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

Outer membrane translocation of the extracellular enzyme pullulanase in Escherichia coli K12 does not require a fatty acylated N-terminal cysteine.

Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids. When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing. Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide. Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini. The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA. Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane. The smaller processed form was also found extracellularly. The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme. Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide. Processing and secretion only occur when the pullulanase secretion functions are expressed.     

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.     

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.     

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.     

Fatty acids covalently bound to alpha-hemolysin of Escherichia coli are involved in the molten globule conformation: implication of disordered regions in binding promiscuity

Alpha-hemolysin (HlyA) is a pore-forming toxin secreted by pathogenic strains of Escherichia coli. The toxin is synthesized as a protoxin, ProHlyA, which is matured in the cytosol to the active form by acylation at two internal lysines, K563 and K689 (HlyA). It is widely known that the presence of fatty acids is crucial for the hemolytic and cytotoxic effects of the toxin. However, no detailed physicochemical characterization of the structural changes produced by fatty acids in the soluble protein prior to membrane binding has been carried out to date. The effects of chemical denaturants, the ANS binding parameters (Kd and n) and the sensitivity to proteases were compared between the acylated and unacylated protein forms HlyA and ProHlyA. Our results are consistent with a molten globular form of the acylated protein. Moreover, because molten globule proteins are intrinsically disordered proteins, using disorder prediction analyses, we show that HlyA contains 9 regions composed of 10-30 natively disordered amino acids. We propose that this conformation induced by covalently bound fatty acids might provide HlyA with the ability to bind to a variety of molecules during its action mechanism.     

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.     

Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate

There is a considerable potential of cold-active biocatalysts for versatile industrial applications. A psychrophilic bacterial strain, Shewanella arctica 40-3, has been isolated from arctic sea ice and was shown to exhibit pullulan-degrading activity. Purification of a monomeric, 150-kDa pullulanase was achieved using a five-step purification approach. The native enzyme was purified 50.0-fold to a final specific activity of 3.0 U/mg. The enzyme was active at a broad range of temperature (10–50 °C) and pH (5–9). Optimal activity was determined at 45 °C and pH 7. The presence of various metal ions is tolerated by the pullulanase, while detergents resulted in decreased activity. Complete conversion of pullulan to maltotriose as the sole product and N-terminal amino acid sequence indicated that the enzyme is a type-I pullulanase and belongs to rarely characterized pullulan-degrading enzymes from psychrophiles.     

Proteome characterization of the culture supernatant of Mycobacterium bovis in different growth stages

This study aimed to identify proteins secreted by Mycobacterium bovis into culture medium at different stages of bacterial growth. A field strain of M. bovis was grown in Middlebrook 7H9 media and culture supernatant was collected at three-time points representing three different phases of growth (early exponential, late exponential, and stationary phases). Supernatants were double filtered, digested by trypsin and analyzed by LC-MS/MS. The study found 15, 21, and 16 proteins in early, mid and late growth phases, respectively. In total, 22 proteins were identified, 18 of which were reported or predicted to have a cell wall or extracellular localization. To our knowledge, this is the first study to identify proteins secreted into the culture medium by a field strain of M. bovis in three different stages of growth. The dataset generated here provides candidate proteins with the potential for the development of serological diagnostic reagents or vaccine for bovine tuberculosis. Data are available via ProteomeXchange with identifier PXD017817.     

Dependence of Escherichia coli hyperbaric oxygen toxicity on the lipid acyl chain composition.

This study examines certain membrane-related aspects of oxygen poisoning in Escherichia coli K1060 (fabB fadE lacI) and its parent strain, K-12 Ymel. Cells were grown to exponential or stationary phase in a minimal medium and exposed to air plus 300 lb/in2 of O2 as a suspension in minimal salts. After an initial lag, both strains lost viability with apparent first-order kinetics. Hypebaric oxygen was more toxic to cells harvested during the exponential phase of growth than to cells harvested from the stationary phase of growth for both strains K-12 Ymel and K1060. Control suspensions exposed to air plus 300 lb/in2 of N2 did not lose viability during a 96-h exposure. The sensitivity of the unsaturated fatty acid auxotroph, strain K1060, to hyperbaric oxygen increased as the degree of unsaturation of the fatty acid supplement increased. Cells grown with a cyclopropane fatty acid (9,10=methylenoctadecanoate) were the most resistant; cells grown with a monounsaturated fatty acid (oleate) were intermediate; and those grown with polyunsaturated fatty acids (linoleate and linolenate) were most sensitive to hyperbaric oxygen. The parent strain, K-12 Ymel, lost viability in hyperbaric oxygen most similarly to strain K1060 supplemented with oleate. To determine the relative effect of hyperbaric oxygen on the survival of E. coli with saturated membranes, substrains of K1060 were selected for growth on 12-methyltetrade-canoate or on 9 or 10-monobromostearate. Substrains grown with a saturated fatty acid supplement were equally or more sensitive to hyperbaric oxygen than when the same substrains were grown with a cyclopropane fatty acid supplement. The lipid acyl chain composition was determined in E. coli K1060 before and after exposure to hyperbaric oxygen or hyperbaric nitrogen. The proportion of nonsaturated acyl chain lipid of either the oleate- or the 9,10-methyleneoctade-canoate-supplemented K1060 remained unchanged after hyperbaric gas exposure. In strain K1060 supplemented with linoleate and grown to stationary phase, however, the relative unsaturated acyl chain content after hyperbaric exposure decreased in both gases. This finding prompted an investigation of the role of lipid oxidation in hyperbaric oxygen toxicity. Assays of potential lipid oxidation products were performed with linoleate-grown cells. The lipid hydroperoxide and peroxide content of the lipid extract increased by 6.9 times after 48 h of air plus 300 lb/in2 of O2; malondialdehyde and fluorescent complex lipid oxidation products showed much smaller or no changes. Lipid extracts from hyperbaric oxygen-exposed cells were not toxic to viable E. coli K1060, nor did they increase the rate of loss of viability in cells simultaneously exposed to hyperbaric oxygen. Linoleic acid hydroperoxide at 1.0 mM had no effect on the viability of E. coli K-12 Ymel and only marginally decreased the viability of E. coli K1060 supplemented with linoleate. We conclude that the kinetics of oxygen toxicity in E...     

The role of the lipoprotein sorting signal (aspartate +2) in pullulanase secretion

The analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino-proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extracellular secretion is an aspartate at position +2, immediately after the fatty acylated amino-terminal cysteine. To see whether the requirement for this amino acid is related to its proposed role as a cyto-plasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase-specific secretion genes. A non-secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin. In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightty lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase. Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c. 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase. When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak. When this peak fraction was applied to a floatation gradient, the pullulanase activity remained at the bottom while the NADH oxidase floated to the top. Thus, there is no evidence that lipoproteins that cofractionate with the cytoplasmic membrane under certain conditions are actually associated with the membrane. Instead, the results support our previous proposal that lipoproteins with an aspartate +2 residue are specifically enriched in a distinct domain of the cell envelope that contains material from both the cytoplasmic and the outer membranes. Possible explanations for the requirement for the aspartate residue in pullulanase secretion are discussed.     

Characterization of Altered Forms of Glycyl Transfer Ribonucleic Acid Synthetase and the Effects of Such Alterations on Aminoacyl Transfer Ribonucleic Acid Synthesis In Vivo

The glycyl transfer ribonucleic acid (tRNA) synthetase (GRS) activities of several Escherichia coli glyS mutants have been partially characterized; the K(m) for glycine and the apparent V(max) of several of the altered GRS differ significantly from the parental GRS. Paradoxically, some of the altered forms exhibit more activity in vitro than the GRS from a prototrophic strain (GRS(L)); several parameters of these activities have been studied in an attempt to resolve this problem. The amount of acylated tRNA(Gly) in vivo was examined to assess the GRS activities inside the cells. During exponential growth in media containing glycine, moderate amounts of acylated tRNA(Gly) occur in the glyS mutants; glycine deprivation leads to a dramatic drop in the amount of acylated tRNA(Gly). An alternative measure of the in vivo activities of the altered enzymes is the efficiency of suppression of the trpA36 locus by su(36) (+); glyS mutants grown with added glycine exhibit one-third to one-fourth the suppression efficiency of the prototrophic glyS(H) parent, presumably because they are less efficient, even in the presence of high levels of glycine, in charging the tRNA(Gly) species which functions as the translational suppressor.     

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.     

Fatty acid synthesis in Escherichia coli

1. Fatty acid formation by cells of a strain of Escherichia coli has been studied in the exponential, post-exponential and stationary phases of growth. 2. During the exponential phase of growth, the metabolic quotient (mmumoles of fatty acid synthesized/mg. dry wt. of cells/hr.) for each fatty acid in the extractable lipid was constant. 3. The newly synthesized fatty acid mixtures produced during this phase contained hexadecanoic acid (41%), hexadecenoic acid (31%), octadecenoic acid (21%) and the C(17)-cyclopropane acid, methylenehexadecanoic acid (4%). 4. As the proportion of newly synthesized material increased, changes in the fatty acid composition of the cells during this period were towards this constant composition. 5. Abrupt changes in fatty acid synthesis occurred when exponential growth ceased. 6. In media in which glycerol, or SO(4) (2-) or Mg(2+), was growth-limiting there was a small accumulation of C(17)-cyclopropane acid in cells growing in the post-exponential phase of growth. 7. Where either NH(4) (+) or PO(4) (3-) was growth-limiting and there were adequate supplies of glycerol, Mg(2+) and SO(4) (2-), there was a marked accumulation of C(17)-cyclopropane acid and C(19)-cyclopropane acid appeared. 8. Under appropriate conditions the metabolic quotient for C(17)-cyclopropane acid increased up to sevenfold at the end of exponential growth. Simultaneously the metabolic quotients of the other acids fell. 9. A mixture of glycerol, Mg(2+) and SO(4) (2-) stimulated cyclopropane acid formation in resting cells.     

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.     

Thermostable Amylolytic Enzymes from a New Clostridium Isolate

A new Clostridium strain was isolated on starch at 60°C. Starch, pullulan, maltotriose, and maltose induced the synthesis of α-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 α-amylase and pullulanase into the culture broth; but also by a decrease of total activity. α-Amylase, pullulanase, and α-glucosidase were active in a broad temperature range (40 to 85°C) and displayed temperature optima for activity at 60 to 70°C. During incubation with starch under aerobic conditions at 75°C for 2 h, the activity of both enzymes decreased to only 90 or 80%. The apparent K_m values of α-amylase, pullulanase, and α-glucosidase for their corresponding substrates, starch, pullulan, and maltose were 0.35 mg/ml, 0.63 mg/ml, and 25 mM, respectively.     

Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end.

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.     

Post-translational modifications of apolipoprotein A-I and Po proteins in the avian peripheral nerve

Apolipoprotein A-I (apo A-I), a soluble lipid transporter, and Po, the major glycoprotein of myelin, are actively synthesized during myelination. To explore the status of post-translational modifications of these proteins in the avian PNS during rapid myelination, endoneurial slices from one day old chick sciatic nerves were incubated with various radioactive precursors that could serve as indicators of such processes. The proteins were isolated from the incubation medium (secreted fraction), the 1% Triton-X-100-soluble intracellular-endoneurial (intracellular) fraction, and myelin-related and purified compact myelin fractions by immunoprecipitation with monospecific anti-apo A-I or anti-Po antisera. Our results demonstrated that secreted apo A-I is fatty acylated, but not phosphorylated or sulfated. Avian Po protein was phosphorylated by a phorbol ester sensitive protein kinase. Sulfation, as well as fatty acylation, of avian Po protein was observed in organ culture using highly sensitive methods of detection. These results indicate that fatty acylation of secreted apo A-I and phosphorylation, sulfation and fatty acylation of Po have been conserved during evolution, and that these post-translational modifications may play a common function in various species.     

Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183

Summary A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The K _m for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca²⁺. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups. Offprint requests to: B. C. Saha