Degradation Of Pullulan Research Articles

Structural and Functional Analysis of Pullulanase Type 1 (PulA) from Geobacillus thermopakistaniensis.

Pullulanase type I (PulA) is a debranching enzyme that specifically cleaves α-1,6-glycosidic linkages in pullulan. Pullulan has not only diverse applications in food industry but also has immune-stimulatory effects on B and T cells, and found to enhance the production of various anti-inflammatory cytokines in human. Moreover, pullulan has been suggested as a possible anti-cancer drug delivery agent without adjuvant due to its unique structure. The process of pullulan degradation is unresolved due to imprecise pullulanase structural characteristics. Therefore, the present study aimed to understand the structural and functional characteristics of pullulanase enzyme fromGeobacillus thermopakistaniensis MAS1 strain using various computational approaches. The physio-chemical topographies and secondary structure of GT_PulA were explored using ProPram, InterPro and SMART. Various tools like I-TASSER, ModRefiner, RAMPAGE, PROCHECK and MOE 2009.10 were used to construct and verify the 3D structural model. The structural elucidation confirmed the significant domains, i.e., CBM48, CBM2, and TIM barrel having catalytically active residues, and conserved region YNGWDP. CBM2 domain along with TIM barrel has a capacity to bind different ligands and proved favorable for multiple substrate catalyses. These structural properties can have a potential effect on enhancing enzymatic activity of GT_PulA enzyme.

An enzymatically controlled mucoadhesive system for enhancing flavour during food oral processing

While a good mucoadhesive biopolymer must adhere to a mucus membrane, it must also have a good unloading ability. Here, we demonstrate that the biopolymer pullulan is partially digested by human salivary α-amylase, thus acting as a controlled release system, in which the enzyme triggers an increased release of flavour. Our oral processing simulations have confirmed an increase in the bioavailability of aroma and salt compounds as a function of oral pullulan degradation, although the release kinetics suggest a rather slow process. One of the greatest challenges in flavour science is to retain and rapidly unload the bioactive aroma and taste compounds in the oral cavity before they are ingested. By developing a cationic pullulan analogue we have, in theory, addressed the “loss through ingestion” issue by facilitating the adhesion of the modified polymer to the oral mucus, to retain more of the flavour in the oral cavity. Dimethylaminoethyl pullulan (DMAE-pullulan) was synthesised for the first time, and shown to bind submaxillary mucin, while still retaining its susceptibility to α-amylase hydrolysis. Although DMAE-pullulan is not currently food grade, we suggest that the synthesis of a sustainable food grade alternative would be a next generation mucoadhesive targeted for the oral cavity.

Sodium chloride improves pullulan production by Aureobasidium pullulans but reduces the molecular weight of pullulan.

The effect of sodium chloride (NaCl) on pullulan production by batch culture of Aureobasidium pullulans CCTCC M 2012259 was investigated. NaCl at 3g/L improved the pullulan titer by 26.7% but reduced the molecular weight of pullulan to only 46.8% of that obtained in the control without NaCl. In order to elucidate the physiological mechanism underlying the effect of NaCl on pullulan production, assays of key enzyme activity, gene expression, energy metabolism, and intracellular uridine diphosphate glucose (UDP-glucose) content were performed. Results indicated that NaCl increased the activities of α-phosphoglucose mutase and glucosyltransferase involved in pullulan biosynthesis, increased the activities of α-amylase being responsible for pullulan degradation, upregulated the transcriptional levels of pgm1, fks, and amy2 genes, enhanced the driving force for ATP supply, and helped to maintain intracellular UDP-glucose at a high level in A. pullulans CCTCC M 2012259. All these results illuminate the reason by which NaCl increases pullulan titer but reduces the molecular weight of pullulan.

Novel system for pulse radiolysis with multi-angle light scattering detection (PR-MALLS) – concept, construction and first tests

Time-resolved pulse radiolysis, utilizing short pulses of high-energy electrons from accelerators, is an effective method for rapidly generating free radicals and other transient species in solution. Combined with fast time-resolved spectroscopic detection (typically in the ultraviolet/visible/near-infrared), it is invaluable for monitoring the reactivity of species subjected to radiolysis on timescales ranging from picoseconds to seconds. When used for polymer solutions, pulse radiolysis can be coupled with light-scattering detection, creating a powerful tool for kinetic and mechanistic analysis of processes like degradation or cross-linking of macromolecules. Changes in the light scattering intensity (LSI) of polymer solutions are indicative of alterations in the molecular weight and/or in the radius of gyration, i.e., the dimensions and shape of the macromolecules. In addition to other detection methods, LSI technique provides a convenient tool to study radiation-induced alterations in macromolecules as a function of time after the pulse. Pulse radiolysis systems employing this detection mode have been so far constructed to follow light scattered at a single angle (typically the right angle) to the incident light beam. Here we present an advanced pulse radiolysis & multi-angle light-scattering-intensity system (PR-MALLS) that has been built at IARC and is currently in the phase of optimization and testing. Idea of its design and operation is described and preliminary results for radiation-induced degradation of pullulan as well as polymerization and crosslinking of poly(ethylene glycol) diacrylate are presented. Implementation of the proposed system provides a novel research tool, which is expected to contribute to the expansion of knowledge on free-radical reactions in monomer- and polymer solutions, by delivering precise kinetic data on changes in molecular weight and size, and thus allowing to formulate or verify reaction mechanisms. The proposed method is universal and can be applied for studying both natural and synthetic polymers. The developed system can be also valuable in studies of the border of biology and medicine, especially on radical reactions of biopolymers and their conformational transitions. Furthermore, capability to follow fast changes in mass and dimensions of nanobjects may be of significant importance for nanoscience and nanotechnology.

A novel cold-adapted type I pullulanase of Paenibacillus polymyxa Nws-pp2: in vivo functional expression and biochemical characterization of glucans hydrolyzates analysis.

BackgroundPullulanase is an important debranching enzyme and has been widely utilized to hydrolyse the α-1,6 glucosidic linkages in starch/sugar industry. Selecting new bacterial strains or improving bacterial strains is a prerequisite and effective solution in industrial applications. Although many pullulanase genes have been cloned and sequenced, there is no report of P. polymyxa type I pullulanase gene or the recombinant strain. Meanwhile most of the type I pullulanase investigated exhibit thermophilic or mesophilic properties. There are just few reports of cold-adapted pullulanases, which have optimum activity at moderate temperature and exhibit rather high catalytic activity at cold. Previously, six strains showing distinct pullulan degradation ability were isolated using enrichment procedures. As containing novel bacterium resource and significant pullulanase activity, strain Nws-pp2 was selected for in-depth study.MethodsIn this study, a type I pullulanase gene (pulN) was obtained from the strain P. polymyxa Nws-pp2 by degenerate primers. Through optimization of induced conditions, the recombinant PulN achieved functional soluble expression by low temperature induction. The enzyme characterizations including the enzyme activity/stability, optimum temperature, optimum pH and substrate specificity were also described through protein purification.ResultsThe pullulanase gene (named pulN), encoding a novel cold-adapted type I pullulanase (named PulN), was obtained from isolated strain Paenibacillus polymyxa Nws-pp2. The gene had an open reading frame of 2532-bp and was functionally expressed in Escherichia coli through optimization of induced conditions. The level of functional PulN-like protein reached the maximum after induction for 16 h at 20 °C and reached about 0.34 mg/ml (about 20 % of total protein) with an activity of 6.49 U/ml. The purified recombinant enzyme with an apparent molecular mass of about 96 kDa was able to attack specifically the α-1,6 linkages in pullulan to generate maltotriose as the major product. The purified PulN showed optimal activity at pH 6.0 and 35 °C, and retained more than 40 % of the maximum activity at 10 °C (showing cold-adapted). The pullulanase activity was significantly enhanced by Co2+ and Mn2+, meanwhile Cu2+ and SDS inhibited pullulanase activity completely. The Km and Vmax values of purified PulN were 15.25 mg/ml and 20.1 U/mg, respectively. The PulN hydrolyzed pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity and products analysis proved that the purified pullulanase from Paenibacillus polymyxa Nws-pp2 belong to a type I pullulanase.ConclusionsThis report of the novel type I pullulanase in Paenibacillus polymyxa would contribute to pullulanase research from Paenibacillus spp. significantly. Also, the cold-adapted pullulanase produced in recombinant strain shows the potential application.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0215-z) contains supplementary material, which is available to authorized users.

A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine: Functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization

Using enrichment procedures, the strain showing distinct pullulan degradation ability was isolated from the stinky tofu brine and identified as Bacillus cereus (named B. cereus Nws-bc5) by 16S rRNA sequence analysis. Meanwhile, the pullulanase gene (named pulBC) involved in pullulan degradation was obtained from Nws-bc5. The gene has an open reading frame of 2142-bp, and shows highest identity with the pullulanase from B. cereus Q1 (CP000227.1). The gene pulBC was expressed in Escherichia coli and Bacillus subtilis WB800 respectively. The expression level of recombinant PulBC expressed in E. coli and B. subtilis was 5.6 and 0.8mg/ml respectively. The purified recombinant enzyme (molecular weight 81.4kDa) was able to attack the α-1,6-linkages in pullulan specifically to generate maltotriose as the major product. The pH and temperature optima of the recombinant enzyme were pH 6.0 and 40°C respectively. Moreover, PulBC showed much higher stability under alkaline conditions and was stable at pH 6.0–9.0. The pullulanase activity was enhanced significantly by Ca2+. The specific activity of purified PulBC was 44.7U/mg (pullulan) and 29.3U/mg (soluble starch). The Km and Vmax values of purified PulBC were 0.45mg/ml and 45.3μmol/min, respectively.

Chemiluminescence related to degradation of thermally oxidized pullulans. Comparison with cellulose and dextran

The degradation of pullulan, cellulose and dextran in the presence of oxygen was examined by chemiluminescence and by size-exclusion chromatography (SEC) methods. While 1,4-α glycosidic bonds in pullulan are split thermally only with a slight effect of oxygen, thermolysis of 1,4-β bonds in cellulose and 1,6-α bonds in dextran is preceded by free radical oxidation of carbon 6 of glucopyranose unit with formation of carboxyl groups and water contributing to the subsequent induced degradation of polysaccharide. MgO which remains in polysaccharide after its impregnation by methoxy magnesium methyl carbonate (MMMC) promotes the reaction of terminal semiacetal groups and/or hydroperoxides which has considerably higher quantum yield of light emission.

Limit dextrinase from germinating barley has endotransglycosylase activity, which explains its activation by maltodextrins.

Limit dextrinase (EC 3.2.1.41) from germinating barley (Hordeum vulgare L) can be activated by millimolar concentrations of linear maltodextrins with a degree of polymerisation > or = 2. The activation was assay-dependent; it was detected using assays based on the solubilisation of cross-linked dyed pullulan but not in assays that directly measured cleavage events such as the formation of new reducing termini. This strongly suggested that maltodextrins did not increase the catalytic rate of limit dextrinase i.e. this is not a true activation. On the other hand, considerable activation was noted in assays that measured pullulan degradation by reduction in viscosity. Taken together, this suggested that maltodextrins altered the mode of action of limit dextrinase, causing more rapid decreases in viscosity or greater solubilisation of dye-linked pullulan fragments per cleavage event. The proposed mechanism of activation by alteration in action pattern was reminiscent of initial work in the discovery of xyloglucan endotransglycosylase. Therefore, the ability of limit dextrinase to catalyse transglycosylation reactions into pullulan was tested and confirmed by an assay based on the incorporation of a fluorescently labelled maltotriose derivative into higher-molecular-weight products. The transglycosylation reaction was dependent on limit dextrinase activity and was enhanced in more highly purified preparations of limit dextrinase. Transglycosylation was inhibited by unlabelled maltotriose. How transglycosylation accounts for the apparent activation of limit dextrinase by maltodextrins and the physiological relevance of this novel reaction are discussed.

A pullulan‐degrading enzyme activity of Aureobasidium pullulans

AbstractA pullulan‐degrading enzyme activity was detected in the culture medium after growth of the fungus Aureobasidium pullulans on corn syrup as a carbon source. The product of pullulan catabolism by this activity was determined to be glucose. The highest specific activity of this enzyme was found after 7 days of fungal growth. Although α‐amylase activity was not detectable, the presence of a glucoamylase activity was indicated. It appeared that the enzyme responsible for pullulan degradation was likely glucoamylase B.

Enzymatic Degradation of Pullulan and Pullulan Derivatives

Pullulan was modified using three different methods: a) chloroformate activation and subsequent reaction with 2-hydroxypropylamine, b) succinoylation and c) partial periodate oxidation and subsequent reduction of the aldehyde groups. Degradation of pullulan and pullulan derivatives by α-amylase, β-amylase and lysosomal enzymes was investigated. It was observed that the rate of degradation decreased with increasing degree of chemical modification of the parent polysaccharide. The stability of pullulan and dextran was tested in serum and liver homogenate.

Purification and properties of the cyclodextrinase of Bacillus sphaericus ATCC 7055

Bacillus sphaericus ATCC 7055 produced an intracellular cyclodextrinase (EC 3.2.1.54). It was purified by solublilising with Triton X-100, Q-Sepharose ion-exchange chromatography, phenyl-Sepharose CL-4B hydrophobic interaction chromatography and Superose-12 gel filtration and gave a single band on SDS-PAGE and preparative isoelectric focusing. The maxima for pH and temperature of the purified enzyme were pH 6.0–6.5 and 40°C. The enzyme had a relative molecular mass of 91 200–95 000 and an isoelectric point of 5.3. The amino-linked pseudotetrasaccharide, acarbose, inhibited activity. As well as cyclodextrins the enzyme was active on a broad range of substrates ranging in size from maltooligosaccharides (G3) to polysaccharides such as starch and pullulan, and branched cyclodextrins. End-product profiles of the cyclodextrinase on various substrates revealed that, upon hydrolysis of 1% (w/v) α-, β- and λ-cyclodextrin and maltoheptaose, glucose and maltose were the dominant end-products. Pullulan degradation resulted in panose (92%, w/v) as the main end-product, and glucose (27%, w/v) and maltose (37%, w/v) were the sole products formed from starch degradation.

Studies of the degradation of pullulan by the decycling maltodextrinase of Flavobacterium sp.

The mode of action on pullulan of the decycling maltodextrinase of Flavobacterium sp. was studied. The enzyme specifically split (1 → 4)-α- d-glucosidic linkages and, preferentially through cleavage of its tetraose units, degraded the α- d-glucan into larger fragments, which were further hydrolysed. About 39% of the carbohydrate was found to be the branched trisaccharide panose, established by 1H NMR spectroscopy and by the mp of the corresponding alditol dodecaacetate. At concentrations > 56 mM, panose exhibited product inhibition of further hydrolysis. Remarkable amounts of a dimer and a tetramer of panose, and of a branched tetrasaccharide, 6 3- O-α- d-glucosylmaltotriose, that arose from the tetraose units and the reducing ends of the pullulan molecules, were found among the larger hydrolysis products. The probable structures of some minor fragments were deduced from the debranching pattern obtained with pullulanase. The formation of glucose (6% of the carbohydrate) observed during pullulan degradation was most probably due to multiple transglycosylation reactions between the branched tetrasaccharide and a branched pentasaccharide, with subsequent splitting off of glucose from the transglycosylation products.

Characterization of the amylolytic system ofCandida strains

Some physical and chemical properties ofα-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) produced in semisolid fermentation byCandida fennica FTPT-1829,C. famata FTPT-1539 andC. fennica FTPT-8903 were determined. The optimum temperature values were 42, 44, 48 °C and 60, 50, 50 °C forα-amylase and glucoamylase excreted byC. fennica 8903,C. fennica 1829 andC. famata 1539, respectively. The optimum pH values for all strains were 5.0 and 6.0 forα-amylase and glucoamylase, respectively. The degradation of pullulan by all the yeast species indicates debranching activity.

Catalytic Properties of Bacillus megaterium Amylase

AbstractA new amylolytic enzyme from Bacillus megaterium is described, which is produced as a trace contaminant in a complex protein mixture, with β‐amylase as the major enzyme. By genetic engineering, the productivity could be increased by a factor 10000, and the enzyme is free of other amylases. Compared to pullulanase, which catalyses pullulan degradation to maltotriose, B. megaterium amylase (BMA) does not attack 1,6‐linkages and hydrolyses pullulan exclusively to panose. Furthermore, it hydrolyses crosslinked Phadebas starch, used for determination of α‐amylases and it also catalyses transfer reactions. Those are strongly enhanced in presence of suitable acceptors, which can be sugar molecules with the same configuration as dextrose in C2, C3, C4‐position. In the industrial dextrose process, about 1% of oligosaccharides remains from starch which is difficult to be hydrolysed, thus reducing yield and quality of the final product. The structure elucidation showed 35% 1,6‐linkages being present. These oligosaccharides can be degraded and eliminated by BMA through panosyl transfer to dextrose, under formation of 63‐α‐glucosylmaltotriose, which is degraded by glucoamylase to dextrose.

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

Starch-degrading enzymes in isolated pea (Pisum sativum L. cv. Laxton's Progress No. 9) chloroplasts were investigated and compared with those in crude pea leaf and stipule preparations. End-product analysis of amylopectin degradation by chloroplast and crude extracts indicates that maltose is the major product of both. Two multiforms of β-amylase (EC 3.2.1.2) were detected in pea chloroplasts using an electrophoretic transfer technique. A starch-debranching enzyme (EC 3.2.1.10) was detected in chloroplasts by electrophoretic transfer and the degradation of pullulan. A different multiform of debranching enzyme was found in crude preparations. α-Amylases (EC 3.2.1.1) were detected by electrophoretic transfer through gels containing starch and starch azure, and by change in viscosity of a starch solution, but were only found in crude preparations indicating an extrachloroplastic location. Incubation of maltotriose with chloroplast extracts gave high levels of glucose production and formation of oligosaccharides with degrees of polymerization larger than that of maltotriose indicating transglycosylase (EC 2.4.1.25) activity. Neither α-glucosidase (EC 3.2.1.20) nor maltose-phosphorylase (EC 2.4.1.1) activity were found in either chloroplast or crude preparations, whereas starch-phosphorylase (EC 2.4.1.1) activity was in both. The possible role of these enzymes in starch degradation by pea chloroplasts is discussed.

A morphological analogue of japanese lacquer. Grafting of p‐hydroxystyrene onto pullulan by ammonium persulfate initiation in dimethylsulfoxide

AbstractPullulan‐g‐poly(p‐acetoxystyrene) (I) was prepared using ammonium persulfate (APS) as an initiator in dimethylsulfoxide (DMSO) solution. I was deacetylated by hydrazine hydrate to yield pullulan‐g‐poly(p‐hydroxystyrene) (II). The degree of polymerization of the grafted poly(p‐hydroxystyrene) chain (III) was 15–20. Despite the effective grafting (%), this method caused the degradation of pullulan. The number of branch polymer chains per pullulan molecule were evaluated to be 0.9 to 2.5 from number average molecular weights of the permethylated II and III. The glass transition temperature of II was higher than that of III, indicating that the polar hydroxyl groups of pullulan in the graft polymer caused an increase in the rigidity of the grafted polymer chains. Pullulan‐g‐poly(o‐hydroxystyrene) and pullulan‐g‐poly(MMA) were also readily prepared by this method.