Thermostable pullulanase type I from new isolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli

Messaoud, Ezzedine Ben; Ammar, Youssef Ben; Mellouli, Lotfi; Béjar, Samír

doi:10.1016/s0141-0229(02)00185-0

Cited by 57 publications

(36 citation statements)

References 20 publications

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“…The size of the authentic A. gottschalkii pullulanase and the full-length recombinant form (rPulAg) (about 96 kDa) is common among the type I pullulanases, Also, the monomeric quaternary structure resembles that of the other debranching enzymes described so far (1,9,13,25,34,36,43,47,53) with the exception of F. pennivorans pullulanase, which has a dimeric structure (8). The temperature and pH profiles of rPulAg and its remarkable resistance to thermal inactivation at temperatures up to 70°C (half-life at this temperature, 22 h) are in agreement with the optimal growth conditions of A. gottschalkii (48).…”

Section: Discussionmentioning

confidence: 99%

“…, 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%

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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: Discussionmentioning

confidence: 99%

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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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“…A large number of microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase (10,11). Although numerous pullulanases have been identified and heterologously expressed (12)(13)(14), reports have shown that the secretion ratio of recombinant pullulanase is low.…”

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confidence: 99%

Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Duan

2015

Appl Environ Microbiol

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Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of ␣-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072-4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8-and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 ؋ 10 8 and 449.3 ؋ 10 8 U · mol ؊1, respectively, which are 0.94-and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7-and 1.3-fold that of the DM enzyme. Kinetic studies showed that the K m values of DM-T1 and DM-T2 were 1.5-and 2.7-fold higher and the K cat /K m values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced D-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. P ullulanase (EC 3.2.1.41), which catalyzes the hydrolysis of ␣-1,6-glucosidic linkages in pullulan, amylopectin, and the dextrins of amylopectin, is a well-known starch-debranching enzyme (1). It belongs to the ␣-amylase family, which is identified as glycoside hydrolase family 13 in the CAZy database. When combined with ␣-amylase, ␤-amylase, glucoamylase, or cyclodextrin glucosyltransferase, pullulanase can be used in the production of glucose, fructose, maltose, cyclodextrins, and amylose (2, 3). The addition of pullulanase allows the reaction time to be reduced, the substrate concentration to be increased, and the conversion rate to be improved (4, 5). Recently, pullulanase has been used as a biocatalyst for the production of fuel ethanol, low-calorie beers, resistant starch, maltotriose, and other important products (6, 7). With this plethora of potential industrial applications, pullulanase production has attracted a great deal of attention in recent years (8,9).A large number of microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase (10, 11). Although numerous pullulanases have been identified and heterologously expressed (12)(13)(14), reports have shown that the secretion ratio of recombinant pullulanase is low. Low extracellular prod...

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“…Maximum DE of the hydrolysate was obtained at 46°C. 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: 97%

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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Thermostable pullulanase type I from new isolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli

Cited by 57 publications

References 20 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

Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Preparation of Maltotriose Syrup from Microbial Pullulan by using Pullulanase Enzyme

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