Direct Detection of Pullulanase Activity in Electrophoretic Polyacrylamide Gels

Furegon, Luca; Curioni, Andrea; Peruffo, A. Dal Belin

doi:10.1006/abio.1994.1397

Cited by 15 publications

(9 citation statements)

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“…After electrophoresis the gels were rinsed in water or in 0.25% (vol/vol) Triton X-100 at 4°C for 1 h to remove the SDS and incubated for 10 to 30 min under optimal assay conditions to detect the pullulanase activity. Zymogram staining for pullulytic activity was performed as described by Furegon et al (16). Native PAGE was performed with 4 to 20% polyacrylamide gradient gels purchased from Novex (MBI Fermentas, St. Leon Rot, Germany).…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…Pullulanase activity was determined spectrophotometrically at 37°C by measuring dye released after 30 min at 534 nm by using red pullulan (Megazyme, Bray, Ireland) as substrate in 50 mM citrate-phosphate buffer (pH 5.2) (24). Pullulanase also was assayed on native activity gels of 7.5% acrylamide, 1.5 mm thickness, containing 1% red pullulan (25). Pullulanase inhibitor activity was determined on fractions heated to inactivate pullulanase (70°C for 15 min) by measuring the inhibition of the fractions on added purified barley malt pullulanase.…”

Section: Preparation Of Endosperm Extracts From Imbibed Grainmentioning

confidence: 99%

Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain

Cho

Wong

Marx

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

122

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Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endospermspecific B1-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B1-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves ␣-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.

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“…The enzyme activity (zymography) of ApuASK was evaluated using a 12% ( w / v ) native-PAGE analysis. Zymography to determine the pullulytic activity was conducted according to Furegon et al [45], except that the gel was immersed in 100 mM potassium phosphate buffer (pH 7.5) and then incubated at 60 °C for 24 h. Zymography to determine the amylolytic activity was conducted as described by Yang et al [46], except that the starch solution was prepared in 100 mM potassium phosphate buffer (pH 7.5) and the incubation temperature was 60 °C.…”

Section: Methodsmentioning

confidence: 99%

A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization

Kahar

Chan

Salleh

et al. 2013

IJMS

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An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.

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Direct Detection of Pullulanase Activity in Electrophoretic Polyacrylamide Gels

Cited by 15 publications

References 0 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

Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain

A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization

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