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PubMed1987

Production of Thermostable alpha-Amylase, Pullulanase, and alpha-Glucosidase in Continuous Culture by a New Clostridium Isolate.

The production of alpha-amylase, pullulanase, and alpha-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60 degrees C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of alpha-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.

Journal Article↗
PubMed1988

Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis.

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.

Journal Article↗
PubMed1995

Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.

The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.

Archaea↗
PubMed2000

Antigenicity, expression, and molecular characterization of surface-located pullulanase of Streptococcus pneumoniae.

A putative pullulanase-encoding gene from Streptococcus pneumoniae was identified by screening a genomic expression library with human convalescent-phase serum. The 3,864-bp gene encoded a 143-kDa protein. Surface location and pullulanase activity of the protein, designated SpuA, was demonstrated. SpuA was present in all investigated pneumococcal isolates of different serotypes. The spuA 5' end was highly conserved among clinical isolates except for a 75-bp region. The properties of SpuA reported here indicate that this novel immunogenic surface protein might have potential as a vaccine target.

Amino Acid Sequence↗
PubMed1985

Structure of two divergent promoters located in front of the gene encoding pullulanase in Klebsiella pneumoniae and positively regulated by the malT product.

Pullulanase is an extracellular starch-debranching enzyme produced by Klebsiella pneumoniae. When its structural gene, pulA, is introduced into Escherichia coli, it is controlled by malT, the positive regulator gene of the maltose regulon. Characterization of the region 5' to pulA and of the beginning of the gene described herein demonstrate that (i) pullulanase is probably a lipoprotein; (ii) an additional malT-controlled promoter (the malX promoter) lies adjacent to the pulA promoter and is oriented in the opposite direction; (iii) in common with the three previously described malT-controlled promoters, the pulA and malX promoters have a conserved hexanucleotide (consensus sequence, 5'-GGATGGA) 35 base pairs upstream from the transcription initiation site; and (iv) upstream from this conserved hexanucleotide the pulA and malX promoters differ from the other mal promoters in that they lack any detectable binding site for the cyclic AMP-binding protein.

Amino Acid Sequence↗
PubMed1988

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.

Bacillus subtilis↗
PubMed1999

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

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.

Amino Acid Sequence↗
PubMed2000

Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE.

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.

Bacterial Proteins↗
PubMed1992

Effect of substitution at C-6 on the susceptibility of pullulan to pullulanases. Enzymatic degradation of modified pullulans.

Pullulan, with all of the primary hydroxyl groups modified, is an excellent substrate for defining the effect of degree of substitution on biodegradability because of the uniform distribution of substituents on the polysaccharide. 6-Chloro-6-deoxypullulan and 3,6-anhydropullulan are highly resistant to hydrolysis by the four different types of pullulanase. 6-Azido-6-deoxypullulan is resistant to three types but susceptible to hydrolysis by the fourth, isopullulanase. Neopullulanase is strongly inhibited by 6-chloro-6-deoxypullulan and 6-azido-6-deoxypullulan, the other pullulanases much less so.

Biodegradation, Environmental↗
PubMed1993

Purification and biochemical properties of an alkaline pullulanase from alkalophilic Bacillus sp. S-1.

A novel extracellular pullulanase (PUL-E, pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified from the alkalophilic Bacillus sp. S-1. The purified enzyme had a molecular mass of about 140 kDa on denaturated and natural conditions. The pI was 5.5. The pullulanase, when resolved by SDS-PAGE, was negative for Schiff staining, suggesting that the enzyme is not a glycoprotein. The N-terminal amino acid sequence of the enzyme was Phe-Leu-Asn-Met-Ser-(Trp-Phe). The enzyme displayed a temperature optimum of around 60 degrees C and a pH optimum of around pH 9.0. The enzyme was stable to incubation from pH 4.0 to pH 11.0 at 4 degrees C for 24 h. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ions. Ca2+ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the alpha-1,6-linkages of amylopectin, glycogens, alpha,beta-limited dextrin, and pullulan. The enzyme had an apparent Km of 7.92 mg/ml for pullulan, a Km of 1.63 mg/ml for amylopectin, and a Km of 3.1 mg/ml for alpha,beta-limited dextrin, when measured at pH 9.0 and 50 degrees C. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was not inhibited by alpha, beta, or gamma-cyclodextrins. The western blotting analysis with mouse anti-serum against PUL-E showed that PUL-E is produced as a single enzyme form during bacterial cultivation.

Amino Acid Sequence↗
PubMed2001

Cloning and nucleotide sequence of the pullulanase gene of Thermus thermophilus HB8 and production of the enzyme in Escherichia coli.

A 3.4-kb SphI fragment carrying the pullulanase gene of Thermus thermophilus HB8 was cloned. Based on the nucleotide sequence of it and the flanking region analyzed by direct sequencing of the inverse PCR product, an expression vector was constructed. The E. coli cells harboring the plasmid produced an about 80-kDa protein having pullulanase activity, the optimum temperature of which was 70 degrees C.

Amino Acid Sequence↗
PubMed1989

Protein secretion by gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12.

Pullulanase secretion in Escherichia coli depends on the expression of a MalT-regulated operon called pulC. Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins are required for pullulanase secretion. The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane. A representative PulC-PhoA hybrid protein fractionated mainly with the inner membrane upon isopycnic sucrose gradient centrifugation of membrane vesicles. This, together with sequencing data, suggests that PulC is an inner membrane protein. Antibodies raised against a purified PulD-PhoA hybrid protein were used to show that PulD was enriched in low density outer membrane vesicles.

Alkaline Phosphatase↗
PubMed1999

Fermentation of starch by Klebsiella oxytoca p2, containing plasmids with alpha-amylase and pullulanase genes.

Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 degrees C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12-24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower alpha-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.

Enzyme Stability↗
PubMed1997

Intracellular and extracellular forms of alkaline pullulanase from an alkaliphilic Bacillus sp. S-1.

Bacillus sp.S-1 alkaline pullulanase (AP) exists in two forms: a precursor form (PUL-Ia, M(r) 180,000) and a processed form (PUL-Ib, M(r) 140,000). PUL-Ia was accumulated intracellularly in large amounts, and PUL-Ib was detected in both the membrane fraction and the fraction trapped between the cytoplasmic membrane and the cell wall. Two forms of AP were purified to homogeneity and their properties were compared with previously purified PUL-E (140 kDa). PUL-Ib showed similar properties, such as the M(r) value, the pI value (5.7), specific activity, substrate specificity, the NH2-terminal amino acid sequence (Phe-Leu-Asn-Met-Ser), and biophysical characters. However, in the case of PUL-Ia, even though the patterns of optimum pH and temperature, substrate specificity, and enzyme inhibition and activation were similar to those for PUL-Ib and PUL-E, the M(r) value and the pI value (5.97) were different. Furthermore, the NH2-terminal amino acid sequence of PUL-Ia was completely blocked, and the stabilities over pH and temperature ranges were decreased. The catalytic activities of PUL-Ia were distinguishable in the Km and Vmax values for various substrates and in the specific activity (71.4 U/mg) for pullulan hydrolysis. PUL-Ib and PUL-E showed 10-fold higher specific activities (744.6 for PUL-E and 736 for PUL-Ib) than PUL-Ia. However, PUL-Ia was immunologically identical to PUL-E and PUL-Ib. Therefore, it was concluded that PUL-Ib and PUL-E are the same form of the enzyme, suggesting that PUL-Ia is initially synthesized and proteolytically processed to the mature form of PUL-E. On the other hand, the translocation of AP required processing of the AP protein and the processing facilitated enzymatic activation and stabilization through a complete conformational change, resulting in an increase in affinity for substrates of PUL-E.

Azides↗
PubMed1990

Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable alpha-amylases and pullulanases.

For the production of cell-free thermostable alpha-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full as well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60 degrees C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10(12) cells up to 700 U/10(12) cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450,000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes.

Alginates↗
PubMed1997

Pullulanase: model protein substrate for the general secretory pathway of gram-negative bacteria.

Pullulanase of Klebsiella oxytoca is one of a wide variety of extracellular proteins that are secreted by Gram-negative bacteria by the complex main terminal branch (MTB) of the general secretory pathway. The roles of some of the 14 components of the MTB are now becoming clear. In this review it is proposed that most of these proteins form a complex, the secretion, that spans the cell envelope to control the opening and closing of channel in the outer membrane. Progress toward the goal of testing this model is reviewed.

Bacterial Outer Membrane Proteins↗
PubMed1975

Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism.

Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.

Amylopectin↗
PubMed1995

Molecular mimicry and ankylosing spondylitis: possible role of a novel sequence in pullulanase of Klebsiella pneumoniae.

Molecular mimicry has been shown between two sequences of Klebsiella pneumoniae pulD secretion protein (DRDE) with HLA-B27 (DRED) and pulA (pullulanase) enzyme (Gly-X-Pro) with types I, III and IV collagen respectively. IgG antibody levels in AS patients were elevated against 16mer synthetic peptides of HLA-B27 and pulD by enzyme immunosorbent assay (ELISA) compared to controls (P < 0.001). ELISA assays against K. pneumoniae grown in the absence and presence of pullulan demonstrated significant levels of IgA antibody in AS patients compared to controls (P < 0.001). Increased IgA and IgG antibody levels to pulA and types I and IV collagen were observed in AS patients compared to controls (P < 0.001). These observations could be relevant in the sequence of molecular events in AS.

Amino Acid Sequence↗