Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24

Kim, C

doi:10.1016/0378-1097(96)00089-4

Cited by 8 publications

(8 citation statements)

References 4 publications

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“…, 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). Sequence information has revealed that there is low overall conservation among type I enzymes; however, a highly conserved region consisting of seven amino acids, YNWGYDP, is found in all type I pullulanases that have been described to date (7,8).…”

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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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“…Moderate thermophilic gram-positive bacteria such as Bacillus flavocaldarius [51], Bacillus thermoleovorans [30], Clostridium sp. [72], and Thermos caldophilus [27] also have ability to secrete pullulanase type I while pullulanase type I from hyperthermophilic bacterium, Fervidobacterium pennavorans, has also been reported [28]. …”

Section: Starch-debranching Enzymesmentioning

confidence: 99%

Pullulanase: Role in Starch Hydrolysis and Potential Industrial Applications

et al. 2012

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The use of pullulanase (EC 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially those aimed for glucose production. Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. The industrial manufacturing of glucose involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action of α-amylase; saccharification, which results in further transformation of maltodextrins into glucose. During saccharification process, pullulanase has been used to increase the final glucose concentration with reduced amount of glucoamylase. Therefore, the reversion reaction that involves resynthesis of saccharides from glucose molecules is prevented. To date, five groups of pullulanase enzymes have been reported, that is, (i) pullulanase type I, (ii) amylopullulanase, (iii) neopullulanase, (iv) isopullulanase, and (v) pullulan hydrolase type III. The current paper extensively reviews each category of pullulanase, properties of pullulanase, merits of applying pullulanase during starch bioprocessing, current genetic engineering works related to pullulanase genes, and possible industrial applications of pullulanase.

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“…Conversely, different temperatures like 50°C, 75°C, 90°C for the hydrolysis of pullulan with pullulanase were reported by various researchers 9,10,11,12 . The variation in the optimum temperature for the maltotriose production by pullulanase enzyme may be due to differences in the source of the enzymes 13,14 .…”

Section: Effect Of Temperature On Hydrolysismentioning

confidence: 99%

Preparation of Maltotriose Syrup from Microbial Pullulan by using Pullulanase Enzyme

Mishra¹,

Manikanta²,

Zamare³

2016

Biosci., Biotech. Res. Asia

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The present work deals with the preparation and single point optimization of maltotriose syrup prepared from microbial pullulan. The enzyme pullulanase was used in this process for hydrolysis of pullulan. Production of pullulan was carried out in a shake flask fermenter with isopropanol precipitation. Various parameters like time, temperature, pH, amount of ethanol and effect of amount of enzyme on the hydrolysis of pullulan were optimized by single point optimization studies. The optimized values for the process parameters were 8h, 46°C, 5.5, 6 (v/v) and 10 ASPU/g for time of hydrolysis, temperature, pH , amount of ethanol and amount of pullulanase enzyme respectively. The results obtained from HPLC, confirmed that major component present in the hydrolysate were composed of maltotriose. Results of experiments indicated that this was a promising way of preparation of maltotriose.

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Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24

Cited by 8 publications

References 4 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

Pullulanase: Role in Starch Hydrolysis and Potential Industrial Applications

Preparation of Maltotriose Syrup from Microbial Pullulan by using Pullulanase Enzyme

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