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PubMed2002

Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E.

The bifunctional activities of alpha-amylase and pullulanase are found in the cloned recombinant amylopullulanase. It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E. An estimated 109-kDa recombinant protein was obtained from the cloned gene under the prokaryotic expression system. The optimum pH of the recombinant amylopullulanase was 6.0. The most stable pH for the alpha-amylase and pullulanase activity was 5.5 and 5.0, respectively. The optimum temperature for the alpha-amylase activity was 90 degrees C, while its most stable temperature was 80 degrees C. Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75 degrees C, respectively. Pullulan was found to be the best substrate for the enzyme. The enzyme was activated and stabilized by the presence of Ca2+, whereas EDTA, N-bromosuccinimide, and alpha-cyclodextrin inhibited its bifunctional activities. A malto-2-4-oligosaccharide was the major product obtained from the enzymatic reaction on soluble starch, amylose, amylopectin, and glycogen. A single maltotriose product was found in the pullulan hydrolysis reaction using this recombinant amylopullulanase. Kinetic analysis of the enzyme indicated that the Km values of alpha-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the Vmax values were 39 and 98 micromol/(min x mg of protein), respectively.

Bacterial Proteins↗
PubMed2002

Chemoenzymatic syntheses of linear and branched hemithiomaltodextrins as potential inhibitors for starch-debranching enzymes.

Oligosaccharides embodying the S-maltosyl-6-thiomaltosyl structure have been readily synthesised by using convergent chemoenzymatic approaches. The key steps for the preparation of these molecules involved: 1) transglycosylation reactions of maltosyl fluorides onto suitable acceptors catalysed by the bacterial transglycosylase, cyclodextrin glycosyltransferase (CGTase), and 2) the SN2-type displacement of a 6-halide from acetylated acceptors by activated 1-thioglycoses. The target molecules, which were obtained in good overall yields, proved to be useful for investigating substrate binding in the active sites of several enzymes that act upon the alpha-1,6-linkage of pullulan and/or amylopectin. The compounds exhibit Ki values in the 2.5-1350 microM range with the different enzymes, and the highest affinity found by using these molecules was seen for the pullulanase from Bacillus acidopullulyticus. Both barley-malt limit dextrinase and pullulanase type II from Thermococcus hydrothermalis only recognised the longest linear thiooligosaccharide, while a branched heptasaccharide was the strongest inhibitor of pullulanase from Klebsiella planticola.

Acetylation↗
PubMed1978

Action of Pseudomonas isoamylase on various branched oligo and poly-saccharides.

Pseudomonas isoamylase (EC 3.2.1.68) hydrolyzes (1 linked to 6)-alpha-D-glucosidic linkages of amylopectin, glycogen, and various branched dextrins and oligosaccharides. The detailed structural requirements for the substrate are examined qualitatively and quantitatively in this paper, in comparison with the pullulanase of Klebsiella aerogenes. As with pullulanase, Ps. isoamylase is unable to cleave D-glucosyl stubs from branched saccharides. Ps. isoamylase differs from pullulanase in the following characteristics: (1) The favored substrates for Ps. isoamylase are higher-molecular-weight polysaccharides. Most of the branched oligosaccharides examined were hydrolyzed at a lower rate, 10% or less of the rate of hydrolysis of amylopectin. (2) Maltosyl branches are hydrolyzed off by Ps. isoamylase very slowly in comparison with maltotriosyl branches. (3) Ps. isoamylase requires a minimum of three D-glucose residues in the B- or C-chain.

Glycoside Hydrolases↗
PubMedDate not supplied

Adhesion of Aureobasidium pullulans is controlled by uronic acid based polymers and pullulan.

Aureobasidium pullulans is a potentially pathogenic microfungus that produces and secretes the polysaccharide pullulan and other biomacromolecules, depending on the microbe's physiological state. The role of these macromolecules in mediating adhesion and attachment were examined. Interfacial forces and adhesion affinities of A. pullulans were probed for early-exponential phase (EEP) and late-exponential phase (LEP) cells, using atomic force microscopy (AFM). Biochemical assays showed that A. pullulans produces both pullulan and a uronic acid based polymer. The pullulan is not produced until the LEP, and it can be removed by treatment with pullulanase. Both adhesion forces between the microbe and the AFM tip (silicon nitride) and attachment of the cells to quartz sand grains were controlled by the density of the uronic acid polymer. Uronic acid polymers doubled in density between the EEP and the LEP and were unaffected by the enzyme pullulanase. Retention to quartz in a packed column was quantified using the collision efficiency (alpha), the fraction of collisions between the microbes, and the sand grains, that result in attachment. Adhesion forces and retention on glass were well correlated, with these values being higher for EEP cells (F(adh) = 7.65 +/-4.67 nN; alpha = 1.15) than LEP (F(adh) = 2.94 +/- 0.75; alpha = 0.49) and LEP + pullulanase cells (F(adh) = 2.33 +/-2.01 nN; alpha = 0.43). Steric interactions alone do not describe the adhesion behavior of this fungus, but they do provide information regarding the length and density of the macromolecules studied.

Cell Adhesion↗
PubMed1996

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions.

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fattyacylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins.

Acylation↗
PubMed1970

A study of sieve element starch using sequential enzymatic digestion and electron microscopy.

The fine structure of plastids and their starch deposits in differentiating sieve elements was studied in bean (Phaseolus vulgaris L.). Ultrastructural cytochemistry employing two carbohydrases specific for different linkages was then used to compare the chemical nature of "sieve tube starch" (the starch deposited in sieve elements) with that of the ordinary starch of other cell types. Hypocotyl tissue from seedlings was fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon-Araldite. Treatment of thin sections on uncoated copper grids with alpha-amylase or diastase at pH 6.8 to cleave alpha-(1 --> 4) bonds resulted in digestion of ordinary starch grains but not sieve element grains, as determined by electron microscopy. Since alpha-(1 --> 6) branch points in amylopectin-type starches make the adjacent alpha-(1 --> 4) linkages somewhat resistant to hydrolysis by alpha-amylase, other sections mounted on bare copper or gold grids were treated with pullulanase (a bacterial alpha-[1 --> 6] glucosidase) prior to digestion with diastase. Pullulanase did not digest sieve element starch, but rendered the starch digestible subsequently by alpha-amylase. Diastase followed by pullulanase did not result in digestion. The results provide evidence that sieve element starch is composed of highly branched molecules with numerous alpha-(1 --> 6) linkages.

Aldehydes↗
PubMed1987

Purification and properties of starch hydrolyzing enzymes in mature roots of sugar beets.

Mature roots of sugar beets, which accumulate large amounts of sucrose but not starch, nevertheless contained acid and neutral amylases, judging from their pH optima, as well as pullulanase. Acid and neutral amylases were partially purified by procedures including fractionation with ammonium sulfate, ion exchange column chromatography, and gel filtration. Acid amylase was classified as an exoamylase, since it produced only glucose from soluble starch, amylopectin. beta-limit dextrin, and rabbit liver glycogen. Neutral amylase was classified as an endoamylase, since it liberated maltose as the main product plus a small amount of glucose and oligosaccharides, and was capable of hydrolyzing beta-limit dextrin. Pullulanase was purified to apparent homogeneity by procedures including fractionation with ammonium sulfate, Diethylaminoethyl-cellulose column chromatography and affinity chromatography. Pullulanase was capable of hydrolyzing soluble starch, amylopectin, beta-limit-dextrin, and pullulan. Debranching of amylopectin was further evident by an increase in extinction coefficient, and by a shift of lambda(max) from 530 to 560 nm when the debranched amylopectin formed a complex with I(2)-KI.

Journal Article↗
PubMed1996

The cryptic general secretory pathway (gsp) operon of Escherichia coli K-12 encodes functional proteins.

Systematic sequencing of the Escherichia coli K-12 chromosome (GenBank entry U18997) has revealed the presence of an apparently complete operon of genes (the gspC-0 operon) similar to genes coding for components of the main terminal branch of the general secretory pathway (e.g., the Klebsiella oxytoca pulC-0 pullulanase secretion operon) and to related genes required for type IV pilus biogenesis. For example, the last gene in the gsp operon, gspO (formerly hopD), encodes a protein which is similar to several type IV prepilin peptidases. Expression of gspO from lacZp promotes cleavage of two known prepilin peptidase substrates in E. coli K-12: Neisseria gonorrhoeae type IV prepilin and K. oxytoca prePulG protein. gspO also complements a mutation in the corresponding gene (pulO) of the pullulanase secretion operon when it is expressed from lacZp. Another gene in the gsp operon, gspG (formerly hopG), encodes a protein similar to prePulG, a component of the pullulanase secretion pathway. Expression of gspG from lacZp leads to production of a protein which (i) is recognized by PulG-specific antiserum (and by antiserum against the Pseudomonas aeruginosa PulG homolog XcpG [formerly XcpT]), (ii) is processed in cells expressing gspO, and (iii) restores secretion in cells carrying a pulG mutation. The chromosomal copies of gspG and gspO are apparently not expressed, probably because of very weak transcription from the upstream region, as measured by using a chromosomal gspC-lacZ operon fusion. Thus, the gsp operon of E. coli K-12 includes at least two functional genes which, together with the rest of the operon, are probably not expressed under laboratory conditions.

Bacterial Outer Membrane Proteins↗
PubMed1988

Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15.

The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase.

Amino Acid Sequence↗
PubMed1997

Antibodies to Klebsiella, Proteus, and HLA-B27 peptides in Japanese patients with ankylosing spondylitis and rheumatoid arthritis.

OBJECTIVE: To determine whether patients with ankylosing spondylitis (AS) and patients with rheumatoid arthritis (RA) from Japan have antibodies to Klebsiella pneumoniae and Proteus mirabilis and to assess whether such antibodies are activated against peptides sharing sequences with HLA-B27. METHODS: Serum samples from 152 Japanese patients, 52 with AS, 50 with RA, and 50 healthy controls, were tested against 3 bacteria (K. pneumoniae, P. mirabilis, and Escherichia coli) and 3 synthetic peptides (HLA-B27, pullulanase-D, and scrambled pullulanase-D control peptide) by ELISA under coded conditions. Samples were tested for elevations in IgG, IgA, and IgM antibody classes in patients with active AS or RA, in patients with RA with probable disease, and in patients with inactive AS. Disease activity was determined by an elevated serum C-reactive protein (> 10 mg/l) level and elevated erythrocyte sedimentation rate (> 20 mm/h). RESULTS: Patients with active AS showed specific elevations in serum IgA antibody levels against K. pneumoniae compared to patients with RA and controls (p < 0.001). No such elevation was seen in the IgG and IgM antibody classes. Patients with inactive AS showed no elevation in any class of antibody against K. pneumoniae compared to controls or patients with RA. Patients with active or probably active RA showed significant elevations in IgG antibody levels against P. mirabilis compared to AS and controls (p < 0.001). Patients with AS (active or inactive), RA (active or probably active), and controls showed no elevations in any antibody class to E. coli. Both active and inactive AS patients had specific autoantibodies against HLA-B27 peptide compared to patients with RA and controls (active AS: IgG, IgA, IgM, p < 0.001; inactive AS: IgG and IgA, p < 0.001). Patients with active AS had IgG and IgA antibodies against pullulanase-D peptide, which contains a sequence that cross reacts with HLA-B27 compared to controls (p < 0.001). CONCLUSION: These results provide the first evidence of AS and RA patients in Japan having specific elevations of antibody to K. pneumoniae and P. mirabilis, respectively. This suggests that K. pneumoniae in AS and P. mirabilis in RA may play a role in triggering and/or exacerbating these diseases.

Adult↗
PubMed1993

Regional sequence homologies in starch-degrading enzymes.

The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by alpha-, beta- and glucoamylases (gamma-amylases), cyclodextrinases, alpha-glucosidases, and debranching enzymes. Saccharomyces cerevisiae cannot utilize starch. Our laboratory has previously co-expressed the Bacillus amyloliquefaciens alpha-amylase (AMY) and the Saccharomyces diastaticus glucoamylase (STA2) genes in S. cerevisiae. A gene encoding a debranching enzyme (pullulanase) from Klebsiella pneumoniae ATCC15050 was cloned and its nucleotide sequence determined. This gene will be co-expressed with the alpha- and gamma-amylase to produce an amylolytic S. cerevisiae strain. Extensive data base comparisons of the K. pneumoniae pullulanase amino-acid sequence with the amino-acid sequences of other debranching enzymes and alpha-, beta- and gamma-amylases (from bacteria, yeasts, higher fungi and higher eukaryotes), indicated that these debranching enzymes have amino-acid regions similar to those found in alpha-amylases. The conserved regions in alpha-amylases comprise key residues that are implicated in substrate binding, catalysis, and calcium binding and are as follows. Region 1: DVVINH; region 2: GFRLDAAKH and region 4: FVDNHD. When comparing conserved regions, no similarity could be detected between debranching enzymes and beta- and gamma-amylases.

Amino Acid Sequence↗
PubMed1977

[Cyclodextrin glucanotransferase from Klebsiella pneumoniae. 1. Formation, purification and properties of the enzyme from Klebsiella pneumoniae M 5 al (author's transl)].

1. The strain M 5 al of Klebsiella pneumoniae grows excellently with starches. We were able to show that besides the pullulanase associated with the external membrane of the cells the bacterium produces an inducible, extracellular cyclodextrin glucanotransferase [1,4-alpha-D-glucan-4-alpha-(1,4-alpha-glucano)-transferase (cyclising) (EC 2.4.1.19)]. Potato starch and cyclohexaamylose or cycloheptaamylose were found to be the best "inducing" carbon sources for the synthesis of the enzyme. When the bacteria are grown batchwise, maltose is a poorly "inducing" carbon source; larger quantities of the enzyme are synthesized by continuous cultivation with maltose as growth limiting factor. 2. For the determination of the cyclodextrin glucanotransferase-activity an assay method wsa worked out. 3. The enzyme could be separated from the culture filtrate and purified to more than 90% in few steps. At a total yield of 61.2% related to the activity of the culture filtrate employed we received an enzyme solution with the specific activity of 26.6 units/mg protein. Some properties of the enzyme are described. 4. The products formed from amylopectin by the enzyme were analyzed. Somewhat more than half the amylopectin was found as cyclodextrins. 29.3% of the cyclodextrin fraction were cycloheptaamylose, 47.2% cyclohexaamylose and 10.7% exo-branched cyclohexaamylose. 12.8% of cyclohexaamylose were obtained from a cyclodextrin glucanotransferase-limit dextrin after debranching by pullulanase and exposing the product to the action of the glucanotransferase again. 5. The importance of the cyclodextrin glucanotransferase for the utilization of starches by this strain of Klebsiella pneumoniae is discussed. After a first characterization the enzyme is compared to the amylase of Bacillus macerans.

Amylopectin↗
PubMed1993

Starch- and glycogen-debranching and branching enzymes: prediction of structural features of the catalytic (beta/alpha)8-barrel domain and evolutionary relationship to other amylolytic enzymes.

Sequence alignment and structure prediction are used to locate catalytic alpha-amylase-type (beta/alpha)8-barrel domains and the positions of their beta-strands and alpha-helices in isoamylase, pullulanase, neopullulanase, alpha-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes--all enzymes involved in hydrolysis or synthesis of alpha-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of beta-->alpha loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved beta-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.

Amino Acid Sequence↗
PubMed1990

Substrate competition and specificity at the active site of amylopullulanase from Clostridium thermohydrosulfuricum.

A highly thermostable pullulanase purified from Clostridium thermohydrosulfuricum strain 39E displayed dual activity with respect to glycosidic bond cleavage. The enzyme cleaved alpha-1,6 bonds in pullulan, while it showed alpha-1,4 activity against malto-oligosaccharides. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as the two competing substrates were used to distinguish the dual specificity of the enzyme from the single substrate specificity known for pullulanases and alpha-amylases.

Binding Sites↗
PubMed1992

The substrate specificity of isoamylase and the preparation of apo-glycogenin.

A new facet of the specificity of the glycogen-debranching enzyme, isoamylase, namely, the hydrolysis of a carbohydrate-amino acid linkage, is described. This bond joins the terminal, reducing-end D-glucose unit of glycogen to the hydroxyl group of tyrosine in glycogenin, the primer protein for glycogen biogenesis. The specificity was further defined by demonstrating that 4-nitrophenyl alpha-maltotrioside and higher homologs also act as substrates. The splitting of the glycogen-glycogenin bond by isoamylase indicates the alpha-anomeric configuration of the terminal D-glucose unit. It also provides a means of preparing apo-glycogenin. Pullulanase, a somewhat similar starch- and glycogen-debranching enzyme, does not split these new isoamylase substrates, permitting the 4-nitrophenyl saccharides to be used in distinguishing between isoamylase and pullulanase.

Glucosyltransferases↗
PubMed1993

On-line immunoanalysis for bioprocess control.

Immunoanalytical techniques such as ELISA are often used for the detection of proteins produced in cultivation processes. Owing to the difficulty of automating of the time-consuming traditional ELISA, there is an intense demand for a suitable on-line monitoring method. Combining well-known immunoassays with the FIA technique, we present the heterogeneous and the turbidimetric immuno-FIA methods. The following proteins were investigated with these FIA methods: thermostable pullulanase, IgG, antithrombin III, and recombinant tissue-type plasminogen activator. In the cases of pullulanase and monoclonal mouse IgG, the turbidimetric immuno-FIA was used for on-line analysis of the cultivation process. Results are presented here to demonstrate the effectiveness and application of these immunoanalysis.

Antithrombin III↗
PubMed1995

Purification and characterization of an alkaline amylopullulanase with both alpha-1,4 and alpha-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378.

The novel alkaline amylopullulanase produced by alkalophilic Bacillus sp. KSM-1378 was purified to an electrophoretically homogeneous state from culture medium. The purified enzyme was a glycoprotein with an apparent molecular mass of about 210 kDa and an isoelectric point of pH 4.8. The N-terminal amino acid sequence was Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and showed no homology to the N-terminal regions of other amylopullulanases reported to date. The enzyme was able to attack specifically the alpha-1,6 linkages in pullulan to generate maltotriose as the major end product, as well as the alpha-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and alpha-amylase activities were pH 9.5 and 50 degrees C and pH 8.5 and 50 degrees C respectively. Both activities were strongly inhibited by well characterized inhibitors, such as diethyl pyrocarbonate and N-bromosuccinimide. The pullulanase activity was specifically inactivated by Hg2+ ions, alpha-cyclodextrin and beta-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of alpha-1,4-linked substrates and one for the cleavage of alpha-1,6-linked substrates.

Amino Acid Sequence↗
PubMed1993

Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae.

Pleiotropic transposon insertion mutants of Vibrio cholerae that are unable to secrete enterotoxin, HA/protease and chitinase through the outer membrane have been isolated. The gene, epsM, responsible for complementation of two of the Tn5 insertion mutations was sequenced. It encodes a putative cytoplasmic membrane protein of 18.5 kDa that exhibits similarity to proteins required for extracellular secretion of pullulanase, pectate lyase or elastase in other Gram-bacteria. It is present on a 15-kb DNA fragment from the V. cholerae genome, containing the epsE gene that was previously shown to be required for secretion of cholera toxin [Sandkvist et al., Gene 123 (1993) 81-86]. Partial reading frames flanking epsM also demonstrated similarity to genes required for extracellular secretion of pullulanase in Klebsiella oxytoca.

Amino Acid Sequence↗