Purification and properties of a thermostable pullulanase from Clostridium thermosulfurogenes EM1 which hydrolyses both ?-1,6 and ?-1,4-glycosidic linkages

Spreinat, Andreas; Antranikian, Garabed

doi:10.1007/bf00172543

Cited by 72 publications

(39 citation statements)

References 32 publications

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“…Most enzymes from thermophilic and hyperthermophilic microorganisms are type II pullulanases (35,42,50,52) and have been isolated mainly from thermophilic archaea (11,14,17,32,40,49). Thermostable type I pullulanases have been characterized from the aerobic moderately thermophilic bacteria Bacillus acidopullulolyticus (25,29,36) and Bacillus flavocaldarius KP 1228 (53), the thermophilic bacteria Thermus aquaticus YT-1 (47), Thermus caldophilus GK-24 (31), and Bacillus thermoleovorans (43), and the extreme anaerobic thermophilic bacteria Caldocellulosiruptor saccharolyticus (1), Fervidobacterium pennivorans (8,12,33), and Thermotoga maritima (9, 34).…”

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confidence: 99%

Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

Bertoldo

Armbrecht

Becker

et al. 2004

Appl Environ Microbiol

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The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg). The N-terminal sequence of purified rPulAg was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, ␤-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono-or bivalent cations did not have a stimulating effect. In addition, rPulAg was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.Pullulanases (pullulan 6-glucanohydrolase; EC 3.2.1.41) are debranching enzymes that are able to hydrolyze the ␣-1,6 bonds of the linear ␣-glucan pullulan, producing maltotriose as the final product. Pullulanases are divided into two classes based on substrate specificity: (i) type I pullulanases or debranching enzymes, which specifically hydrolyze the ␣-1,6 linkages in branched oligosaccharides, such as starch, amylopectin, and glycogen, forming linear ␣-1,4-linked oligomers; and (ii) type II pullulanases or amylopullulanases, which cleave ␣-1,6 glycosidic linkages in pullulan and branched substrates in addition to the ␣-1,4 glycosidic linkages in polysaccharides other than pullulan (7).A number of pullulanases have been purified from different bacterial and archaeal sources and characterized. Most enzymes from thermophilic and hyperthermophilic microorganisms are type II pullulanases (35,42,50,52) and have been isolated mainly from ...

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confidence: 99%

Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

Bertoldo

Armbrecht

Becker

et al. 2004

Appl Environ Microbiol

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“…Hydrolysis products arising after the action of pullulanase on various linear and branched polysaccharides were analyzed by high-performance liquid chromatography (HPLC) using an Aminex HPX-42A column (300 by 78 mm; Bio-Rad, Hercules, Calif.). Double-distilled water was used as the mobile phase at a flow rate of 0.3 ml/min (27,42). The purified pullulanase was incubated at different temperatures with 0.2% (wt/vol) pullulan, starch, glycogen, amylopectin, maltodextrin, panose, or amylose.…”

Section: Methodsmentioning

confidence: 99%

A New Thermoactive Pullulanase from Desulfurococcus mucosus : Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in Bacillus subtilis

et al. 2000

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The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed ␣-1,6 glycosidic linkages of pullulan, producing maltotriose, and also ␣-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.Pullulanases (pullulan-6-glucanohydrolase [EC 3.2.1.41]) are classified as type I or type II (amylopullulanase) depending on their ability to degrade ␣-1,4 glycosidic linkages in starch, amylopectin, and related oligosaccharides. Unlike type II, pullulanase type I is unable to attack ␣-1,4 glycosidic linkages. Both pullulanase type I and type II attack ␣-1,6 glycosidic linkages in pullulan, producing maltotriose. All pullulanases known to date are unable to degrade cyclodextrins. In contrast, pullulan hydrolase type I (neopullulanase) and type II (isopullulanase) are able to cleave ␣-1,4 glycosidic linkages in pullulan, releasing panose and isopanose, respectively, and are highly active on cyclodextrins (32). Enzymes that degrade cyclodextrins faster than starch are designated cyclodextrinases (EC 3.2.1.54; cyclomaltodextrinase) (30).In recent years, a large number of pullulanases have been isolated, particularly from thermophilic microorganisms (32). The research on pullulanases from thermophiles is interesting not only for understanding enzyme stability but also for discovering improved enzymes for application in the industrial starch hydrolysis process. The first step in the conversion of starch to glucose is liquefaction, which runs at temperatures of 95 to 105°C in the presence of a thermostable ␣-amylase. Due to the absence of thermoactive enzymes, saccharification follows at a temperature...

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“…A number of pullulanase with dual specificities have been investigated, including pullulanase from Bacillus subtilis [5] , Thermoanaerobium brockii [6] , Clostridium thermohydrosulfuricum [7,8] , Bacillus circulans [9] , Bacillus sp. [10] , Clostridium thermosulfurogenes [11] , Thermus aquaticus [12] , Thermoanaerobacterium saccharolyticum [13] , Pyrococcus furiosus and Thermococcus litoralis [14] , an alkalophilic Bacillus sp. [15] and Bacillus sp.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Pullulanase Type II from Bacillus cereus H1.5

Ling¹,

Ling²,

Mohamad³

et al. 2009

American J. of Biochemistry and Biotechnology

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Problem statement: Pullulanase is one of the important enzymes in starch industry. Search for the pullulanase with distinct features, possibly from easily grown bacterium, is of interest for industrial applications Approach: The extracellular pullulanase produced by Bacillus cereus HI.5 was purified by chromatographic method of DEAE-Sepharose, followed by Superdex gel filtration. The enzyme was characterized in terms of the optimal pH and temperature for activity as well as substrate specificity. Results: The enzyme showed optimal activity at 55°C and pH 6.0. The thermostability and the thermoactivity of the enzyme were increased considerably in the presence of Ca 2+. In the present of 2 mM Ca 2+ , the enzyme had half-life duration of more than 2 h at 50°C. Almost all metal ions had a strong inhibitory effect, except Ca 2+ and Mn 2+. The Ca 2+ had a very strong stimulating effect on the enzyme, increasing its activity by 170%. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, where as N-bromosuccinimide and Schardinger dextrins were inhibitors, suggesting that tryptophan and thiol residues may be important for the activity. The apparent K m and V max value for pullulan was 1.1 mg mL −1 and 0.275 µmol min −1 , respectively. A relative substrate specificity for hydrolysis of pullulan, amylopectin and soluble starch by this pullulanase was 100%, 28.5% and 20.4%, respectively. Conclusion: The enzyme was able to attack specifically the α-1,6 linkages in pullulan to generate maltotriose as the major end product, as well as the α-1,4 linkages in amylopectin and soluble starch leading to the formation of a mixture of maltose and glucose and therefore be classified as a type II pullulanase or an amylopullulanase.

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Purification and properties of a thermostable pullulanase from Clostridium thermosulfurogenes EM1 which hydrolyses both ?-1,6 and ?-1,4-glycosidic linkages

Cited by 72 publications

References 32 publications

Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

A New Thermoactive Pullulanase from Desulfurococcus mucosus : Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in Bacillus subtilis

Characterization of Pullulanase Type II from Bacillus cereus H1.5

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