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At least 19 recordsLinked to original sources
PubMed1992

Role of lipid modification on a starch-debranching enzyme, Klebsiella pullulanase: comparison of properties of lipid-modified and unmodified pullulanases.

Klebsiella pullulanase is a lipoprotein synthesized as a precursor with a signal peptide, which is processed by lipoprotein signal peptidase. To clarify the role of lipid modification of pullulanase, we purified lipid-modified wild-type and the unmodified (mutant) pullulanases and compared their properties. The Km and Vmax values of both pullulanases for pullulan were the same. The optimal pH and temperature, the stabilities over pH and temperature ranges, the specificity of substrates, and the patterns of inhibition of the lipid-modified and unmodified pullulanases were also the same. However, we found that the wild-type pullulanase formed trimers whereas the unmodified enzyme did not, and that the migrations of the two enzymes on sodium dodecyl sulphate/electrophoresis were different when the samples were applied on the gel without heating. The results presented in this paper and in previous work show that the correct processing and translocation of pullulanase in K. aerogenes require modification of lipid. However, the enzymatic properties and physical stabilities of pullulanase were not affected by the lipid modification.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes.

The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.

Cell Membrane↗
PubMed1989

Cell-associated pullulanase from Bacteroides thetaiotaomicron: cloning, characterization, and insertional mutagenesis to determine role in pullulan utilization.

We have cloned a pullulanase gene from Bacteroides thetaiotaomicron. The pullulanase expressed from this clone in Escherichia coli was cell associated and soluble and had a molecular mass of 72 kilodaltons by gel filtration. Maxicell analysis of proteins coded by the cloned insert showed that a 71.6- to 73.2-kilodalton doublet was associated with pullulanase activity. Thus, the pullulanase is probably a monomer. The cloned pullulanase produced maltotriose as an end product of pullulan digestion. In B. thetaiotaomicron the pullulanase activity was cell associated. Approximately 80% of the activity was soluble, and 16 to 18% was membrane associated. The molecular mass of the soluble pullulanase was 77 kilodaltons by gel filtration. To determine whether the cloned pullulanase gene was essential for pullulan utilization, we used directed insertional mutagenesis to inactivate the B. thetaiotaomicron pullulanase gene. The pullulanase specific activity of the mutant was approximately 45% of that of wild-type B. thetaiotaomicron. However, the pullulanase-negative insertional mutant 95-1 was still able to grow on pullulan at a rate similar to that of wild-type B. thetaiotaomicron. Thus, there must be a second pullulanase in B. thetaiotaomicron.

Bacteroides↗
PubMed1989

Biosynthesis and secretion of pullulanase, a lipoprotein from Klebsiella aerogenes.

We constructed, by site-directed mutagenesis, a mutant pullulanase gene in which the cysteine residue in a pentapeptide sequence, Leu16-Leu-Ser-Gly-Cys20 within the NH2-terminal region of pullulanase from Klebsiella aerogenes, is replaced by serine (Ser20). The modification, processing, and subcellular localization of the mutant pullulanase were studied. Labeling studies with [3H]palmitate and immunoprecipitation with mouse antiserum raised against pullulanase showed that the wild form of both the extracellular and intracellular pullulanases contained lipids, whereas the mutant enzyme was not modified with lipids. Only the Cys20 was modified with glyceryl lipids. The bulk of the mutant pullulanase was located in the periplasm, but a portion of the unmodified, mutant pullulanase was secreted into the medium. Mutant pullulanases from the extracellular and the periplasm were purified and their NH2-terminal sequences were determined. Both the mutant pullulanases were cleaved between residues of Ser13 and Leu14 which is 6-amino acid residues upstream of the lipid modified pullulanase cleavage site. This new cleavage was resistant to globomycin, an inhibitor of the prolipoprotein signal peptidase of Escherichia coli. These results indicate that the pentapeptide sequence plays an important role in maturation and translocation of pullulanase in K. aerogenes. However, the modification of pullulanase with lipids seems to be not essential for export of the enzyme across the outer membrane.

Amino Acid Sequence↗
PubMed1990

Analysis of the subcellular location of pullulanase produced by Escherichia coli carrying the pulA gene from Klebsiella pneumoniae strain UNF5023.

Three different techniques, protease accessibility, cell fractionation and in situ immunocytochemistry, were used to study the location of the lipoprotein pullulanase produced by Escherichia coli K12 carrying the cloned pullulanase structural gene (pulA) from Klebsiella pneumoniae, with or without the K. pneumoniae genes required to transport pullulanase to the cell surface (secretion-competent and secretion-incompetent, respectively). Pullulanase produced by secretion-competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked the N-terminal fatty-acylated cysteine residue (and probably also a short N-terminal segment of the pullulanase polypeptide), confirming that the N-terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion-incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electronmicroscopy of vesicles labelled with antipullulanase serum and protein A-gold confirmed that pullulanase was associated with both types of vesicles. When thin-sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion-competent cells and mainly in the proximity of the inner membrane in the secretion-incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility and in situ immunocytochemistry) show that pullulanase is transported to the cell surface of secretion-competent cells, this could not be confirmed by cell-fractionation techniques. Possible explanations for this discrepancy are discussed.

Bacterial Proteins↗
PubMed1987

Export and secretion of the lipoprotein pullulanase by Klebsiella pneumoniae.

Pullulanase, a secreted lipoprotein of Klebsiella pneumoniae, is initially localized to the outer face of the outer membrane, as shown by protease and substrate accessibility and by immunofluorescence tests. Freeze-thaw disruption of these cells released both membrane-associated and apparently soluble forms of pullulanase. Membrane-associated pullulanase co-fractionated with authentic outer membrane vesicles upon isopycnic sucrose-gradient centrifugation, whereas the quasi-soluble form had the same equilibrium density as inner membrane vesicles and extracellular pullulanase aggregates. The latter also contained outer membrane maltoporin, but were largely devoid of other membrane components including LPS and lipids. K. pneumoniae carrying multiple copies of the pullulanase structural gene (pulA) produced increased amounts of cell-associated and secreted pullulanase, but a large proportion of the enzyme was neither exposed on the cell surface nor released into the medium, even after prolonged incubation. This suggests that factors necessary for pullulanase secretion were saturated by the over-produced pullulanase. When pulA was expressed under lacZ promotor control, the pullulanase which was produced was not exposed on the cell surface at any time, suggesting that pullulanase secretion genes are not expressed constitutively, and raising the possibility that they, like pulA, may be part of the maltose regulon.

Cell Membrane↗
PubMed1988

Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum mutant Z 21-109 produced intracellular thermostable pullulanase and glucoamylase activities. The glucoamylase activity was inactivated by treating C. thermohydrosulfuricum cells with 10% (v/v) propan-1-ol at 85 degrees C in the presence of 5 mM-CaCl2. Pullulanase activity was selectively solubilized from cells by treatment with detergent and lipase. The solubilized pullulanase was purified by treatment with streptomycin sulphate and (NH4)2SO4 and by DEAE-Sephacel, octyl-Sepharose and pullulan-Sepharose chromatography. Pullulanase was purified 3511-fold and displayed homogeneity on SDS/polyacrylamide-gel electrophoresis. The pullulanase was a monomeric glycoprotein with an apparent Mr of about 136,500, and it displayed a pI of 5.9. The enzyme was enriched in both acidic and hydrophobic amino acids. The purified pullulanase was stable and optimally active at 90 degrees C. The optimum pH for activity and pH-stability ranges were 5.0-5.5 and 3.0-5.0 respectively. The enzyme was inhibited by cyclodextrins, EDTA and N-bromosuccinimide, but not by p-chloromercuribenzoate and acarbose. The pullulanase displayed a relative substrate specificity for hydrolysis of pullulan (100%) versus 75% for glycogen and 50% for soluble starch. The apparent Km, Vmax. and Kcat. values for enzyme activity on pullulan at 60 degrees C were 0.675 mg/ml, 122.5 mumol of reducing sugar formed/min per mg of protein and 16,240 min-1 respectively. The novel properties of this extremely thermostable pullulanase are discussed in relation to other purified starch-debranching enzymes.

Amino Acids↗
PubMed1990

The normally periplasmic enzyme beta-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surface enzyme pullulanase.

Hybrid proteins were constructed in which C-terminal regions of the bacterial cell surface and extracellular protein pullulanase were replaced by the mature forms of the normally periplasmic Escherichia coli proteins beta-lactamase or alkaline phosphatase. In E. coli strains expressing all pullulanase secretion genes, pullulanase-beta-lactamase hybrid protein molecules containing an N-terminal 834-amino-acid pullulanase segment were efficiently and completely transported to the cell surface. This hybrid protein remained temporarily anchored to the cell surface, presumably via fatty acids attached to the N-terminal cysteine of the pullulanase segment, and was subsequently specifically released into the medium in a manner indistinguishable from that of pullulanase itself. These results suggest that the C-terminal extremity of pullulanase lacks signal(s) required for export to the cell surface. When beta-lactamase was replaced by alkaline phosphatase, the resulting hybrid also became exposed at the cell surface, but exposition was less efficient and specific release into the medium was not observed. We conclude that proteins that do not normally cross the outer membrane can be induced to do so when fused to a permissive site near the C-terminus of pullulanase.

Biological Transport↗
PubMed1990

Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsiella pneumoniae UNF5023.

The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.

Amino Acid Sequence↗
PubMed1989

A new regulatory locus of the maltose regulon in Klebsiella pneumoniae strain K21 identified by the study of pullulanase secretion mutants.

This study has shown that Klebsiella pneumoniae strain K21 differs from the previously characterized and closely related K. pneumoniae strain PAP996 in that expression of the pullulanase gene (pulA) and other genes of the maltose regulon is partially independent of exogenous inducer (maltose/maltotriose). Mutants of strain K21 which are defective in pullulanase synthesis and/or secretion were isolated following Tn10 mutagenesis. Three phenotypic classes of mutants were identified. Class I mutants were defective in the surface localization and secretion of pullulanase. Class II mutants did not secrete detectable levels of pullulanase but were able to export pullulanase to the cell surface. Class II mutants also expressed pullulanase and other maltose-regulated genes at markedly lower levels than those found in the parent strain under non-inducing conditions. The single class III mutant was intermediate between K21 and class I mutants; most of the cell-associated pullulanase was localized at the cell surface whilst a significant amount was secreted into the medium. Mapping indicated that all but three of the Tn10 insertions were adjacent to, and at either side of, pulA. One class II mutant carried a Tn10 insertion in or close to malT whereas in the remaining class II mutants the insertions were located at least 4 kb upstream of pulA in a region which may define a new regulatory locus of the maltose operon.

Blotting, Southern↗
PubMed1968

Metabolism of the reserve polysaccharide of Streptococcus mitis. Some properties of a pullulanase.

1. A pullulanase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by fractionation with ammonium sulphate. 2. Pullulanase was produced in the absence of inducers, and addition of glucose or maltose to the broth did not increase the yield of enzyme. 3. The pullulanase acted rapidly on alpha-(1-->6)-bonds in substrates having the structure alpha-maltodextrinyl-(1-->6)-maltodextrin, but had no action on isomaltose, 6-alpha-glucosylmaltodextrins or 6-alpha-maltodextrinylglucoses. 4. 6-alpha-Maltotriosylmaltodextrins were hydrolysed over 10 times faster than 6-alpha-maltosylmaltodextrins. 5. The branch linkages of amylopectin phosphorylase limit dextrin, glycogen phosphorylase limit dextrin and glycogen beta-amylase limit dextrin were hydrolysed. The action of pullulanase on amylopectin and glycogen was accompanied by a rise in the iodine stain of 50% and 30% respectively. 6. A reversal of pullulanase action occurred on incubation with high concentrations of maltotriose. Condensation of maltosyl units to form a branched tetrasaccharide occurred less readily. 7. S. mitis pullulanase was rapidly inactivated at temperatures higher than 40 degrees , and the enzyme did not recover activity on storage at room temperature.

Chemical Precipitation↗
PubMedDate not supplied

[Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli].

Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C. thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E. coli cells has been identified. Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined. The pul enzyme was shown to be a protein with molecular weight of approximately 60,000. It was found that both pullulanase and glucoamylase activities resides in pullulanase. The intracellular distribution of pullulanase was studied. An E. coli strain that produces large amounts of thermostable pullulanase has been constructed.

Amino Acid Sequence↗
PubMed1974

Pullulanase synthesis in klebsiella (aerobacter) aerogenes strains growing in continuous culture.

1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.

Culture Media↗
PubMed1989

Cloning and expression of the Clostridium thermohydrosulfuricum alpha-amylase-pullulanase gene in Escherichia coli.

An alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7.0 kb EcoRI fragment using a lambda vector. The gene produced, from an indigenous promoter, active thermostable alpha-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E. coli. Gel filtration separated the active enzyme produced into three peaks, each having both alpha-amylase and pullulanase activities. Immunoblotting after SDS-PAGE revealed more than ten alpha-amylase-pullulanase specific polypeptides; the biggest of these had an Mr of about 165,000, whereas the smallest enzymically active polypeptide had an Mr of about 100,000. Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80-85 degrees C) was only some 5 degrees C lower and the heat stability the same as that of the extracellular alpha-amylase-pullulanase produced by the native host. Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7.0 kb DNA insert.

Bacterial Proteins↗
PubMed1987

A gene fusion approach to the study of pullulanase export and secretion in Escherichia coli.

A series of fusions between the gene for the Klebsiella pneumoniae secreted lipoprotein pullulanase (pulA) and the genes for cytoplasmic beta-galactosidase (lacZ) or periplasmic alkaline phosphatase (phoA) were created by transposon mutagenesis using mini-MudII1681 or TnphoA, respectively. The hybrid genes were expressed in Escherichia coli K-12 with or without the K. pneumoniae genes that promote pullulanase secretion in E. coli. We characterized seven different pulA-lacZ gene fusions encoding hybrid polypeptides containing from 14 to c. 1060 residues of pro-pullulanase. All but the smallest hybrid were fatty acylated and were toxic to producing cells, causing the accumulation of precursors of other exported proteins. Four different pulA-phoA gene fusions encoded hybrids with alkaline phosphatase activity. All four hybrids were fatty acylated, but were not toxic. Although the hybrids were apparently membrane-associated, they were not secreted into the medium either by E. coli carrying pullulanase secretion genes or by K. pneumoniae. Immunofluorescence tests indicated that the pullulanase secretion genes promoted the localization of one of these hybrids to the outer face of the E. coli outer membrane, which may have important implications for the design of live vaccine strains and of immobilized enzymes.

Amino Acid Sequence↗
PubMed1989

Klebsiella pneumoniae strain K21: evidence for the rapid secretion of an unacylated form of pullulanase.

Klebsiella pneumoniae strain PAP996 was previously shown to secrete fatty acylated, aggregated (micellar) pullulanase only after the end of exponential growth. Here we show that the closely related strain K21 secretes large amounts of unacylated, non-aggregated (monomeric) pullulanase during exponential growth. Only a small amount (less than 10%) of the secreted pullulanase was initially retained by the exponentially growing cells to be subsequently secreted in a fatty acylated, aggregated form. Despite the absence of fatty acids in secreted monomeric pullulanase, the effects of the antibiotic globomycin on pullulanase maturation indicated that all of the enzyme synthesized by strain K21 is processed by lipoprotein signal peptidase.

Acylation↗
PubMed1976

Extracellular and protease-released pullulanases.

The extracellular form of pullulanase (EC 3.2.1.41) from Klebsiella aerogenes has been purified to homogeneity by successive chromatography through diethylaminoethyl-cellulose, Sephadex G-200, and 1,6-diaminohexane-Sepharose. In addition, the cell-bound form of pullulanase has been released by the action of a serine endopeptidase obtained from Pronase and purified to apparent homogeneity. Protease-released pullulanase has a slightly larger molecular weight and a specific activity over twice that of the extracellular protein. The properties of each of these forms of pullulanase have been compared with those reported for the detergent-released form. Each form has different features as examined by amino acid composition, specific activity, molecular weight, or inhibition pattern, which distinguish it from the other pullulanases. It is hypothesized that a single gene product consisting of a single polypeptide chain generates these different enzyme forms after selective cleavages by endogenous or applied proteases.

Amino Acids↗
PubMed1986

Extracellular pullulanase of Klebsiella pneumoniae is a lipoprotein.

Pullulanase is a starch-debranching enzyme produced by the gram-negative bacterium Klebsiella pneumoniae. In this organism, the enzyme is first exported to the outer membrane and is subsequently released into the growth medium. Evidence reported here indicates that pullulanase is a lipoprotein. It is apparently synthesized as a precursor with a 19-residue-long signal sequence and modified by the covalent attachment of palmitate to the cysteine residue which becomes the amino terminus after cleavage of the signal sequence. In this respect, pullulanase is similar to some penicillinases produced by gram-positive bacteria which are initially exported to the cell surface and subsequently released into the medium. However, pullulanase and the penicillinases differ in one important aspect, namely, that the extracellular pullulanase still carries the covalently attached fatty acyls, whereas extracellular penicillinases lack the modified amino-terminal cysteine together with a limited number of other residues from the amino terminus.

Amino Acid Sequence↗