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The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.

ABSTRACT: The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.

SUBMITTER: Erra-Pujada M

PROVIDER: S-EPMC93789 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.

Erra-Pujada M M Debeire P P Duchiron F F O'Donohue M J MJ

Journal of bacteriology 19990501 10

The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a ...[more]

PMID: 10322035

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Project description: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 degrees 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, beta-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.

Project description:Pullulanase is an important debranching enzyme and has been widely utilized to hydrolyse the ?-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.In 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.The 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 ?-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 Co(2+) and Mn(2+), meanwhile Cu(2+) 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.This 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.

Project description:BACKGROUND:Plant P-type II Ca2+ATPases are formed by two distinct groups of proteins (ACAs and ECAs) that perform pumping of Ca2+ outside the cytoplasm during homeostasis, and play vital functions during development and stress management. In the present study, we have performed identification and characterisation of P-type II Ca 2+ ATPase gene family in an important crop plant Triticum aestivum. RESULTS:Herein, a total of 33 TaACA and 9 TaECA proteins were identified from the various chromosomes and sub-genomes of Triticum aestivum. Phylogenetic analysis revealed clustering of the homoeologous TaACA and TaECA proteins into 11 and 3 distinct groups that exhibited high sequence homology and comparable structural organization as well. Both TaACA and TaECA group proteins consisted of eight to ten transmembrane regions, and their respective domains and motifs. Prediction of sub-cellular localization was found variable for most of the proteins; moreover, it was consistent with the evolutionarily related proteins from rice and Arabidopsis in certain cases. The occurrence of assorted sets of cis-regulatory elements indicated their diverse functions. The differential expression of various TaACA and TaECA genes during developmental stages suggested their roles in growth and development. The modulated expression during heat, drought, salt and biotic stresses along with the occurrence of various stress specific cis-regulatory elements suggested their association with stress response. Interaction of these genes with numerous development and stress related genes indicated their decisive role in various biological processes and signaling. CONCLUSION:T. aestivum genome consisted of a maximum of 42 P-type II Ca 2+ ATPase genes, derived from each A, B and D sub-genome. These genes may play diverse functions during plant growth and development. They may also be involved in signalling during abiotic and biotic stresses. The present study provides a comprehensive insight into the role of P-type II Ca 2+ ATPase genes in T. aestivum. However, the specific function of each gene needs to be established, which could be utilized in future crop improvement programs.

Dataset Information

The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.

Publications

The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.

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