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PubMed1985

[Pullulanase].

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Enterobacter↗
PubMed1991

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1.

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.

Amylases↗
PubMed1988

Collagen-like sequences stabilize homotrimers of a bacterial hydrolase.

Pullulanase from Klebsiella pneumoniae strain FG9 has an unusual N-terminal amino acid sequence that includes six repeats of the tripeptide Gly-X-Pro. This type of sequence is characteristic of animal collagens and collagen-like proteins which form triple helical structures. We have investigated the molecular organization of this bacterial pullulanase isolated from the cell surface of Escherichia coli cells that carry the cloned FG9 pulA (pullulanase encoding) gene. Non-denaturing polyacrylamide gel analysis shows that pullulanase exists as higher order, apparently homogeneous, structures. We have used highly purified bacterial collagenase to probe the role of the collagen-like region and we demonstrate that this feature is essential for non-covalent association of pullulanase homotrimers. In addition we show collagenase-specific release of cell-bound pullulanase.

Amino Acid Sequence↗
PubMed1987

Formation of 6-O-alpha-maltosylcyclomalto-oligosaccharides by transfer action of three debranching enzymes.

O-Maltosylcyclomaltohexaoses (G2-cG6) were formed in yields of 24.3 and 23.2 mmol from 40 mmol of alpha-maltosyl fluoride (alpha-G2F) and 90 mmol of cyclomaltohexaose (cG6) by the transfer action of pullulanase from Aerobacter aerogenes (A-pullulanase) and isoamylase from Pseudomonas amyloderamosa, respectively. These yields were three times that given by pullulanase from Bacillus acidopullulyticus (B-pullulanase). The yields of O-maltosylcyclomalto-oligosaccharides were changed according to the origin of the enzymes and the kind of cyclomalto-oligosaccharide (cG6, cG7, or cG8) used as the acceptor. By the reaction with 40 mmol of alpha-G2F and 90 mmol of cG6, 20 mmol of alpha-G2F and 30 mmol of cG7, or 40 mmol of alpha-G2F and 90 mmol of cG8, the amounts of O-maltosylcyclomalto-oligosaccharides produced and the transfer ratios of alpha-G2F to the acceptors were as follows. By A-pullulanase, 24.3 mmol of G2-cG6 was produced in a 60.8% transfer ratio, whereas the yields of G2-cG7 and G2-cG8 were 1.7 mmol (8.5%) and 8.4 mmol (21.0%), respectively. The yields of G2-cG6, G2-cG7, and G2-cG8 by B-pullulanase were 8.8 mmol (22.0%), 1.2 mmol (6.0%), and 11.7 mmol (29.3%), respectively. In the case of isoamylase, G2-cG7 (9.2 mmol, 46.0%) and G2-cG8 (20.9 mmol, 52.3%) were produced, as much as for G2-cG6 (23.2 mmol, 58.0%). It was suggested that the difference in the amounts of G2-cG6 produced by these three debranching enzymes is based on the difference in the mode of action on the alpha-G2F used as the substrate, either a transfer action or a hydrolytic action.

Bacillus↗
PubMed1993

Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

The secretion of the Klebsiella oxytoca cell surface lipoprotein pullulanase involves translocation across the cytoplasmic and outer membranes of the Gram-negative bacterial cell envelope. A variant of pullulanase was created by fusing the signal peptide-encoding 5' region of the Escherichia coli gene for periplasmic MalE protein to the 3' end of the pulA gene encoding almost the entire mature part of pullulanase. When produced in E. coli carrying the malE-pulA gene fusion on a high copy number plasmid and the complete set of genes specifically required for pullulanase secretion on a second plasmid, the hybrid protein differed from wild-type pullulanase as follows: (i) it was not fatty-acylated; (ii) it was apparently processed by LepB signal peptidase rather than by LspA lipoprotein signal peptidase; (iii) it was released into the periplasm and was only slowly transported across the outer membrane, and (iv) it was released directly into the medium rather than via the usual surface-anchored intermediate. The hybrid protein was secreted more rapidly when malE-pulA was expressed from a low copy number plasmid. The two steps in the secretion pathway could be totally uncoupled by expressing first the malE-pulA gene fusion and then the cognate secretion genes. These results show that fatty-acylation of wild-type PulA is not essential for secretion but may improve its efficiency when large amounts of the protein are produced, that the two steps in secretion can occur quite independently and that the periplasmic intermediate can persist for long periods under certain circumstances.

ATP-Binding Cassette Transporters↗
PubMed1991

Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes.

An on-line assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of 1 mg l-1 together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.

Antigen-Antibody Complex↗
PubMed1992

Translocation of a folded protein across the outer membrane in Escherichia coli.

A mutation in the Escherichia coli dsbA gene (coding for a periplasmic disulfide oxidoreductase) reduces the rate of disulfide bond formation in the enzyme pullulanase and also reduces the rate at which the enzyme is secreted to the cell surface, as measured by protease accessibility. The enzyme did not become protease accessible when disulfide bond formation was completely prevented in the mutant strain by carboxymethylation. These results indicate that a disulfide bond may be required for, and certainly does not impede, the translocation of pullulanase across the outer membrane. Since it is unlikely that a disulfide bond could be formed and then reduced again in the periplasm, these results would appear to strengthen the argument that pullulanase polypeptides fold into or close to their final conformation before they are transported across the outer membrane. It is suggested that this might be a feature common to all proteins that are secreted by other Gram-negative bacteria by a pullulanase-like pathway.

Bacterial Proteins↗
PubMed2000

Pilus formation and protein secretion by the same machinery in Escherichia coli.

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram-negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus-like bundles. Assembly of the 'secreton pilus' required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane-associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K-12 gene ppdD, led to secreton-dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus-like structures.

Bacterial Proteins↗
PubMed1993

Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca.

The signal sequence of the Klebsiella oxytoca puIG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the puIO gene product processes prePuIG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PuIG signal sequence. Like most type IV pilins, processed PuIG was found to have a methylated amino-terminal phenylalanine residue. Site-directed mutagenesis was used to replace amino acids in prePuIG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePuIG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11(+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently N-methylated. Cleavage of prePuIG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the carboxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePuIG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.

Amino Acid Sequence↗
PubMed1987

Thermostable amylolytic enzymes from a new clostridium isolate.

A new Clostridium strain was isolated on starch at 60 degrees C. Starch, pullulan, maltotriose, and maltose induced the synthesis of alpha-amylase and pullulanase, while glucose, ribose, fructose, and lactose did not. The formation of the amylolytic enzymes was dependent on growth and occurred predominantly in the exponential phase. The enzymes were largely cell bound during growth of the organism with 0.5% starch, but an increase of the starch concentration in the growth medium was accompanied by the excretion of alpha-amylase and pullulanase into the culture broth; but also by a decrease of total activity. alpha-Amylase, pullulanase, and alpha-glucosidase were active in a broad temperature range (40 to 85 degrees C) and displayed temperature optima for activity at 60 to 70 degrees C. During incubation with starch under aerobic conditions at 75 degrees C for 2 h, the activity of both enzymes decreased to only 90 or 80%. The apparent K(m) values of alpha-amylase, pullulanase, and alpha-glucosidase for their corresponding substrates, starch, pullulan, and maltose were 0.35 mg/ml, 0.63 mg/ml, and 25 mM, respectively.

Journal Article↗
PubMed1990

Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A.

A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60 degrees C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75 degrees C and pH 5.0, respectively. Both the enzyme activities were stable up to 70 degrees C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70 degrees C and 45 min at 75 degrees C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by beta- and gamma-cyclodextrins but not by alpha-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in alpha1,6 linkages and produced multiple saccharides from cleavage of alpha-1,4 linkages in starch. The K(m)s for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.

Journal Article↗