the purification to homogeneity of ferredoxin-NADP⁺ oxidoreductase (EC 1.18.1.2) from the nitrogen-fixing filamentous cyanobacterium Anabaena sp. strain 7119 is described. A crude extract prepared by solubilizing the cells with a detergent was first partially purified on a DEAE-cellulose column and then chromatographed on 2′,5′-ADP-Sepharose 4B. Ligand-bound ferredoxin-NADP⁺ oxidoreductase was eluted by a linear gradient of NaCl. The overall procedure provided an enzyme purified about 400-fold with a yield of 60 to 70%. The final enzyme preparation exhibited a specific activity of 120 units/mg protein and an absorbance ratio $\text{A}{}_{280}\text{A}{}_{458}$ of 8.26. The enzyme protein migrated as a single band when subjected to polyacrylamide gel electrophoresis and chromatographed as a single isoelectric species under chromatofocusing.     

Continuous production of panose by immobilized neopullulanase

Panose is a mildly sweet trisaccharide composed of three glucose units which has the nature of an anti-cariogenic sugar in foods. Continuous production of panose from pullulan was investigated by using a column of immobilized neopullulanase. Although enzyme immobilization by ionic bond is one of the most general methods for the production of non-ionic materials from non-ionic substrates, the activity of the neopullulanase immobilized on DEAE-cellulose significantly decreased. When the neopullulanase was immobilized on a carrier with spacer arms by covalent bond through the formation of the schiff base, the activity was fully expressed. Optimum temperature and pH for the reaction of the immobilized neopullulanase were 55–60°C and 6.0, which were almost the same as those of the free enzyme. The immobilized neopullulanase column was employed for the continuous production of panose from pullulan. More than 92% of pullulan was converted by this system into the final products; panose, maltose, and glucose. The ratio of panose in the mixture of products was 84%, which was significantly higher than that (around 70%) of a batch system using the free enzyme. The immobilized neopullulanase was very stable, and more than 90% of the initial activity was retained after 150-h continuous reaction at 55°C.     

Characterization of pullulan-hydrolysing enzyme from an alkalopsychrotrophic Micrococcus sp.

Summary A pullulan-hydrolysing enzyme of Micrococcus sp. 207 was purified to an electrophoretically homogenous state by chromatography on DEAE-Toyopearl, -cyclodextrin-Sepharose and Asahipak GS-520P. The purified enzyme was free of -amylase activity. The molecular weight of the enzyme as estimated by SDS-PAGE was 120,000 and the pI value as determined by isoelectric focusing was 4.9. The enzyme was most active at pH 8.0 and 50°C. The enzyme was activated by the addition of CaCl₂, but its thermoresistance increased after removing free Ca²⁺ ions. The enzyme could hydrolyse the -1,6-linkages of amylopectins, glycogens and pullulan and the K _m value for pullulan was about 0.018%. Pullulan at concentrations above 0.012% inhibited the enzyme activity and the activity was competitively inhibited by cyclodextrins. Offprint requests to: K. Horikoshi     

Purification and characterization of a soluble,cytoplasmic decycling maltodextrinase from aLactobacillus species,strain 26X,isolated from kitchen waste water

A soluble, cytoplasmic decycling maltodextrinase with a relative molecular mass (M _r) of around 62 000, and an isoelectric point (pI) of about 4 was purified to 93% homogeneity from aLactobacillus species, strain 26X, that was isolated from the waste water of a kitchen and resembledLactobacillus plantarum in its physiological and biochemical characteristics, but differed from this species in producing exclusively L( + )-lactic acid. The enzyme exhibited higher activities with maltotetraose to maltoheptaose than with cyclodextrins, in which cyclomaltohexaose was hydrolysed fastest, and the amounts of the linearized cyclic substrates were in the order cyclomaltohexaose < cyclomaltoheptaose < cyclomaltooctaose. The substituents of cyclomaltoheptaose derivatives considerably blocked the enzymic activity, and the accessibility to enzymic attack depended on the degree of substitution. The main product of hydrolysis proved to be maltose. The maltodextrinase exhibited, above all with maltotetraose, some transglycosylation activity. Except for maltopentaose, maltosyl transfer predominated. Starch and pullulan were degraded at low rates, the main products from starch being glucose and maltose, and from pullulan a branched trisaccharide, presumably panose.     

Cloning and Sequencing of an Original Gene Encoding a Maltogenic Amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. US149 Strain and Characterization of the Recombinant Activity

A gene encoding maltogenic amylase from acidic Bacillus sp. US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. The alignment of deduced amino acid sequence revealed a relatively low homology with the already reported maltogenic amylases. In fact, its highest identity, of only 60%, was found with the maltogenic amylase of Thermus sp. IM6501. The recombinant enzyme (MAUS149) was found to be intracellular and was purified to homogeneity from the cell crude extract with a yield of 23%. According to PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The maximum activity was obtained at 40°C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose from starch, maltose and glucose from β-cyclodextrin, and panose from pullulan.     

Purification and Some Properties of a Novel α-Amylase Produced by a Strain of Thermoactinomyces vulgaris

An α-amylase[α-l,4-glucan 4-glucanohydrolase, EC 3.2.1.1.], found in the culture filtrate of a strain of Thermoactinomyces vulgaris, was purified by ammonium sulfate fractionation, and DEAE-cellulose and CM-cellulose chromatographies. The purified enzyme showed a single band on disc gel electrophoresis. The optimum reaction pH and temperature were determined to be around pH 5.0 and 70°C. The isoelectric point was determined to be pH 5.2. The α-amylase was stabilized by Ca²⁺.

The α-amylase was found to hydrolyze pullulan to panose. Therefore, the hydrolytic pattern of this enzyme is different from those of pullulanase and isopullulanase.