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PubMed1994

Molecular characterization of PulE, a protein required for pullulanase secretion.

pulE, one of 14 genes specifically required for pullulanase secretion in Klebsiella oxytoca, codes for a putative nucleotide-binding protein. Subcellular fractionation indicated that the majority of PulE in Escherichia coli cells expressing all 14 secretion genes is mainly associated with the cytoplasmic membrane through both hydrophobic and non-hydrophobic interactions. Mutational analysis revealed that one of the two regions of PulE that are conserved in many nucleotide-binding proteins (Walker box A) is essential for pullulanase secretion. Likewise, mutations that removed aspartate residues from each of two regions immediately downstream from the Walker box A also reduced secretion. These aspartate-rich regions are highly conserved in all 16 known PulE homologues but not in any other nucleotide-binding proteins. Altogether, these results indicate that PulE might belong to a new family of nucleotide-binding proteins. The protein could not be cross-linked to the photoactivatable ATP analogue azido-ATP, however. Most pulE point or deletion mutations which prevented pullulanase secretion exhibited transdominance when expressed at high levels in cells producing wild-type PulE protein. Evidence presented suggests that PulE might be a homodimer.

Adenosine Triphosphatases↗
PubMed1994

Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved. These include an active center and common substrate-binding sites of various amylases. In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times. The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter. Purification of AapT from this recombinant E. coli was performed, and it was shown that the aapT gene product exhibits both alpha-amylase and pullulanase activities with one active site. The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively. Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch. Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. The enzyme also hydrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9).

Amino Acid Sequence↗
PubMed1985

Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae.

Some strains of Klebsiella pneumonia secrete pullulanase, a debranching enzyme which produces linear molecules (maltodextrins, amylose) from amylopectin and glycogen. pulA, the structural gene for pullulanase, was introduced into Escherichia coli, either on a multiple-copy-number plasmid or as a single copy in the chromosome. When in E. coli, pulA was controlled by malT, the positive regulatory gene of the maltose regulon. Indeed, pulA expression was undetectable in a malT-negative mutant and constitutive in a malTc strain. Furthermore, the plasmid carrying pulA titrated the MalT protein. When produced in E. coli, pullulanase was not localized in the same way as in K. pneumoniae. In the latter case it was first exported to the outer membrane, with which it remained loosely associated, and was then released into the growth medium. In E. coli the enzyme was distributed both in the inner and the outer membranes and was never released into the growth medium.

Cell Membrane↗
PubMed1985

Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum.

We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum. These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units. Amylase synthesis was more severely repressed by glucose than by xylose. Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates. Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose. In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats. Therefore, we concluded that amylase synthesis in C. thermohydrosulfuricum was inducible and subject to catabolite repression. The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase. The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.

Cellobiose↗
PubMedDate not supplied

[Isolation and characteristics of thermostable pullulanase from Clostridium thermohydrosulfuricum produced by Escherichia coli cells].

The expressed gene (pul) for a thermostable pullulanase from Clostridium thermohydrosulfuricum was cloned into Escherichia coli. The enzyme was purified from cell extracts of E. coli by thermoinactivation, ammonium sulphate precipitation and gel exclusion. The purified enzyme was characterized as monomer with both pullulanase and glucoamylase activities. The general physico-chemical and catalytic properties of this enzyme were obtained. In particular, pullulanase and glucoamylase activities were stable and optimally active at 65 degrees C. The pH optimum for activity was 5.8. The amino acid composition and amino acid sequence of N-terminal end were estimated.

Chromatography, Gel↗
PubMed2001

Free polymeric bioligands in aqueous two-phase affinity extractions of microbial xylanases and pullulanase.

Two reversibly soluble-insoluble polymers (viz. Eudragit S-100 and alginate) were used as free macroaffinity bioligands in polyethylene glycol (PEG)/salt two-phase systems for separation of enzymes. Incorporation of Eudragit S-100 and alginate in the PEG phase led to considerable selectivity in separation of microbial xylanases and pullulanase, respectively. Xylanase from Aspergillus niger was recovered 93% with 56-fold purification, whereas the enzyme from Trichoderma reesei and Bacillus amyloliquefaciens was obtained with 93% activity recovery (31-fold purification) and 90% activity recovery (32-fold purification), respectively. From Bacillus acidopullulyticus pullulanase, 85% enzyme activity recovery with 44-fold purification was obtained. The approach described here shows the potential of developing into a general approach for use of reversibly soluble-insoluble macroaffinity ligand in two-phase affinity extraction.

Alginates↗
PubMed1991

A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability.

A cell-associated pullulanase (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55,000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr- Ala- Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75-80 degrees C and at 80-85 degrees C, respectively. The enzyme was stable up to 90 degrees C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae----B. acidopullulyticus----B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.

Amino Acid Sequence↗
PubMed1996

Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene.

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (Mr) of 102,069 Da, similar in size to its Mr of about 100,000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3'-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.

Amino Acid Sequence↗
PubMed1990

Five additional genes in the pulC-O operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion.

DNA sequence analysis, Tnpho and Tntac-1, mutagenesis, deletion analysis, expression under bacteriophage T7 gene 10 promoter control, subcellular fractionation and complementation tests were used to study the function of DNA located in the centre of the pulC-O operon from Klebsiella oxytoca strain UNF5023. The characterized region of the operon includes five genes (pulG, pulH, pulI, pulJ and pulK) coding for apparently integral inner membrane proteins which are required for pullulanase secretion. The results presented here and previously show that the pulC-O operon contains at least 11 pullulanase secretion genes.

Amino Acid Sequence↗
PubMed2001

Nucleotide and deduced amino acid sequences of an alkaline pullulanase from the alkaliphilic bacterium Bacillus sp. KSM-1876.

The nucleotide sequence of an alkaline pullulanase-encoding gene from alkaliphilic Bacillus sp. strain KSM-1876 was determined. The open reading frame of the gene encoded 1142 amino acids with a calculated molecular mass of 128739 Da. The alkaline pullulanase showed very limited homology (<32% identity) to previously reported debranching enzymes from prokaryotes and eukaryotes. It contained unique tandem repeats in both the N-terminal and the C-terminal regions.

Amino Acid Sequence↗
PubMed2001

Effect of various flours on the production of thermostable beta-amylase and pullulanase by Clostridium thermosulfurogenes SV2.

The effects of various flours on production of thermostable beta-amylase and pullulanase using Clostridium thermosulfurogenes SV2 was studied in submerged fermentation. Among the flours added to PYE basal medium, potato flour was the best substrate for enzyme production, and under optimal conditions C. thermosulfurogenes SV2 produced 0.87 and 0.98 U of thermostable beta-amylase and pullulanase, respectively, per ml culture broth.

Biomass↗
PubMed2003

Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate.

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.

Alginates↗
PubMed1988

Purification and some properties of the extracellular alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum.

The novel alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum E 101-69 was purified as two forms (I and II) from culture medium, by using gel filtration in 6 M-guanidine hydrochloride as the final step. Renatured alpha-amylase-pullulanase I and II had apparent Mr values of 370,000 +/- 85,000 and 330,000 +/- 85,000 respectively, as determined by native polyacrylamide-gradient-gel electrophoresis. Both forms appear to be dimers of two similar subunits, with Mr values of 190,000 +/- 30,000 for enzyme I and 180,000 +/- 30,000 for enzyme II according to SDS/polyacrylamide-gradient-gel electrophoresis. The two forms had similar amino acid compositions, the same N-terminal sequence (Glu-Ile-Asp-Thr-Ala-Pro-Ala-Ile) and the same pI of 4.25. Both forms contained sugars having mobilities identical with those of rhamnose, glucose, galactose and mannose. The amount of neutral hexoses relative to protein was 11-12% (w/w) for both forms.

Amino Acid Sequence↗
PubMed1998

Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme.

Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1. 41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1-7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands ('substrate-induced forms', numbered I-III) with different specific activities, dissociation constants of the enzyme-substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.

Allosteric Regulation↗
PubMed1999

Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain.

Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B(1)-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B(1)-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves alpha-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.

Glycoside Hydrolases↗
PubMed1975

Role of the tryptophan group in the action of pullulanase of Aerobacter aerogenes.

1. Pullulanase [EC 3.2.1.4.] was inhibited by Hg2+, N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, riboflavine 5'-phosphate, histamine, imidazole, and carbodiimide. 2. The tryptophan groups of the pullulanase were modified by N-bromosuccinimede. It was found that one or two of the 25 tryptophan groups seemed to be important for the activity. 3. Studies on difference spectra using various substrates suggested that the tryptophan groups were important for the formation of an enzyme-substrate complex.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1991

Two distinct steps in pullulanase secretion by Escherichia coli K12.

Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide/sec-gene-dependent general export pathway, and then inducing the pulC-O operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.

Bacterial Proteins↗
PubMed1996

Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24.

A thermostable pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified to homogeneity from Thermus caldophilus GK-24 by chromatographic methods, including gel-filtration and ion-exchange chromatography. The specific activity of the enzyme was increased 431-fold with a recovery of 13.2%. The purified enzyme was a monomer, M(r) = 65 kDa as estimated by SDS-PAGE and gel filtration. The pI was 6.1. The enzyme was most active at pH 5.5. The activity was maximal at 75 degrees C and stable up to 95 degrees C for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at 4 degrees C for 24 h. The activity of the enzyme was stimulated by Mn2+ and Mg2+ ions. Ni2+, Ca2+, Co2+ 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 caused the complete hydrolysis of pullulan to maltotriose. The activity was inhibited by alpha-, beta-, or gamma-cyclodextrins. The N-terminal sequence [(AIa-Pro-Gln-(Asp or Tyr)- Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] showed some similarity to those of bacterial pullulanases.

Amino Acid Sequence↗